2. A2! .5. o f” I m7 , $1....th , n ‘ .fi; a: re. fig! 2.“... , y- .. n {th . Mag? .\ 6.1.7,? .. , grands . .HMWevJ 6 ...v:. 312:» I}! is a I»! .3, Griffin. w $5... 1: EU, Ho 3f4a§6é§a3§xz fi.~r.¢§% ‘ figfiéfimfi d. F H3... . 1.2.5: xii... THESIS n.— This is to certify that the dissertation entitled MUTATIONAL ANALYSIS OF PHOTOSYSTEM 1 IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803 presented by Jianping Yu has been accepted towards fulfillment of the requirements for Ph.D. , Botany & Plant Pathology degreein Major professor Date January 23, 1997 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 3 1293 01579 5960 LIBRARY I Mlchlgan State t Unlverslty ,‘ RNBOXtoncnwomtehockMMyourrocord. PLACE ll RETU baton dd. duo. TO AVOID FINES return on or ATE DUE DATE DUE DATE DUE MSU Is An Affirmative Wand Oppomnny Inflation Wan-w MUTATIONAL ANALYSIS OF PHOTOSYSTEM I IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803 By Jianping Yu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1997 ABSTRACT MUTATIONAL ANALYSIS OF PHOTOSYSTEM I IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803 By J ianping Yu The focus of this study is an investigation of the assembly, structure and function of photosystem I (PSI) in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synechocystis sp. PCC 6803 is an excellent organism for the study of PSI because its genome has been completely sequenced and it is readily transfonnable. It also has an active homologous recombination system, and a photosynthetic apparatus much like that of higher plants. Furthermore, it may be grown heterotrophically. The PsaA and PsaB subunits of the PSI complexes form a heterodimer that contains the primary electron donor P700, and the electron carriers A0, A1, and F x. The PsaC subunit provides ligands for two [4Fe-48] clusters, denoted F A and F8. When the gene for PsaC was inactivated and PsaC was absent, subunits PsaD and PsaE did not accumulate in thylakoid membranes, whereas PsaA and PsaB did accumulate and electrons could move between P700 and Fx. A set of site—directed PsaC mutants was made in which a cysteine ligand to F A (C51) or FB (C14) was substituted by aspartate, serine, or alanine. All mutant strains were unable to grow photoautotrophically or mixotrophically (in the presence of glucose) under white light at an intensity of 22 umol m‘2 s". Photoautotrophic and mixotrophic growth may be rescued by red light. Mixotrophic grth may also be rescued by addition of a specific inhibitor of PSH, DCMU, to the growth medium. Mutant cells grown under light-activated-heterotrophic-growth (LAHG) conditions showed lower capacity of whole-chain oxygen evolution than did wild-type cells. Thylakoids from all these mutants have lower or undetectable levels of PsaC, PsaD, and PsaE, and lower PSI capacity. EPR and optical spectroscopic studies showed that the electron transfer efficiency of PSI complex is also decreased by the mutations, and that mixed-ligand [4Fe- 48] clusters that formed at the mutated sites are capable of accepting electrons. Spontaneous pseudorevertants of primary PSI mutant strains have been isolated and genetically characterized. Suppressor mutations in some pseudorevertants have been localized by functional complementation and sequencing. ACKNOWLEDGMENTS I wish to express my deepest gratitude to those people who have shared their precious time, knowledge, experience, and enthusiasm with me in the process of completing this project. I am pleased to have had the opportunity to exchange ideas and techniques with my collaborators at University of Nebraska: Dr. Yean-Sung Jung, Dr. Ilya Vassiliev, and Dr. John Golbeck. I am indebted to Dr. Lawrence Smart for sharing his techniques and helping me to start this project. I have been very fortunate to have had the enthusiastic support and encouragement of my advisory committee, Dr. Kenneth Keegstra, Dr. Barbara Sears, Dr. C. Peter Wolk, and Dr. Charles Yocum. Equally supportive have been past and present members in the McIntosh lab, especially Dr. Neil Bowlby, Dr. Carrie Hiser, Dr. Rudiger Schulz, Dr. Denis Maxwell, Yong Wang, and Roxy Nickels. I must give a great deal of credit for the success of this project to Dr. Lee McIntosh, who has given unending support and guidance. Finally, I would like to thank my wife Yali Li for her loving understanding and support, and our son Shian who has given me so much pride and fun. iv TABLE OF CONTENTS LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ........................................................................................................ ix LIST OF ABBREVIATIONS ......................................................................................... xi CHAPTER 1 INTRODUCTION ........................................................................................................... l Photosynthesis Photosystem I PSI Components Electron Transfer Reactions PSI Structure Genetic Manipulation of PSI in a Model Organism Sjmechocystis sp. PCC 6803 Focus of this Study References CHAPTER 2 ABSENCE OF PsaC SUBUNIT ALLOWS ASSEMBLY OF PHOTOSYSTEM I CORE BUT PREVENTS THE BINDING OF PsaD AND PsaE IN SYNECHOCYSHS SP. PCC 6803 .............................................................................................................................. 21 Introduction Materials and methods Materials Strains and Growth Conditions Nucleic Acid Manipulations psaC Interposon Mutagenesis Transformation of Synechocystis 6803 and Selection Conditions Membrane Isolation and Analysis Thylakoid Membrane Preparations for EPR and Optical Measurements In vitro Reconstitution Electron Paramagnetic Resonance Spectroscopy Flash—induced Absorption Change Results Southern and Northern Analysis Growth Characteristics Detection of Proteins by Antibodies EPR Spectral Characterization of CDK25 Thylakoid Membranes Optical Characterization of Thylakoid Membrane of CDK25 Discussion Assembly of the PSI Core (PsaA-PsaB Dimer) is Independent of PsaC, PsaD and PsaE Subunits in Synechocystis 6803 Assembly of PsaD and PsaE Into the PSI Complex is Dependent on PsaC Acknowledgments References CHAPTER 3 STRAINS OF SYNECHOCYSTIS SP. PCC 6803 WITH ALTERED PsaC. I. MUTATIONS INCORPORATED IN THE CYSTEINE LIGANDS OF THE TWO [4Fe-48] CLUSTERS F A AND FB OF PHOTOSYSTEM I ............................................ 47 Introduction Materials and Methods Strains and Grth Conditions DNA Manipulations Transformation of Synechocystis 6803 and Selection Conditions Western Blot Analysis of Thylakoid Membrane Proteins Oxygen Evolution Membrane Isolation and Purification of PS I Complex Steady State Reductase Activity Time-Resolved Optical Absorption Spectroscopy Results Genetic Characteristics of the PS I Mutants Construction and Characterization of AC-RCPT PsaC Mutagenesis and Genetic Characterization of the Mutants Physiological Characterization of the PS I Mutants Growth Analysis of the Mutants Oxygen Evolution of the Mutant Cells Rates of NADP+ Photoreduction Biochemical Characterization of the PS I Mutants PS I Subunit Composition in the Mutants Single Turnover Flash Studies of Mutant PS I Complexes Discussion PsaC Mutations and PS I Biogenesis PsaC Mutations and Electron Transport References CHAPTER 4 STRAINS OF SYNECHOCYSTIS SP. PCC 6803 WITH ALTERED PsaC. II. EPR AND OPTICAL SPECTROSCOPIC PROPERTIES OF F A AND FB WHEN CYSTEINES 14 AND 51 ARE REPLACED BY ASPARTATE, SERINE AND ALANINE ................... 77 Introduction Results and Discussion Summary of Results Number of Electron Acceptors Present in the C14X—PS I and C5 lX—PS I Mutants Comparison with in vitro Reconstituted C14D-PS I and C51D-PS I Mutant Complexes References CHAPTER 5 ISOLATION AND CHARACTERIZATION OF PSEUDOREVERTANT S FROM SITE- DIRECTED PSI MUTANT S ........................................................................................ 87 Introduction Isolation and Genetic Characterization of Pseudorevertants from PSI Mutants Isolation of Pseudorevertants from the PsaC Mutants Genetic Conformation for Suppressor Mutations Suppressor Mutations Can be Localized to Other Proteins Implication for Position and Function of F A and FB Secondary Mutation Can Be at the Same Site as the Primary Mutation Genetic Transformation in Synechocystis 6803 Comparison of Grth Conditions for Primary PSI Mutants Comparison of Methods for Preparation of Transforming DNA Competency of the PSI Mutant cultures at Various Stages of Log Phase Materials and Methods Strains and Grth Conditions DNA Manipulations References CHAPTER 6 DISCUSSION AND POTENTIAL FUTURE EXPERIMENTS .................................. 106 Summary of the study on the PsaC mutants The Mechanisms of Suppressor Mutations Restoring PSI Activity Light Inhibition of PSI Mutants Identification of Suppressor Mutations in the Genome of Synechocystis 6803 Combinatorial Mutagenesis: Determination of Properties and Function of Fe-S Clusters by Protein Environment References vii LIST OF TABLES Table Page 3.1- Physiological characterization of the PsaC mutants .................................................. 59 5.1- Transformation frequencies for LAI-IG and mixotrophically grown C51D cells ........ 99 viii LIST OF FIGURES Figure Page 1.1- Scheme for electron transport from water to NADP+ ................................................. 3 1.2- A model for the organization of PSI subunits and electron transfer components ......... 6 2.1- Partial restriction map of the plasmid p61-2.4k2 ...................................................... 26 2.2- Southern and Northern analysis of CDK25 .............................................................. 30 2.3- Grth of CDK25 under mixotrophic conditions .................................................... 32 2.4 -Westem blot analysis of CDK25 .............................................................................. 34 2.5- EPR spectra for Fx .................................................................................................. 36 2.6- EPR spectra for F A and FB ....................................................................................... 37 2.7- Optical spectroscopy for CDK25 ............................................................................. 39 3.1- Predicted structure of PsaC ..................................................................................... 50 3.2- PsaC mutagenesis strategy ...................................................................................... 56 3.3- Western blots of thylakoid membranes of PsaC mutants .......................................... 62 3.4- Western blots of purified mutant PSI complexes ..................................................... 64 3.5- Kinetics of AA,20 absorbance changes (I) ................................................................. 66 3.6- Kinetics of AA,20 absorbance changes (11) ............................................................... 68 5.1- A flow chart for isolation and genetic characterization of a ‘typical’ pseudorevertant from a PSI mutant ....................................................... 90 5.2- In situ dot transformation demonstrates suppressor mutation ................................... 94 ix 5.3- Arrangement of the geometrical centers of PSI electron carriers .............................. 96 6.1- Over-reduction of the shared components inhibits grth ...................................... 110 6.2- A shot-gun cloning vector, pJY 4 ........................................................................... 113 2’ ATP chl COX DCBQ DCMU DCIP DM EPR or ESR FA Fa Fd LHC Mb NADPH, NADP+ P680, P700 PAGE LIST OF SYMBOLS AND ABBREVIATIONS the first electron acceptor in PSI, a chlorophyll the secondary electron acceptor in PS I, a phylloquinone adenosine 5'-triphosphate chlorophyll cytochrome oxidase cytochrome 2,6-dichloro-p-benzoquinone 3 -(3 ,4-dichlorophenyl)-1, 1 -dimethyl-urea, diuron 2,6-dichlorophenol-indophenol n-dodecyl B-D-maltoside electron paramagnetic resonance or electron spin resonance [4Fe-4S] center A in PSI [4Fe-48] center B in PSI ferredoxin [4Fe-48] center X in PSI gentamycin kilobases or kilobase pairs kilodalton kanamycin light-activated heterotrophic growth light harvesting complex million basepairs nicotinamide adenine dinucleotide phosphate, reduced and oxidized the primary electron donor in PSH and PS 1, respectively. Chlorophyll a dimers polyacrylarrride gel electrophoresis pas pcc PCR PSI, P811 5135 Wt AAm 1'.’ phycobilisome Pasteur Culture Collection polymerase chain reaction photosystem I or 11 sodium dodecyl sulfate wild type photoinduced absorbance change at 820 nm life time of exponential decay component xii Chapter 1 INTRODUCTION PHOTOSYNTHESIS Light is of fundamental importance for life on earth. Through photosynthesis, light ultimately provides the sole source of energy for the grth and development of almost all life. Photosynthesis, a series of chemical reactions used by plants, algae, and some bacteria to convert light energy into chemical energy, dramatically affects the earth and its atmosphere. This series of chemical reactions is responsible for generating the 02 that we breathe and for fixing CO2 into organic compounds. Plants, algae, and cyanobacteria perform oxygenic photosynthesis, evolving 02 from the light-driven oxidation of water and transferring electrons through a linear series of carriers, eventually yielding reducing equivalents, in addition to performing cyclic photophosphorylation. Anoxigenic photosynthesis is characterized by cyclic electron flow through a single reaction center, driving ATP synthesis but not the generation of reducing equivalents. Anoxigenic photosynthesis is performed by species of bacteria and the reaction center has been best characterized structurally in the purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis (Deisenhofer et al., 1985; Allen et al., 1987). Oxygenic photosynthesis (recently reviewed by Nugent, 1996) involves the light- driven flow of electrons from water to reduce nicotinamide adenine dinucleotide 2 phosphate (N ADP*) to NADPH, used as reductant for many anabolic reactions. The electron transport process is accomplished by three thylakoid membrane-bound, multi- subunit protein complexes: photosystem II (PSII), the cytochrome b, f (Cyt b6 f ) complex, and photosystem I (PSI). As electrons are transferred by certain carriers, a proton gradient is generated across the thylakoid membrane. This gradient is released by the flow of protons through the ATP synthase (also called the coupling factor), driving ATP synthesis (Figure 1.1). ATP and NADPH act as substrates for carbon fixation and sugar synthesis, amino acid synthesis, fatty acid synthesis, and other anabolic reactions. In plants and algae, the thylakoids and many of the enzymes involved in biosynthetic processes are compartmentalized within the chloroplasts. The basic requirements for the light-dependent photosynthetic reactions are: light capture, charge separation, and stabilization of charge separation. To use energy efficiently, the charge separation must be quickly stabilized by a series of oxidation- reduction reactions between spatially separate electron carriers. The initial step is the capture of energy from light by antenna pigments on protein complexes such as PSI, PSII, LHCI, LHCII and phycobilisomes. This energy migrates to a special reaction center chlorophyll dimer, which in P811 is termed P680. The energy captured by P680 excites an electron, which is transferred to a pheophytin, which in turn reduces a bound plastoquinone molecule called Q A. Q A reduces a second quinone, QB, which unlike Q A functions as a two-electron accepter (Figure 1.1). On the donor side of PSII, P68; oxidizes a tyrosine residue TyrZ, which in turn oxidizes a cluster of four manganese atoms. The tetrad of Mn atoms accumulates oxidizing equivalents in a series of oxidation ‘ states or S-states, and finally oxidizes water and releases oxygen (Barry et al., 1994). PSH Cyt bgf PSI ATpase NADPH-14* NADPH H+ H+ r—_i i. FNR r _ ,Qa FAT/PB i Q. J, \ \ PQI-I2 Cyt b6 Fx Phco ‘. ~‘. + I x" if At P5?) pQ .4" PCS + Tyr Z Cyt f 20 - P700 M H¢0 02+H+ Hi 3 '''''''''''''''' 1’. pc Figure 1.1. Scheme for electron transport from water to NADP”. CFl and CFO, major coupling factor components; M, the manganese containing component involved in the reduction of water and the release of oxygen; TyrZ, a tyrosine residue and the first electron donor to P680‘; P680, P811 reaction center Chl a; Pheo, pheophytin; Q A and Q3, first and second quinone electron acceptors, respectively; PQ, plastoquinone; PQHZ, plastoquinol; Fe-S, Rieske iron-sulfur center; Cyt f, cytochrome f; Cyt b6, cytochrome b6; PC, plastocyanin; P700, PSI reaction center Chl a; A0 and A,, primary and secondary PSI electron acceptors, respectively; Fx, F A and F3, iron-sulfirr centers; F d, ferredoxin; FNR, ferredoxin-NADP oxidoreductase. Solid lines indicate electron and proton transfers, dashed lines indicate component diffusion. 4 The electrons on Q8 are carried to the Rieske Fe-S center in the Cyt b6 f complex by a mobile pool of plastoquinone (PQ) molecules which shuttle between P811 and the Cyt b6 f complex. Concomitantly with transferring electrons to the Rieske center, PQ transfers protons fiom the stromal to the lumenal side of the thylakoid (Figure 1.1). Cyt f donates single electron to the second mobile carrier of electron transport, plastocyanin, which may be replaced by cytochrome c, in cyanobacteria. The Cyt b6 f complex is shared between photosynthesis and respiration in the cyanobacteria (Kallas, 1994). Plastocyanin or cytochrome c, donates electrons to P700, the reaction center chlorophyll pair in PSI. The antenna of PSI captures the second photon of light needed to complete reduction of NADP* and transfers the energy to P700, creating P700*, the strongest reductant in a biological system (z-1.2V) (Vos and van Gorkom, 1988). P700 passes an electron to a series of electron carriers in PSI, which then donate electrons to ferredoxin (Figure 1.1). In cyanobacteria, flavodoxin can substitute for ferredoxin under conditions of iron-limitation (Ho and Krogrnann, 1982). Ferredoxin then reduces NADP+ to NADPH, a reaction catalyzed by the ferredoxinzNADP+ oxidoreductase (Ho and Krogrnann, 1982). The detailed nature of the electron carriers in PSI will be discussed below. PSI is also capable of catalyzing cyclic electron flow, in which electrons are returned to the cytochrome complex to cycle back to P700 (Kallas, 1994). This process does not reduce NADPZ but does cause formation of a proton gradient for ATP synthesis. PHOTOSYSTEM I PSI components PSI is a heteromultimeric pigment-protein complex that functions as a light-driven plastocyanin-ferredoxin oxidoreductase (Figure 1.2). It contains polypeptides and cofactors that are required for light absorption, charge separation and stabilization. PSI consists of at least 11 different proteins in cyanobacteria and 13 in chloroplasts (Chitnis, 1996). All PSI proteins are believed to be present as one copy per P700 reaction center. They vary considerably in their molecular weights, hydrophobicities, and locations with respect to the lipid bilayer. In addition to proteins, the PSI complex contains approximately 100 Chl a molecules, several B-carotenes, two phyloquinone molecules, and three [4Fe-4S] clusters. The cofactors of PSI are bound to the PsaA, PsaB, and PsaC proteins. The remaining subunits of PSI do not bind any redox centers. Trimers of PSI have been observed by electron microscopy of the photosynthetic membranes of cyanobacteria and are considered to be the firnctional units in vivo (Boekema etal., 1994). In plants and green algae, P81 is localized in the non-appressed regions of the thylakoid membrane, where it is surrounded by a pigment- protein light harvesting complex (LHCI). LHCI functions as an antenna complex, harvesting light energy and funneling it to the PSI reaction center (Preiss et al., 1993). LHCI contains hydrophobic apoproteins with apparent molecular masses of 11 to 24 kD that bind Chl a, Chl b, violaxanthin, and lutein. Such separate, membrane-embedded, PSI-specific LHCs have not been found in cyanobacteria. However, phycobilisomes serve as additional antennae for PSI in cyanobacteria, although they primarily transfers excitation energy to PSH (Fujita et al, 1994). Stroma Thylakoid Membrane Lumen Figure 1.2. A model for the organization of PSI subunits and electron transfer components. PSI subunits PsaA, PsaB, etc, are indicated using letters A, B, etc. PC, plastocyanin; P700, PSI reaction center Chl a dimer; A0 and A,, primary and secondary electron acceptors, respectively; Fx, F A and F3, iron-sulfirr centers; Fd, ferredoxin. Arrowheads indicate electron transfers. 7 In the past 10 years, multi-disciplinary research efl‘orts have led to characterization of the components, the electron transfer reactions, and the structure of PSI. Detailed recent reviews of the structure and function of PSI have been published and provide the reader with a valuable compilation of the current state of understanding of PSI (Chitnis, 1996; Golbeck, 1994). The primary structures of individual PSI proteins and the overall mechanism of PSI firnction are remarkably conserved among cyanobacteria, green algae, and plants. The PsaA (83.0 kDa for Synechocystis sp. PCC 6803) and PsaB (82.4 kDa) proteins form the photoreactive core of P81. Three peripheral proteins PsaC (8.9 kDa), PsaD (15.6 kDa), and PsaE (8.0 kDa) and five integral membrane proteins PsaL (16.6 kDa), PsaK (8.5 kDa), PsaF (15.7 kDa), PsaI (4.3 kDa), and PsaJ (4.4 kDa) are present in the PSI complexes from the cyanobacteria, green algae, and plants. PsaM (3.4 kDa) had been detected in the cyanobacteria] PSI but has yet to be shown in the PSI preparations from eukaryotes. In contrast, PsaG(10-10.8 kDa, in higher plants), PsaI-I (10.2-111 kDa), and PsaN (9 kDa) have been shown only in the PSI preparation from green algae and plants. Genes for all known subunits of PSI have been cloned fi'om several plants, algae, and cyanobacteria. These genes are designated as psaX, where X is assigned in the order of discovery. The protein subunits of eukaryotic PSI are encoded in both chloroplast and nuclear genomes; the psaA, psaB, psaC, p501, and psaJ genes are localized on the chloroplast DNA, and the others are present in the nucleus. The nuclear-encoded PSI proteins are synthesized as precursors with amino-terrninal transit peptides and are imported posttranslationally into chloroplasts. Functions of some PSI proteins have been studied by biochemical approaches. In 8 recent years, many researchers have also used molecular genetic strategies, or “reverse genetics”, to decipher structure-firnction relations in PS1. The cyanobacteria Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002, Anabaena variabilis ATCC 29413, and the green alga Chlamydomonas reinhardtti have been the most popular and versatile model systems to generate PSI mutants. PsaA, PsaB, and PsaC: Binding to redox centers and cofactors. In the late 19803, Golbeck and colleagues developed resolution-reconstitution assays to identify the PSI proteins that bind redox centers. Treatment with chaotropic agents (urea, NaBr, NaI, NaSCN, and NaClO,), followed by sucrose density ultracentrifirgation, was used to prepare a PsaA-PsaB core lacking peripheral subunits, including PsaC (Golbeck et al., 1988b). Such core complexes contain all pigments in the intact PSI and are capable of charge separation and electron transfer to Fx. F A and F8 can be reconstituted in purified PsaC and subsequently assembled on a PsaA-PsaB core (Golbeck et al., 1988a). These analyses demonstrated that PsaA, PsaB, and PsaC bind all electron transfer centers of PSI. Biochemical evidence for the function of PsaA, PsaB, and PsaC has been corroborated by the phenotypes of cyanobacteria] and algal mutants. Inactivation of psaA or psaB results in the absence of P81 in the mutant thylakoids, showing that these subunits cannot form homodimers and that the presence of the PsaA-PsaB heterodimer is essential for the assembly of the complex (Smart et al., 1991; Smart et al., 1993a). Vigorous site-directed mutagenesis of the PSI core has been focused on identifying the residues that bind different cofactors. Conserved cysteinyl residues have been proposed to firnction as F x ligands (Golbeck and Bryant, 1991). As an experimental proof, the replacement of these residues affects Fx and drastically reduces accumulation of PSI in the membranes (Smart 9 et al., 1993b; Vassiliev et al, 1995). The results also demonstrate that F x is essential for stable assembly of the PSI core (Smart et al., 1993b). The absence of PsaC has different effects on the assembly of the core complex in different organisms. The PSI core is unstable in the PsaC-less mutant of C. reinhardtii (Takahashi etal., 1991) but is assembled and firnctional in charge separation in A. variabilis (Mannan et al., 1991). Conserved cysteinyl residues of PsaC have been proposed to function as ligands for F A and F3. A combination of site-directed mutagenesis and in vitro reconstitution has been used to test the proposal. Mutant PsaC expressed in E. coli was reconstituted with a biochemical preparation of a PSI core. These experiments showed that Cys 21, 48, 51, and 54 in PsaC provide ligands to the F A cluster, whereas Cys 11, 14, 17, and 58 are ligated to the FB cluster (Zhao et al., 1992). Similar mutations of ligands to F A and F B were introduced into A. variabilis in vivo, and the mutant strains were shown to grow under photoautotrophic conditions (Mannan et al., 1996) Interaction with soluble electron acceptors: PsaC, PsaD, PsaE, PsaM, and PsaF. Formation of the PSI-ferredoxin (F d) complex precedes electron transfer; the rate constants for complex formation depend on ionic strength, suggesting that there are electrostatic interactions between Pd and PSI. Fd accepts electrons from the F A and FB clusters of PsaC, implying that Fd and PsaC contact each other. The major obstacle in the association between PsaC and Fd is the unfavorable electrostatic interactions; both proteins have strong electro-negative surfaces at physiological pH. Therefore, docking proteins are required to facilitate the interaction by providing amino acid clusters of opposite charges. Numerous recent studies involving biochemical reconstitutions, 10 chemical cross-linking, and subunit-deficient mutants have revealed crucial roles of PsaD and PsaE in the docking of Fd (reviewed in Chitnis et al., 1995). Thus, PsaC, PsaD, and PsaE form the reducing site of PSI, on which F d can dock and accept electrons. On the oxidizing side, PsaF may form a docking site for plastocyanin (F afah et al., 1995). In addition to the linear electron transfer that results in NADP+ photoreduction, PSI also participates in cyclic electron flow. Characterization of cyanobacterial mutant strains has shown that PsaE and PsaM may be involved in cyclic electron flow around PSI (Yu et al., 1993) Role in PSI organization and its interaction with LHCI. Some PSI subunits, such as PsaL, are essential components of the trimeric quaternary structure of PSI, which has been demonstrated in the photosynthetic membranes of cyanobacteria (Boekema et al., 1994). T rimers of PSI cannot be obtained from cyanobacterial mutants that lack PsaL (Chitnis, et al., 1995). Recent characterization of PsaI- and PsaJ-less mutants of Synechocystis sp. PCC 6803 indicates that these polypeptides assist in the correct organization of PsaL and PsaF, respectively, presumably by stabilizing their transmembrane domains in the lipid bilayer (Xu et al., 1995). Electron transfer reactions The PsaA and PsaB proteins of PSI bind the electron transfer components P700, A0, A1, and Fx. The reaction center chl dimer, P700, is the site of the primary photochemical charge separation in PSI. It is named by the wavelength at the peak of the oxidized-minus-reduced difi‘erence spectrum (Kok, 1956). When a photon is absorbed by a PSI antenna pigment and the energy from the photon is transferred to P700, P700 is ll raised to an excited state, P700*, then donates an electron to A0 and forms the cation P700’, which is detectable by EPR spectroscopy. P700’ is reduced by plastocyanin or in cyanobacteria, by cyt c6. P700 has been assigned to PSI crystal structure as a Chl a dimer (Fromme et al., 1996). Two chl molecules have been assigned to the spectroscopically characterized ‘primary’ electron accepter A0. Two additional monomeric ‘accessory’ chlorophylls were found between P700 and A0; their function remains to be determined. Because of the quasi-symmetrical arrangement, it was not possible to identify either one of the chlorophyll molecules as the primary accepter, A0, that is active in electron transport (F romme et al., 1996) A0 has been characterized best by time-resolved optical spectroscopy, in which the flash-induced absorption changes of P700 are measured under highly reducing conditions, and by flash-induced difference spectra. A0 is identified as a chl a molecule (Mathis, et al., 1988). PSI contains two phylloquinone molecules, either one or both of which serves as A1. The next intermediate acceptor is the [4Fe-4S] cluster Fx. The subsequent path of electrons through the [4Fe-4S] clusters F A and FB remains an unresolved area in the electron transfer pathway in PSI (Jung et al., 1995). The electrons may travel in a series: Fx to F3,A to Fm; to Fd. Alternatively, the electrons from Fx may be transferred to F A or FE and then one or both of these reduced clusters can donate electrons to Fd. The proposed locations of these redox centers in the X-ray crystallographic structure are at different distances from Fx, thus implying a serial flow of electrons between FH and F A (Krauss et al., 1993). Selective inactivation of FB with mercurial such as HgCl2 indicates that photoreduction of F A is independent of FB (Jung et al., 1995). Therefore, F3 may be 12 firnctionally and spatially more distant from Fx than is F A. The electron from F A or FB is used to reduce Fd, and the electron lost by P700 is regained by oxidizing plastocyanin. Thus, the PSI complex interacts with soluble electron carrier proteins on both sides of the thylakoid membrane. The interprotein electron transfer on the reducing side of PSI is a complex process. It involves several different PSI-Fd complexes and three different first-order components with half lives of approximately 500 ns, 13-20 us, and 100-123 us (Setif and Bottin, 1995). The 500-ns phase corresponds to electron transfer from F A or FB to Fd. When grown under iron- deficient conditions, cyanobacteria contain flavodoxin as an additional electron acceptor fiom PSI. The structural requirements for efficient electron transfer to Anabaena F d and flavodoxin are highly dependent on the reaction partner (Navarro et al., 1995). On the oxidizing side of PSI, the interaction between plastocyanin and PSI involves a fast phase with a half life of 12 to 14 us and second slower phase with a half life of 200 as (Haehnel et al., 1994). The fast rate can be attributed to the association of plastocyanin close to P700, whereas the slower phase may represent a distant plastocyanin population. In cyanobacteria and green algae, Cyt c6, a second electron donor, is synthesized during growth in copper-depleted medium. Recently, interaction of PSI with plastocyanin or Cyt c6 proteins from diverse organisms was studied by laser flash absorption spectroscopy (Hervas et al., 1995). PSI reduction by plastocyanin or Cyt shows varying kinetics depending on the organism fi'om which the photosystem and metalloproteins are isolated. These findings suggest that PSI was able to first optimize its interaction with positively charged Cyt and that the evolutionary replacement of the ancestral Cyt by plastocyanin, as well as the appearance of the fast kinetic phase in the l3 plastocyanin/PSI system of higher plants, involved structural modifications in both the donor protein and PSI (Hervas et al., 1995). PSI structure Major advances have been made in the past few years in understanding the structure of PSI. Electron microscopic studies have provided valuable information concerning the global shape and size of PSI complexes (Boekema et al., 1994), X-ray crystallography has permitted a more detailed knowledge of secondary and tertiary structural elements in PS1 (Krauss et al., 1993; Schubert et al., 1995), and biochemical studies have revealed some details of PSI topography and subunit interactions. On the basis of the electron density maps, a structural model for trimeric PSI from Synechococcus elongatus was initially proposed at a resolution of 6 A (Krauss et al., 1993). From new data, many aspects of this model were later refined to 4.5 A resolution (Schubert et al., 1995) The crystal analysis of PSI has indicated the location of the [4Fe-4S] clusters F x , F A, and F3, 86 Ch] molecules, 29 transmembrane a helices, and 4 a-helices that are parallel to the plane of the membrane. A monomer of PSI consists of a “catalytic domain” and a smaller “connecting domain” that links monomers to form a trimer. The connecting domain does not extend significantly beyond the lipid bilayer and contains three transmembrane helices which may belong to PsaL and PsaI. The catalytic domain contains all cofactors and protrudes 15 and 35 A into the lumen and stroma, respectively (Chitnis, 1996) Because of the high electron density, the three iron-sulfirr centers of PSI are clearly 14 identified in the crystal structure of PSI. They form an obtuse-angled triangle oriented roughly perpendicular to the plane of the membrane. FM is located at the edge of the membrane plane. From the top view of PSI, FX is at the center of the PSI complex. The other two clusters are located 15.4 and 22.2 A from F :0 respectively. The F A-FB axis is tilted 54° from the membrane perpendicular axis. Two a-helices are present close to F A and F3, thus resembling two iron-sulfirr centers of the soluble F d from Peptostreptococcus asaccharolyticus (formerly known as Peptococcus aerogenes), which has sequence similarity to PsaC. However, an unambiguous assignment of FA and FH to the respective clusters is not yet possible. A striking feature of the proposed PSI structure is the arrangement of helices around the electron transfer chain. The framework of the inner core of PSI consists of 10 helices of PsaA and PsaB that resemble similar helices of the L and M subunits of the bacterial reaction center. These tilted transmembrane helices, along with four additional helices parallel to the membrane plane, make a cage for coordination and protection of the electron transfer chain. The proposed arrangement of the antenna Chl a in PSI is different from that seen in the bacterial LHC or in LHCII of higher plants (Schubert et al., 1995). Located 8 to 15 A from each other, Chl a molecules are positioned along the wall of an oval bowl with the bottom oriented toward the luminal side. This wall may function as a storage device for rapid delocalization of an excited state. 1 5 GENETIC MANIPULATION OF PSI IN A MODEL ORGANISM SYNECHOC YST IS SP. PCC 6803 Molecular biology offers the researcher the ability to alter an element of a biological system in order to learn more about that element in the normal system. Synechocystis sp. PCC 6803 is an excellent genetic system for the study of oxygenic photosynthesis. It is a unicellular cyanobacterium that is naturally competent, and thus is readily transforrnable. It has an active homologous recombination mechanism, making gene replacement by double recombination a straightforward technique (Williams, 1988). There does not appear to be an active restriction or modification system in this strain that can digest transforming DNA (Williams, 1988). Of great importance is that this strain may be grown photoheterotrophically (with DCMU and glucose) (Williams, 1988) or heterotrophically, under light-activated heterotrophic grth conditions (LAHG), complete darkness except for 5 min of light every 24 h, and in the presence of glucose (Anderson and McIntosh, 1991). Growth under these conditions eliminates the selective advantage conferred by the expression of wild-type photosynthesis genes, allowing for complete segregation of mutations (Smart et al., 1991). This strain has approximately 8- 10 copies of its chromosome per cell (Williams, 1988); thus in order to obtain a homozygous strain, segregation is crucial. The entire 3.57 Mb genome of Synechocystis sp. PCC 6803 has recently been sequenced (Kaneko et al., 1996). Availability of the sequences will undoubtedly simplify genetic studies in this model organism. Inactivation of individual subunits has led to identification of the function of some subunits in the assembly of, stability of, and electron transport in PSI (Smart etal., 1991). Site-directed mutagenesis has been used to study 16 the structure-firnction relationships of particularly interesting regions such as ligands to Fx and the electronic environment near Fx (Smart et al., 1993b). FOCUS OF THIS STUDY The body of the work presented in this thesis addresses the development and utilization of the cyanobacterium Synechocystis sp. PCC 6803 as a system for the study of PSI assembly, structure and function. Two psaC genes, psaC] and psaCZ, have been reported in Synechocystis sp. PCC 6803. The initial stage of the project was the inactivation of the psaCZ gene, in order to find out which gene codes for the PsaC subunit. This stage included the analysis of the assembly of the PSI complex and of electron transport in the absence of PsaC. Mutations were introduced at the proposed cysteine ligands to F A and F8. Analysis of these mutants gives us insight into the assembly of PSI complex and the efficiency of electron transfer, as well as into the functions and properties of iron-sulfur clusters with alternate ligands. Since the PsaC mutants were unable to grow photoautotrophically under white light and their nrixotrophic grth was inhibited by white light of moderate intensity, spontaneous pseudorevertants were isolated to gain insight of the structure of PSI complex and to study its function in cyanobacterial metabolism . 17 REFERENCES Allen JP, Feher G, Yeats TO, Komiya H and Rees DC (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors. Proc. Natl. Acad Sci. USA 84: 5730-5734 Anderson SL, McIntosh L: Light-activated heterotrophic grth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process. J Bacteriol 173: 2761- 2767 (1991). Barry BA, Boemer RJ, and de Paula JC (1994) The use of cyanobacteria in the study of the structure and function of photosystem II. In: The molecular biology of cyanobacteria. Bryant DA, ed. Kluwer Academic Publishers, pp.217-257 Boekema EJ, Boonstra AF, Dekker JP, and Rogner M (1994) Electron microscopic structural analysis of photosystem I, photosystem II, and the cytochrome b6/f complex from green plants and cyanobacteria. J Bioenerg Biomembr 26: 17-29 Chitnis PR, Xu Q, Chitnis VP, and Nechushtai R (1995) Function and organization of photosystem I polypeptides. Photosynth Res 44: 23-40 Chitnis PR (1996) Photosystem I. Plant Physiology 111: 661-669 Deisenhofer J, Epp 0, Miki K, Huber R, and Michel H (1985) Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3 A resolution. Nature 318: 618-624 Farah J, Rappaport F, Choquet Y, Joliot P, and Rochaix JD (1995) Isolation of a psaF- deficient mutant of Chlamydomonas reinhardtii: efficient interaction of plastocyanin with the photosystem I reaction center is mediated by the PsaF subunit. EMBO J 14: 4976- 4984 F romme P, Witt HT, Schubert W-D, Klukas O, Saenger W, and KrauB N (1996) Structure of photosystem I at 4.5 A resolution : a short review including evolutionary aspects. Biochim Biophys Acta 1275: 76-83 Fujita Y, Murakami A, Aizawa K (1994) Short-terrn and long-terrn adaptation of the photosynthetic apparatus: homeostatic properties of thylakoids. In: The molecular biology of cyanobacteria. Bryant DA, ed. Kluwer Academic Publishers, pp. 677-692 Golbeck JH (1994) Photosystem I in cyanobacteria. In: The molecular biology of cyanobacteria. Bryant DA, ed. Kluwer Academic Publishers, pp. 319-360 Golbeck JH and Bryant (1991) Photosystem 1. Curr. Topics Bioenerg. 16: 83-177 18 Golbeck JH, Mehari T, Parrett K, Ikegami I (1988a) Reconstitution of the photosystem I complex from the P700 and F x-containing reaction center core protein and F A/F B polypeptide. FEBS Lett 240: 9-14 Golbeck JH, Parrett K, Mehari T, Jones KL, Brand JJ (1988b) Isolation of the intact photosystem I reaction center core containing P700 and iron-sulfur center Fx. FEBS Lett 228: 268-272 Haehnel W, Jansen T, Gause K, Klosgen RB, Stahl B, Michel D, Huvermann B, Karas M, Herrrnann R (1994) Electron transfer from plastocyanin to photosystem I. EMBO J 13: 1028-1038 Hervas M, Navarro JA, Diaz A, Bottin H, de la Rosa MA (1995) Laser-flash kinetic analysis of the fast electron transfer from plastocyanin and cytochrome c, to photosystem 1. Experimental evidence on the evolution of the reaction mechanism. Biochemistry 34: l 1 32 1- 1 1326 Ho K and Krogmann DW (1982) Photosynthesis. In The Biology of Cyanobacteria, Carr NG and Whitton BA, eds. Los Angeles: University of California Press, pp. 191-214 Jung Y-S, Yu L, Golbeck III (1995) Reconstitution of iron-sulfur center FB results in complete restoration of NADP’ photoreduction in Hg-treated photosystem I complexes from Synechococcus sp. PCC 6301. Photosynth Res 46: 249-255 Kallas T (1994) The cytochrome b, f complex. In: The molecular biology of cyanobacteria. Bryant DA, ed. Kluwer Academic Publishers, pp. 259-317 Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouch T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Tasuda M, and Tabata S (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. 11. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA research 3: 109- 136 Kok B(1956) On the reversible absorption change at 705 my in photosynthetic organisms. Biochim. Biophys. Acta 22. 399-401 KrauB N, Hinrichs W, Witt I, F romme P, Pritzkow W, Dauter Z, Betzel C, Wilson KS, Witt HT, and Saenger W (1993) Three-dimensional structure of system I of photosynthesis at 6 A resolution. Nature 361: 326-331 Mannan RM, He W-Z, Metzger S, Whitmarsh J, Malkin R, and Pakrasi HB (1996) Active photosynthesis in cyanobacterial mutants with directed modifications in the ligands for two iron-sulfirr clusters in the PsaC protein of photosystem I. EMBO J 15: 1826-1833 l9 Mannan RM, Whitmarsh J, Nyman P, and Pakrasi HB (1991) Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. Proc Natl Acad Sci USA 88: 10168-10172 Mathis P, Ikegami I and Setif P (1988) Nanosecond flash studies of the absorption spectrum of the photosystem I primary acceptor A0. Photosynth Res 16: 203-210 Navarro J A, Hevas M, Genzor CG, Cheddar G, Fillat MF, de la Rosa MA, Gomez- Moreno C, Cheng H, Xia B, Chae YK, Yan H, Wong B, Straus NA, Marldey JL, Hurley JK, Tollin G (1995) Site-specific mutagenesis demonstrates that the structural requirements for efficient electron transfer in Anabaena ferredoxin and flavodoxin are highly dependent on the reaction partner: kinetic studies with photosystem I, ferredoxinzNADP+ reductase, and cytochrome c. Arch Biochem Biophys 321: 229-238 Nugent JH (1996) Oxygenic photosynthesis. Electron transfer in photosystem I and photosystem 11. Eur J Biochem 237: 519-531 Preiss S, Peter GF, Anandan S, and Thomber JP (1993) The multiple pigment-proteins of the photosystem I antenna. Photochem Photobiol 57: 152-157 Setif PQ, and Bottin H (1995) Laser flash absorption spectroscopy study of ferredoxin reduction by photosystem I: spectral and kinetic evidence for several photosystem I- ferredoxin complexes. Biochemistry 34: 9059-9070 Schubert WD, Klukas O, Krauss N, Saenger W, Fromme P, Witt HT (1995) Present state of the crystal structure analysis of photosystem I at 4.5 A resolution. In Mathis P, ed, Photosynthesis: From Light to Biosphere, Vol II. Kluwer Academic, Dordrecht, The Netherlands, pp 3-10 Smart LB, Anderson SL, and McIntosh L (1991) Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp PCC 6803. EMBO J 10:3289-3296 Smart LB, and McIntosh L (1993a) Genetic inactivation of the psaB gene in Synechocystis sp PCC 6803 disrupts assembly of photosystem 1. Plant Mol Biol 21: 177- 1 80 Smart LB, Warren PV, Golbeck JH, and McIntosh L (1993b) Mutational analysis of the structure and biogenesis of the photosystem I reaction center in the cyanobacterium Synechocystis sp PCC 6803. Proc Natl Acad Sci USA 90: 1132-1136 Takahashi Y, Goldschmidt-Clermont M, Soen SY, Franzen, and Rochaix JD (1991) Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfirr protein destabilizes photosystem I. EMBOJ 10: 2033-2040 20 Vassiliev IR, Jung YS, Smart LB, Schulz R, McIntosh L, and Golbeck JH (1995) A mixed-ligand iron-sulfur cluster (C556SM, or C565 SM) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I. Biophys J 69: 1544- 1553 Vos MH and van Gorkom HJ (1988) Thermodynamics of electron transport in photosystem I studied by electric field-stimulated charge recombination. Biochim. Biophys. Acta 934: 293-302 Williams JGK (1988) Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Meth Enzymol 167: 766-778 Xu Q, Chitnis PR (1995) Organization of photosystem I polypeptides. Identification of PsaB domains that may interact with PsaD. Plant Physiol 108: 1067-1075 Yu L, Zhao J, Muhlenhofl‘, Bryant DA, and Golbeck JH (1993) PsaE is required for in vivo cyclic electron flow around photosystem I in the cyanobacterium Synechococcus sp. PCC 7002. Plant Physiol 103: 171-180 Zhao J, Li N, Warren PV, Golbeck JH, and Bryant DA (1992) Site-directed conversion of a cysteine to aspartate leads to the assembly of a [3F e-4S] cluster in PsaC of photosystem I. The photoreduction of F A is independent of F3. Biochemistry 31:5093-5099 Chapter 2 ABSENCE OF PsaC SUBUNIT ALLOWS ASSEMBLY OF PHOTOSYSTEM I CORE BUT PREVENTS THE BINDING OF PsaD and PsaE IN S YNECHOCYSTIS SP. PCC 6803 Published in modified form as Jianping Yu, Lawrence B. Smart, Yean-Sung Jung, John Golbeck and Lee McIntosh. Plant Molecular Biology vol. 29, pp. 331-342 (1995). Introduction Photosystem I (PSI) is a multi-subunit, thylakoid membrane-bound pigment-protein complex that catalyzes the light-dependent transfer of electrons fiom plastocyanin or cytochrome c.S to ferredoxin or flavodoxin. Purified PSI typically contains at least eleven polypeptides, approximately 100 chlorophyll a molecules, a pair of phylloquinones, and three [4Fe-4S] clusters. The PSI core consists of a heterodimer of homologous 83 and 82 kDa reaction center polypeptides, encoded by the psaA and psaB genes, respectively. This complex is responsible for binding the electron donor P700 and the acceptors A0, A1, and Fx (Golbeck, 1992). The temrinal PSI electron acceptors FA and FE, both [4F e-4S] clusters, are associated with a single 8.9 kDa polypeptide encoded by the psaC gene (Haj et al., 1987; Oh- oka et al., 1988). The 15.5 kDa PsaD subunit is proposed to be a ferredoxin-binding subunit (Zilber and Malkin, 1988) and is required for stable binding of the PsaC to the reaction center 21 22 core in reconstitution experiments (Li et al., 1991b). The 8 kDa PsaE subunit is suggested to stimulate the reduction of soluble electron acceptors such as ferredoxin and /or flavodoxin, and is also suggested to be associated with cyclic electron transport around PSI (Yu et al., 1993). The 15.7 kDa PsaF subunit is proposed to be a plastocyanin-binding subunit (Wynn and Malkin, 1988), and the 16.6 kDa PsaL subunit is proposed to play a role in PSI trimmer formation (Chitnis et al., 1993). The cyanobacterial PSI complex contains at least 4 additional polypeptides of unknown firnction (Golbeck, 1994; Henry et al., 1990). We are employing site-directed mutagenesis to understand the protein architecture responsible for electron flow in the PSI reaction center. This genetic manipulation is most readily accomplished using the unicellular cyanobacterium Synechocystis sp. PCC 6803, since it has a photosynthetic apparatus much like that of plants, is easily transformed, has an active homologous recombination system, can use bacterial drug resistance genes, and may be grown heterotrophically (Anderson and McIntosh, 1991a). A number of mutants have been made in PsaA and PsaB proteins. Analysis of these mutants has provided new insights into the identity and function of Fx (Smart et al., 1993; Warren et al., 1993). The nucleotide and the protein sequences of several psaC genes and PsaC proteins of cyanobacteria, algae and plants are known (Golbeck, 1992). The psaC gene is located in the chloroplast genome in eukaryotes. The mature PsaC protein has 80 anrino acids, is highly acidic, and has two regions of the CXXCXXCXXXCP binding motif characteristic of 8Fe-8S proteins. The PsaC amino acid sequence is highly conserved among cyanobacteria (>90% identical) and higher plants (>95% identical) (Golbeck, 1992), and the differences are usually conservative. The psaC gene has been previously inactivated in a cyanobacterium, Anabaena 23 variabilis sp. ATCC 29413 (Mannan et al., 1991), and in the alga Chlamydomonas reinhardtii (Takahashi et al., 1991). In Anabaena variabilis 29413, the loss of PsaC prevented autotrophic grth and caused the cells to be light-sensitive, but it did not significantly alter stable assembly of the PSI reaction center; further study showed that PsaD and PsaE were absent in isolated thylakoid membranes (Mannan et al., 1994). However, in Chlamydomonas, inactivation of psaC prevented stable accumulation of the PSI reaction center proteins and of seven additional peripheral PSI polypeptides. Unlike other cyanobacteria, two different psaC genes have been reported in Synechocystis 6803: one (psaC!) (Anderson and McIntosh, 1991b) with a deduced anrino acid sequence identical to that of tobacco, and another (psaC2) (Steinmr‘iller, 1992) with a deduced amino acid sequence similar to those reported for other cyanobacteria. Specific transcripts for psaC] were not detected (Anderson and McIntosh, 1991b). In contrast, the psaC2 gene is transcribed into an abundant mRNA of about 450 nucleotides (Steinmt'rller, 1992). Two questions arise: which gene encodes PsaC protein and what is the role of PsaC in assembly and function of PSI? We describe here the inactivation of the psaC2 gene and characterization of the resulting mutant strain in Synechocystis 6803. Materials and methods Materials Chemicals and antibiotics used were obtained from Sigma Chemical Co. (St. Louis, MO) or Research Organics (Cleveland, OH). Restriction endonucleases and other enzymes were purchased from New England Biolabs (Beverly, MA). Radioactive isotope ([a- 32P]dATP and [f3 P]ATP) was purchased from Amersham (Arlington Heights, IL), 24 nitrocellulose membrane from Schleicher and Schuell (Keene, NH), and Bacto-agar from Difco (Detroit, MI). Strains and growth conditions A glucose-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803 (Williams, 1988) was used in this study. Cells were grown in liquid BG-ll medium with 5 mM glucose or on BG-ll with 5 mM glucose and 1.5% purified Bacto-agar under LAHG conditions, as previously described (Anderson and McIntosh, 1991a). Transformation of Synechocystis 6803 was performed essentially as described (Williams, 1988). The mutant strain CDK25 was isolated and maintained in medium containing 5 mg/l kanamycin sulfate. For analysis of mutants, cells were grown in carboys containing 15 l of medium, were harvested using a $834 continuous flow rotor (DuPont Sorvall, Wilmington, DE), and were frozen at -70 °C in BG-ll with 15% v/v glycerol. Nucleic acid manipulations All nucleic acid manipulations were performed using standard procedures (Sambrook et al., 1989), unless otherwise stated. Plasmid p61-2.4, containing the psaC2 gene, was a kind gift from Klaus Steinmt'rller (Universitat Dusseldorf, Dusseldorf, Germany). Genomic DNA was isolated from Synechocystis 6803 essentially as described (Williams, 1988), fractionated by agarose gel electrophoresis, and transferred to nitrocellulose. RNA isolation and conditions for hybridization were previously described (Smart et al., 1991). Synthetic oligonucleotides designed to recognize both psaC] and psaC2 sequences (CGCAGTATGGGTCTAGCTTA) or to recognize psaC2 specifically (using a probe of two mixed sequences, 25 A=GGCTTGCCCCCTCGATGTT and B=CCGAGTTTATTTGGGTGCCGAA) were end- labeled and used as probes. Plasmid pSLA4-l (Anderson and McIntosh, 1991b), consisting of pUCl 19 containing a 202bp Sau3Al-Taql fragment of psaC] , was random primer-labeled (F einberg et al., 1983) and used as a psaCI-specific probe. psaC Interposon mutagenesis A 2.4 kilobase pair (Kb) Sal I fragment containing psaC2 gene from Synechocystis 6803 (Steinmr'jller, 1992), in pSP65 (plasmid p61-2.4), was digested with XbaI and the resulting single-stranded DNA overhangs were digested briefly with mung bean nuclease. A 1.25-Kb HincH fiagment, encoding an arninoglycoside 3' phosphotransferase from T n903 and conferring resistance to kanamycin(km) (Oka et al., 1981), was purified fi'om pUC4k (Vieira and Messing, 1982) and ligated into the blunt XbaI site of p61-2.4. The product was transformed into Escherichia coli XLl-Blue (Stratagene, La Jolla, CA), and km-resistant (KmR) colonies were recovered. The resulting plasnrid, p61-2.4k, had lost the Xbal site and the HincH sites flanking the KmR cassette. A restriction map of the construct from p61-2.4k is shown in figure 2.1. Transformation of Synechocystis 6803 and selection conditions Synechocystis 6803 that had been maintained under LAHG conditions for two subcultures was transformed with the plasmid p61-2.4k. Selection for KmR colonies was performed under LAHG conditions. Single KmR colonies were streaked to at least five serial transfers to obtain full segregation of the mutation. KmR colony CDK25 was analyzed firrther. 26 =—— '6 — —CUQ. : '— mXU) I (D [II I l l "' psaCZ | R . .* le Hincll Hind Ill Hincll .L 4 500 bp Figure 2.1. Partial restriction map of the plasmid p61-2.4k2. The plasmid was used for inactivation of psaC2. Cloned Synechocystis 6803 DNA in p61-2.4k2 is indicated as a line (insert fiom p61-2.4, a gift from K. Steinmt’rller), The psaC2 gene reading frame and the KmR gene cassette from Tn903 are indicated as boxes. The arrows indicate the direction of transcription. The KmR gene cassette was inserted at the Xbal site. 27 Membrane isolation and analysis Thylakoid membranes were isolated and SDS-PAGE and immunoblotting were performed as described (Smart et al., 1991). To resolve the PsaA/B proteins, 10% SDS- PAGE gels were used; 17% gels were used to resolve PsaC, PsaD, PsaE and PsaF. D2 protein in photosystem II was resolved with 17% gel to serve as a control. Protein assays were performed using the method of Lowry et al. (Lowry et al., 1951). Equal amounts of protein (150 ug) were loaded in each lane. Antibody to PsaA/B proteins from Synechococcus was raised in rabbits as previously described (Henry et al., 1990). Also raised in rabbits were antibodies to PsaC or PsaD protein purified from strains of Escherichia coli expressing, respectively, the psaC gene from Synechococcus sp. PCC 7002 or the psaD gene fi'om Nostoc sp. PCC 8009 (Li et al., 1991a). Antibodies against PsaE and PsaF are gifts from Dr. Parag Chitnis and were raised in rabbit immunized with PsaE and PsaF purified from Synechocystis sp. PCC 6803. Antibodies to the D2 polypeptide from spinach were raised as previously described (Vermaas et al., 1988). Chlorophyll was extracted with methanol and quantified using published extinction coefficients (Lichtenthaler, 1987). Thylakoid membrane preparations for EPR and optical measurements Thylakoid membranes were isolated from Synechocystis sp. PCC 6803 using a modification of the procedure described by Noren et al. (Noren et al., 1991). The cells were broken in six cycles of a prechilled bead-beater (Biospec Products, OK) in buffer containing 20 mM Tris, pH 7.2, 0.8 M sucrose, and protease inhibitor; one cycle consists of a 30-5 "on phase" and a 15-min "ofl‘ phase". The membranes were pelleted by centrifirgation in 40 mM CaCl2 and washed with Tris, pH 8.3, 20 mM CaCl and 20 mM MgCl2 . The thylakoid 28 membranes were resuspended in buffer containing 50 mM Tris, pH 8.3, frozen in liquid nitrogen, and stored at -80°C. In vitro reconstitution In vitro reconstitution was performed as described (Li et al., 1991b), using thylakoid membrane from CDK25 and PsaC from Synechococcus sp. PCC 7002 and PsaD from Nostoc sp. PCC 8009. Both PsaC and PsaD were overexpressed by E. coli (Li et al., 1991b). Electron paramagnetic resonance spectroscopy Electron paramagnetic resonance (EPR) studies were performed with a Bruker ESC-106 X-band spectrometer. Cryogenic temperatures were maintained with an Oxford liquid helium cryostat and an Oxford ITC4 temperature controller. Microwave fi'equency was sampled during run-time with a Hewlett-Packard 5340A frequency counter. The field was calibrated using the position of the “g” = 2.0025 resonance derived from the P700’ cation. Sample temperature was monitored by a calibrated thermocouple situated beneath the 3-mm i.d. quartz sample tube and referenced to liquid nitrogen. Actinic illumination of the sample was provided by a 150-W xenon arc source (Oriel) filtered through 5 cm of water and passed through a heat-absorbing filter to remove the near-IR wavelengths. Sample contained 1.8 mg/ml Chl, 1.7 mM sodium ascorbate, and 0.033 mM DPIP in 50 mM Tris, pH 8.3. F lash-induced absorption change F lash-induced absorption changes in the millisecond time range were determined at 698 nm with a laboratory-built, single-beam spectrometer described previously (Parrett et al., 29 1989). Actinic illumination was provided by a S-us xenon flash (PTI Model 610, London, ON). The absorption transient was captured with a Biomation Model 8100 digitizer interfaced to a Macintosh IIci computer for signal averaging, data storage, and data manipulation. The cuvette contained thylakoid membranes at 8.75 ug/ml Chl, 1.7 mM sodium ascorbate, 0.033 mM DPIP in 50 mM Tris, pH 8.3. The point-by-point spectrum was determined between 400 and 500 nm at a chlorophyll concentration of 10 ug/ml. Results Southern and Northern analysis Southern blots of EcoRI digests of genomic DNA from CDK25 were probed with a synthetic oligonucleotide designed to be specific for psaC2 (C2 probe; Fig. 2.2A), and to recognize both psaC] and psaC2 (Cl/C2 probe; Fig. 2.2B); or probed with pSLA4-l (Anderson and McIntosh, 1991b) which was pUC119 containing a 202bp Sau3A1-Taq1 fragment of psaC 1 (Cl probe; Fig. 2.2C). In all the digests of CDK25 DNA, no wild-type copies of the psaC2 genes were detected (Fig. 2.2A), and the psaC] gene was found unchanged in CDK25, as in wild-type cells. (Fig. 2.2C). The C2 probe hybridized to a 10.2- Kb EcoRI fragment of CDK25 DNA, rather than to the 9.0 Kb wild-type fragment, confirming the insertion of the KmR gene into psaC2. The C1 probe hybridized to a 3.3-Kb EcoRI fragment or to both CDK25 and wild-type DNA, confirming that the psaC I gene was not interrupted in CDK25. These results are firrther supported by C l/C2 probe hybridization. In CDK25, insertion of the KmR cassette caused premature temrination of psaC2 transcription, and its transcripts were not detectable using a synthetic oligonucleotide probe designed to be specific for psaC2 mRN A. In contrast, a 450-bp message was detected in the 30 ‘ “"" -9.4 -6.6 -4.4 Figure 2.2. Southern and Northern analysis of CDK25. Panels A—C: Southern blot analysis of CDK25 (lane 1) and wt-6803 (lane 2) genomic DNA. Each lane contains 5 pg DNA digested by EcoRI. The same blot was hybridized with synthetic oligonucleotide probe designed to be specific for psaC 2 (panel A) and to recognize both psaC] and psaC2 (panel B). Another blot was hybridized with pSLA4-1 which was pUCl 19 containing a 202 bp Sau3A1-Taq1 fragment of psaC 1 (panel C). Panel D: Northern blot analysis with CDK25 (lane 1) and wt-6803 (lane 2) RNA. Synthetic oligonucleotide probe designed to be specific for psaC 2 mRN A was used. CDK25 RNA was isolated from LAHG grown cells. Wt-6803 RNA was isolated from mixotrophically grown cells. Each lane contains 120 pg RNA. 31 wild type by the same probe (Fig. 2.2D). Expression of psaC] was not detected when the same blot was reprobed with oligonucleotide probe designed to be specific for psaC! mRN A (data not shown). Growth characteristics The growth of CDK25 was indistinguishable from that of wild-type cells under LAHG conditions (data not shown). However, after several attempts, CDK25 would not divide under continuous illumination of 20 umol m‘2 8", our standard conditions for growth of wild-type Synechocystis 6803 (Jansson et al., 1987), in both the presence and absence of glucose in the medium. Under mixotrophic conditions with a light intensity of 5 umol m”2 s", CDK25 cells were able to grow, with a doubling time (15 hours) close to that of the wild-type strain (20 hours), although CDK25 started log phase one day later than the wild type, and did not reach the maximum cell density of the wild type (Figure 2.3). However, in the absence of glucose in the medium, CDK25 would not divide under the same light conditions. The results indicated that CDK25 is a photosynthesis-deficient mutant and its grth is more susceptible to photoinhibition than is that of wild type. Shen et al. (Shen et al., 1993) recently reported a PSI deletion strain of Synechocystis 6803 that was also sensitive to higher light intensity (50 umol m”2 s") but was able to grow under photoheterotrophic conditions at 5 umol m‘2 s‘1 (doubling time z 28 hours). A major difference between CDK25 and the PSI deletion strain appears to be that the PSI deletion strain needed about four weeks to go through transition from LAHG condition to continuous light condition, but CDK25 liquid culture needed only one day to grow into log phase during the transition (Fig. 2.3). Moreover, the wild-type strain grew into log phase in the first day of the transition (Fig. 2.3). Our data do not seem to agree 32 2.5 A 2 O ‘3 1.5 D o 1 0'5_ ,x" 00.5;25 wt3§§03 O é 4 6 8 Days Figure 2.3. Growth of CDK25 and wt-6803 under mixotrophic conditions. Light intensity: 5 umolm‘2 5". Medium: BGll + Glucose 5 mM. Kanamycin was added to CDK25 growth medium at 5 mg/l. Starting OD730 for both strains were 0.08. 33 with the speculation (Shen et al., 1993) that under LAHG and continuous dim-light conditions the cells employ dramatically different metabolic pathways, and specific metabolites accumulated to deal with dark growth may be poisonous to cells under continuous dim-light conditions. Detection of proteins by antibodies Thylakoid proteins from wild-type and CDK25 cells grown under LAHG conditions were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies raised to PSI proteins and to the D2 protein of PSII (Fig. 2.4). Antibodies raised to Synechococcus 7002 PsaC cross-reacted to protein of :9 kDa in wild-type sample, but not in CDK25 (panel A). Similarly, antibodies raised to Nostoc 8009 PsaD cross-reacted to protein of z16 kDa in wild-type sample, but not in CDK25 (panel B); antibodies raised to Synechocystis 6803 PsaE reacted to protein of z9kDa in the wild-type sample, but not in CDK25. Antibodies raised to Synechococcus PsaA/B cross-reacted to adjacent bands with apparent molecular mass of 60-65 kDa in both wild-type and CDK25 samples. This anomalous behavior of the P700-apoproteins in SDS-PAGE, migration at a lower molecular weight than the predicted 83 kDa, has been observed previously and is probably due to the hydrophobic nature of these proteins (Fish et al., 1985). Antibodies raised to Synechocystis 6803 PsaF reacted to protein of z 16kDa in both wild-type and CDK25 samples. Antibodies raised to spinach D2 polypeptide cross-reacted to protein of z34 kDa in both wild-type and CDK25 samples, which indicates the assembly of photosystem II is not interrupted in this photosystem 1 mutant. 34 C D E A/B F m 1 2 1 2 1 2 1 2 1 2 1 2 ~ 1 i .. l i. - g i - 'ré‘fl'. m ' * '- Figure 2.4. Western blot analysis of CDK25. Thylakoid membranes were isolated from LAHG grown cells. SDS-PAGE and immunoblotting were performed as described (Smart et al., 1991). To resolve the PsaA/B proteins, 10% SDS—PAGE gels were used; 17% gels were used to resolve PsaC, PsaD, PsaE and PsaF. D2 protein in photosystem II was resolved with 17% gel to serve as a control. Lane 1: CDK25; lane 2: wt-6803. Protein assays were performed using the method of (Lowry et al., 1951). Equal amounts of protein (150 11g) were loaded in each lane. Antibodies used were against PsaC, PsaD, PsaE, PsaF, PsaA/B and D2. 35 EPR spectral characterization of C DK 25 thylakoid membranes Figure 2.5a shows a low temperature EPR spectrum of the CDK25 thylakoid membranes after freezing during illumination. The presence of resonances at g = 2.079 (partly obscured by the P700* radical), 1.903 and 1.784, the broad linewidths, and the requirement for high microwave powers and low temperatures are diagnostic of iron-sulfur center Fx. As shown in figure 2.6a, there is no evidence for the presence of iron-sulfur centers F A or F B in the CDK25 membranes (Fig. 2.6a). The Fx spectrum differs only slightly from that when PsaC, PsaD and PsaE are removed from a dodecyl maltoside Synechococcus sp. PCC 6301 complex using chaotropic agents (Figure 2.5b). Within the limits imposed by the signal to noise ratio, the highfield resonance appears somewhat broader in the CKDZS mutant, and the midfield resonance a g = 1.903 in the CDK25 membranes is located upfield at g = 1.885 in the P700-Fx core. This difference may be species related, or it may be due to the presence of detergents or secondary action of the chaotropic agents. When the CDK25 membranes are frozen in darkness, the resonances at g = 2.079, 1.903 and 1.784 appear during illumination and disappear in subsequent darkness, indicating complete reversibility of the signal. This behavior is diagnostic of low temperature electron transfer between P700 and F x. There are no other signals that can be attributed to F A or F3; however, the resonances are about 70% as intense as when the sample is frozen during illumination (data not shown). When PsaC from Synechococcus sp. PCC 7002 and PsaD from Nostoc sp. PCC 8009 are added to the thylakoid membranes, resonances at g = 2.046, 1.936, 1.922 and 1.880 appear when the sample is illuminated during freezing to 15 K (Figure 2.6b). The resonance at 3440 G is present in all reconstituted PS 1 complexes, and is probably not derived from either F A or F8. The Rieske iron-sulfur center is present at g = 1.922, but one or more of the 36 A CDK25 Membranes g = 2.079 1 .903 3 1.784 g = 2.065 P700‘Fx Core Figure 2.5. EPR spectra for Fx. 2.5a) Electron paramagnetic resonance (EPR) spectrum of the thylakoid membrane of CDK25 under conditions optimal for observing iron-sulfur centers Fx. The sample contained 1.7 mM ascorbate and 0.033 mM DPIP in 50 mM Tris, pH 8.3. The spectrum shown is the difference between a dark frozen sample and one that was frozen during illumination. 2.5b) Electron paramagnetic resonance spectrum of the dodecyl-maloside P700-Fx core isolated from Synechococcus sp. PCC 6301. Spectrometer conditions: microwave power, 40 mW; microwave frequency, 9.151 GHz; receiver gain, 2.0 x 10‘; modulation amplitude, 32 G at 100 kHz, magnetic field, 3600 G with scan width of 1000 G. The temperature was 9 K; the Chl concentration was 200 rig/ml. 37 A CDK25 Membranes g = 1.922 B 3 = 2.046 CDK25 Membranes + PsaC and PsaD Figure 2.6. EPR spectra for PA and F3. 2.6a) Electron paramagnetic resonance (EPR) spectrum of the thylakoid membrane of CDK25 under conditions optimal for observing iron-sulfur centers F A and F3. The sample contained 1.7 mM ascorbate and 0.033mM DPIP in SOmM Tris, pH8.3. The spectrum shown is the difference between a dark frozen sample and one that was frozen during illumination. 2.6b) EPR spectrum after the addition of recombinant PsaC from Synechococcus sp. PCC 7002 and PsaD fiom Nostoc sp. PCC 8009 to the whole thylakoid membrane of CDK25. The resonances at g= 2.046, l.936.(1.922), and 1.880 are characteristic of an interaction-type spectrum of F A‘ and FB‘. Spectrometer conditions: microwave power, 20 mW; microwave frequency, 9.128 GHz; receiver gain, 2.0 X 10‘; modulation amplitude, 10 G at 100 kHz, magnetic field, 3400 G with scan width of 1000 G. The temperature was 15 K; the Chi concentration was 700 jig/ml Chl. The large derivative-shaped resonance at 3525 G (g = 1.92) is the midfield peak of the Rieske iron-sulfirr center. 38 of the midfield resonances of the F A'/F 13° interaction spectrum also contributes in this region. Vlfrth the single qualification that the midfield resonances are obscured by the Rieske center, these signals are characteristic of an interaction spectrum of F A’ and FB'. As expected, the Fx spectrum can be observed in the PsaC/PsaD-reconstituted membranes only when F A and FB are prereduced prior to illumination. These data indicate that the in vitro reconstitution leads to a PS I complex with photochemical properties that are nearly identical to that of the wild- type. Optical characterization of thylakoid membrane of CDK25 Figure 2.7 shows the flash-induced absorption transient at 698 nm in thylakoid membranes isolated from the CDK25 mutant (solid line) and the theoretical fit (dashed line). The decay of the P700’ transient is biphasic; the two kinetic phases have half-time of 3 10 us and 1.048 ms representing 29% and 54% of the total absorption change, respectively (the remaining 17% is a firnction of the concentration of DPIP). Previously, we reported monophasic decay with a half-time of 1.2 ms in the P700’Fx' backreaction from a P700-Fx core isolated from Synechococcus sp. PCC 6301 (Parrett et al., 1989). On reexamining these data, we found that the P700+ transient also fits better to two exponentials, with half-times similar to that reported in the CDK25 mutant above. The presence of the two kinetic phases in the P700-FX core is therefore not a result of damage caused by the use of chaotropic agents. As a consequence, there may be several kinetic components to the backreaction from Fx'; however, the origin of the heterogeneity is unknown. The inset shows the point-by-point differences in spectra of the electron donor and acceptor in the CDK25 mutant from 400 to 500 nm. We measured the absorption change at 39 ”‘1 CDK25 Membranes Figure 2.7. Optical spectroscopy for CDK25. Flash-induced absorption change at 698 nm in thylakoid membranes of CDK25. The measurement was performed at 8.75 rig/ml Chl in 50 mM Tris, pH 8.3, containing 1.7 mM ascorbate and 0.033 mM DPIP. The dashed line represents the curve-fit to a biexponential decay. The deviation between experimental and fit points immediately after the flash represents the settling time of the amplifier due to the use of filtering with a 10 kHz high frequency rolloff. Inset: Point-by-point difference spectrum of P700/P700+ and Fx/Fx' in CDK25 thylakoid membranes. The difi‘erence spectnrm for P700/P700" (open circles) was obtained on a sample containing 0.033 mM DPIP, 1.7 mM ascorbate and 450 mM methyl viologen. The difference spectrum for F x/F x' (open squares) was obtained by subtracting the absorption change in a sample containing 0.033 mM DPIP, 1.7 mM ascorbate and 450 mM methyl viologen from one lacking methyl viologen. The measurements were performed at 10 ug/ml Chl in 50 mM Tris, pH 8.3. 40 a single wavelength to obtain the composite absorption change for P700+ Fx', then added methyl viologen to obtain only the P700+ absorption change. The difference between the two samples is the absorption change due to the electron acceptor. The negative-going absorption change from 400 to 500 nnr, with the broad peak at 430 nm is characteristic of the weak charge-transfer bands of an iron-sulfirr cluster. The two kinetic phases show the same spectrum fiom 400 to 500 nm, hence, only the composite is shown. In sum, the kinetics and the spectral properties are compatible with the identity of the room temperature electron acceptor as Fx. The addition of recombinant PsaC from Synechococcus sp. PCC 7002 and recombinant PsaD from Nostoc sp. PCC 8009 to CDK25 thylakoid membrane leads to the replacement of the 1.2 ms backreaction with a long-lived kinetic transient (not shown). The decay of this transient shows a dominant phase with a half-time of about 30 ms, and the minority phase with a half-time that depends on the concentration of DPIP. The 30 ms transient is characteristic of the P700’[FA/FB] 'backreaction, and indicates that the room temperature electron flow is re-established to the terminal iron-sulfur clusters by the addition of PsaC and PsaD. In summary, the EPR and optical spectral characterizations clearly show that the terminal electron acceptor in CDK25 is iron-sulfur center F x. This agrees with the molecular and biochemical data, which indicate that the mutant lacks PsaC, and as a consequence, F A and F3. The data show that an intact F x cluster is able to form and fimction in Synechocystis sp. PCC 6803 in the absence of the PsaC and PsaD. 41 Discussion Assembly of the PSI core (PsaA -PsaB dimer) is independent of PsaC, PsaD and PsaE subunits in Synechocystis 6803 In this study we inactivated the me2 gene by inserting a KmR cassette into its coding region, thus creating a PsaC deletion mutant in Synechocystis 6803. The mutant CDK25 lacked the PsaC, PsaD and PsaE subunits in the thylakoid membrane, and thus lacked the F A and FB iron-sulfirr clusters. However, the PsaA/PsaB dimer assembled in thylakoid membranes in the absence of PsaC; and primary electron donor P700 and electron carriers A0, A1, and F x inserted and were functional in the PsaC-PsaD-PsaE minus photosystem I complex. Evidence supporting this finding includes (a) the presence of PsaA/B polypeptides detected through irnrnunoblot analysis (F ig 2.4), (b) EPR and time-resolved optical spectroscopic data showing formation of P700+ and E; (Fig 2.5), and (c) in vitro reconstitution of CDK25 thylakoid membrane with PsaC and PsaD proteins restores near wild-type PSI EPR signals (Fig 2.6). PsaF was also present in CDK25 thylakoid membranes, suggesting its incorporation into PSI is also independent of PsaC, PsaD, and PsaE. It was observed in an in vitro reconstitution experiment that PsaD and PsaE did not bind to the PSI core in the absence of the PsaC holoprotein (Li et al., 1991a). Assembly of PsaA/B dimer in the absence of PsaC was also reported in another cyanobacterium, Anabaena variabilis (Mannan et al., 1991), in which it was also found that the PsaC is necessary for the stable association of the PsaD and PsaE in PSI complex (Mannan et al., 1994). But in green alga Chlamydomonas reinhardtii, inactivation of the psaC gene prevented stable assembly of all other PSI subunits into the thylakoid membrane (Takahashi et al., 1991). The contrasting results between cyanobacteria and green alga suggest an intrinsic difference in subunit interaction and PSI biogenesis in 42 these different organisms. Assembly of PsaD and PsaE into the PSI complex is dependent on PsaC When PsaC was absent in Synechocystis 6803, the PsaD and PsaE subunit could not stably assemble into the thylakoid membrane, even though PsaA/B and PsaF subunits did so. PsaD is proposed to be located at the cytoplasmic side of the PSI complex, firnctions to bind ferredoxin, and is required for stable binding of PsaC to the reaction center core in in vitro reconstitution experiments (Li et al., 1991b). In the presence of PsaC, reconstitution experiments also showed that the PsaD and PsaE proteins rebind to the PSI core, but there is little or no rebinding of either PsaD or PsaE in the absence of the F A/F B iron-sulfirr clusters (Li et al., 1991b). These results indicate that binding of PsaD and PsaE to the PSI core is mediated by PsaC. A reasonable speculation might be that the binding of PsaC induced a conformational change in PsaA/B, thus providing the binding sites for PsaD and PsaE. psaC] Two different psaC genes have been reported in Synechocystis 6803. In this study we demonstrate that the psaC2 gene encodes the PsaC subunit in PSI. When psaC2 is inactivated, no PsaC polypeptide is found in the thylakoid membrane, which shows that the psaC] does not encode a PsaC protein. Moreover, the protein it encodes, if any, cannot substitute PsaC function. We did not find any transcription from psaC] (data not shown). However, efforts to inactivate psaC] were not successfirl (Anderson and McIntosh, unpublished results). The deduced amino acid sequence of psaC I more closely resembles PsaC sequences of higher plants than of cyanobacteria. It is identical to the tobacco PsaC sequence. At the 43 nucleotide level, the psaC] sequence is 93% identical to the tobacco sequence (Anderson and McIntosh, 1991b), but only 75% to psaC2 (Steinmiiller, 1992). The psaC I flanking sequences resemble nth and ndhE sequences, respectively, of higher plants, and the gene organization is conserved relative to that in chloroplast genomes. It is possible that the psaC] gene and its flanking regions were derived from a transformation of Synechocystis 6803 with higher plant chloroplast DNA, before the strain was isolated (Steinmt‘rller, 1992). Our results are consistent with this speculation, and firrther suggest the psaC] is a pseudogene in the Synechocystis 6803 genome. Why this sequence is maintained in the cyanobacterial genome remains unknown. Acknowledgments I thank L. Smart for helping me start this work. The EPR and optical spectrocopy was performed by Y.-S. Jung in J. Golbeck lab. I thank N. Bowlby for helpfirl advice. Gift of antibodies from J. Guikema, P. Chitnis, and W. Vermaas is appreciated. I also wish to thank K. Steinmt'rller for the generous gift of psaC2 and helpfirl discussions. 44 References Anderson SL, McIntosh L (1991a) Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process. J Bacteriol 173: 2761-2767 . Anderson SL, McIntosh L (1991b) Partial conservation of the S'ndhE-psaC-nth 3' gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts. Plant Mol Biol 16: 497-499. Chitnis VP, Xu Q, YU L, Golbeck JH, Nakamoto H, Xie DL, and Chitnis PR (1993) Targeted inactivation of the gene psaL encoding a subunit of photosystem I of the cyanobacterium Synechocystis sp PCC 6803. J Biol Chem 268: 11678-1168. Feinberg AP and Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6-13. Fish LE, Kuck U, and Bogorad L (1985) Two partially homologous adjacent light-inducible maize chloroplast genes encoding polypeptides of the P700 chlorophyll a-protein complex of photosystem I. J Biol Chem 260: 1413-1421. Golbeck JH (1992) Structure and function of photosystem 1. Annu Rev Plant Physiol Plant Mol Biol 43: 293-324. Golbeck JH (1994) Photosystem I in Cyanobacteria. In Bryant DA (ed), The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers, Dordrecht, Netherlands. pp. 179- 220. Henry RL, Li M, and Guikema JA (1990) A comparison of the PSI polypeptide organization on soybean and the cyanobacterium, Anacystis nidulans. In Baltscheffsky M(ed), Current Research in Photosynthesis, Vol. II. Kluwer Academic Publishers, Dordrecht, Netherlands. pp.567-570. Haj PB, Svendsen I, Scheller HV, and Muller BR (1987) Identification of a chloroplast- encoded 9-kDa polypeptide as a 2[4Fe-4S] protein carrying centers A and B of photosystem I. J Biol Chem 262: 12676-12684. Jansson C, Debus RJ, Osiewacz HD, Gurevitz M, and McIntosh L (1987) Construction of an obligate photoheterotrophic mutant of the cyanobacterium Synechocystis 6803. Plant Physi0185: 1021-1025. Li N, Warren PV, Golbeck JH, Frank G, Zuber H, and Bryant DA (1991a) Polypeptide composition of the photosystem 1 complex and the photosystem I core protein fiom Synechococcus sp. PCC 6301. Biochim Biophys Acta 1059: 215-225. 45 Li N, Zhao J, Warren PV, Warden JT, Bryant DA, and Golbeck JH (1991b) PsaD is required for the stable binding of PsaC to the photosystem I core protein of Synechococcus sp. PCC 6301. Biochemistry 30: 7863-7872. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148: 350-382. Lowry OH, Rosebrough NJ, Farr AL, and Randall R] (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-275. Mannan RM, Pakrasi HB, and Sonoike K (1994) The PsaC protein is necessary for the stable association of the PsaD, PsaE, and PsaL proteins in the photosystem 1 complex: analysis of a cyanobacterial mutant strain. Arch Biochem Biophys 315: 68-73. Mannan RM, Whitmarsh J, Nyman P, and Pakrasi HB (1991) Directed mutagenesis of an iron-sulfirr protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. Proc Natl Acad Sci USA 88: 10168-10172. Noren GH, Boemer RJ, and Bany BA (1991) EPR characterization of an oxygen-evolving photosystem H preparation from the transfonnable cyanobacterium Synechocystis 6803. Biochemistry 30: 3943-3950. Oh-oka H, Takahashi Y, Kuriyama K, Saeki K, and Matsubara H (1988) The protein responsible for center MB in spinach photosystem I: isolation with iron-sulfirr cluster(s) and complete sequence analysis. J Biochem 103: 962-968. Oka A, Sugisaki H, and Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 174: 217-226. Parrett KG, Mehari T, Warren PV, and Golbeck JH (1989) Purification and properties of the intact P700 and Fx-containing photosystem 1 core protein. Biochim Biophys Acta 973: 324-332. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Shen G, Boussiba S, and Vermaas WFJ (1993) Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function. Plant Cell 5: 1853-1863. Smart LB, Anderson SL, and McIntosh L (1991) Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp PCC 6803. EMBO J 10:3289-3296. Smart LB, Warren PV, Golbeck JH, and McIntosh L (1993) Mutational analysis of the structure and biogenesis of the photosystem I reaction center in the cyanobacterium 46 Synechocystis sp PCC 6803. Proc Natl Acad Sci USA 90: 1132-1136. Steinmt‘rller K (1992) Identification of a second psaC gene in the cyanobacterium Synechocystis sp PCC 6803. Plant Mol Biol 20: 997-1001. Takahashi Y, Goldschmidt-Clennont M, Soen SY, Franzen, and Rochaix JD (1991) Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfirr protein destabilizes photosystem I. EMBO J 10: 2033- 2040. Vermaas WFJ, Ikeuchi M, and Inoue Y (1988) Protein composition of the photosystem 11 core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp PCC 6803. Photosynth Res 17: 97-113. Vreira J and Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259-268. Warren PV, Smart LB, McIntosh L, and Golbeck JH (1993) Site-directed conversion of cysteine-565 to serine in PsaB of photosystem 1 results in the assembly of [3F e-4S] and [4Fe- 48] clusters in FX. A mixed-ligand [4Fe-4S] cluster is capable of electron transfer to F A and F a. Biochemistry 32: 441 1-4419. Williams JGK (1988) Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol 167: 766-77 8. Wynn RM and Malkin R (1988) Interaction of plastocyanin with photosystem I : a chemical cross-linking study of the polypeptide that binds plastocyanin. Biochemistry 27: 5863-5869. Yu L, Zhao J, Muhlenhoff, Bryant DA, and Golbeck JH (1993b) PsaE is required for in vivo cyclic electron flow around photosystem I in the cyanobacterium Synechococcus sp. PCC 7002. Plant Physiol 103: 171-180. Zilber AL and Malkin R (1988) Ferredoxin cross-links to a 22kD subunit of photosystem 1. Plant Physiol 88: 810-814. Chapter 3 STRAINS OF S YNECHOC YSTIS SP. PCC 6803 WITH ALTERED PsaC. I. MUTATIONS INCORPORATED IN THE CY STEINE LIGANDS OF THE TWO [4FE-4S] CLUSTERS F A AND F3 OF PHOTOSYSTEM I A modified form is in press (JBC) as Jianping Yu, Ilya R. Vassiliev, Yean-Sung Jung, John H. Golbeck and Lee McIntosh Introduction Photosystem 1 (PS 1) functions as a plastocyaninzferredoxin oxidoreductase in the thylakoid membranes of chloroplasts. In cyanobacteria, cytochrome c, and flavodoxin serve as alternate donors and acceptors under conditions of low copper and low iron. The PS 1 complex contains the photosynthetic pigments, the primary donor P700, and five electron transfer centers (A0, A1, Fx, F A, and F3) that are bound to the PsaA, PsaB, and PsaC proteins. In cyanobacteria, the PS I complex contains at least eight other polypeptides (Chitnis et al., 1995). The cofactors of PS I participate in electron transport across the membrane, oxidizing plastocyanin and reducing ferredoxin according to the following sequence: plastocyanin (Cu) or cytochrome c, (heme) ~ P700 (Chl a dimer) ~A0(Chl a)~A1(phylloquinone)-o F x (a [4F e-48] cluster)- F A or FB ([4Fe-4S] clusters}. ferredoxin ([2Fe-ZS] cluster) or flavodoxin (flavin). The PsaC subunit, encoded by the psaC gene, provides the ligands for two [4F e-4S] clusters, F A and F8. Previous studies showed that the introduction of aspartic acid in position 14 (C14D) and in position 51 (C51D) led to the introduction of [3F e-48] 47 48 and nrixed-ligand [4Fe-48] clusters in the modified F3 and F A sites, respectively, of Escherichia coli-expressed PsaC proteins (Yu L. et al., 1993). However, when the mutant PsaC proteins were rebound to P700-Fx cores, only mixed-ligand [4F e-4S] iron-sulfur clusters were found in the modified sites of the reconstituted C14D-PS I (Yu et al., 1995a) and C51D-PS I (Yu et al., 1995b) complexes. In both mutant PS I complexes, electrons could be transferred to the mixed-ligand iron-sulfur cluster at 15 K, and room temperature NADP+ photoreduction was supported at rates similar to the wild-type. The formation of mixed-ligand [4Fe-48] clusters with altered spectral and redox properties in vitro provided a rationale for probing the firnctions of FA and FB in vivo. The ability to support [4Fe-4S] clusters which are able to transfer electrons at both 15 K and 298 K suggests that the putative oxygen-ligated iron-sulfur clusters should be fimctional in living organisms. The step taken in this work is to move mutations in these ligands into a genetic system such as Synechocystis sp. PCC 6803 and to study the in vivo consequences on grth and electron transfer. Unlike other cyanobacteria, two different psaC genes have been reported in Synechocystis sp. PCC 6803. One (psaC 1) (Anderson and McIntosh, 1991a) has a deduced amino acid sequence identical to that of tobacco, while the other (psaC2) (Steinmt’rller, 1992) has a deduced arrrino acid sequence similar to those reported for other cyanobacteria. The psaC! gene is not involved in PS I, nor can it substitute for psaC2 when the latter is insertionally inactivated. The amino acid sequence of Synechocystis sp. PCC 6803 PsaC matched that predicted from psaC2 (J. Golbeck, personal communication). Insertional inactivation of psaC2 prevented the formation of PsaC, thus demonstrating that this gene encodes the PS I-bound polypeptide. Further work showed that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PS I complex in vivo, and that PsaC, PsaD and PsaE are not needed for assembly of PsaA/PsaB dimer and electron transport from P700 to Fx (Chapter 2). In this chapter the term psaC refers to the gene psaC2. Site-directed mutagenesis and transformation of Synechocystis sp. PCC 6803 has 49 been used successfirlly in the study of PS I biogenesis and firnction (Smart et al., 1993; Warren et al., 1993). In the work presented here, a set of strains with mutations in PsaC has been created in which a cysteine ligand to F A and/or FB is substituted by aspartate, serine or alanine in positions 14 and/or 51 as shown in figure 3.1. The genetic, physiological, and biochemical characterization of the PS I mutants will be presented. Chapte 4 will describe the EPR and optical kinetic properties of the mixed-ligand F A and FE clusters in site-modified PS I complexes. Materials and Methods Strains and Growth Conditions Experiments were performed using a glucose-tolerant strain of Synechocystis sp. PCC 6803, which was acclimated for growth on solid medium in the dark. Except for tests for photoautotrophic and mixotrophic growth, cells were grown at 30°C under light-activated heterotrophic grth (LAHG) conditions, as previously described (Anderson and McIntosh, 1991b). Antibiotics were added in the following concentrations: kanamycin (Km), 5 mg/liter; gentamicin (Gm), 1mg/1iter. Transformations were performed essentially as described (Williams, 1988), except in the case of strain AC-RCPT, which was carefirlly maintained in dim light (5 umol m’2 5'1 during 2 hour incubation) throughout the procedure, since it is light-sensitive (its mixotrophic growth in the presence of glucose is inhibited at white light greater than 10 umol m'2 s"). Tests for photoautotrophic and rrrixotrophic growth were performed using solid media with or without supplemental glucose in a chamber providing continuous light. Cool white fluorescent bulbs made by General Electric were used. The light intensity was varied by covering plates with layers of cheese cloth and were monitored using a L1-185A photometer (LICOR, Lincoln, NE). Large cultures were grown in carboys (15 I) under LAHG conditions and were bubbled with air. 50 Figure 3.1. Predicted structure of PsaC. Based on its sequence similarity with bacterial ferredoxin, showing identity of FA and PB relative to their cysteine ligands C51 and C14, which were changed to aspartate, serine, or alanine in this work. Modified from Golbeck (1994) 51 DNA Manipulations Nucleic acids were manipulated using standard methodology (Sambrook et al., 1989), unless otherwise stated. Site-directed mutagenesis was performed using an oligonucleotide-directed in vitro mutagenesis kit as directed by the manufacturer (Amersham, Arlington Heights, IL). For amplification of psaC gene from cyanobacterial strains, cells picked from a medium-size colony or equivalent amount of cells collected from liquid culture were washed once with water and used as template. Amplification products were purified using a PCR purification kit (Promega Corporation, Madison, WI). Procedure for preparation of cyanobacterial DNA was adapted fiom Ohad and Hirschberg (1992), with two “loopfirls” of cells scraped from plates or cells from 10 ml liquid culture being used to extract DNA. Transformation of Synechocystis 6803 and Selection Conditions Synechocystis sp. PCC 6803 that had been maintained under LAHG conditions for at least two subcultures (cells were subcultured once a week) was transformed with plasmids containing resistance genes to kanamycin or gentamycin. Selection for antibiotic- resistant colonies was performed under LAHG conditions. Resistant colonies were restreaked to single colonies with at least five serial transfers to obtain full segregation of the mutation, as verified by restriction enzyme analysis of PCR products, direct sequencing of PCR products, Southern hybridizations, and growth tests. Western Blot Analysis of Thylakoid Membrane Proteins Thylakoid membranes were isolated and SDS-PAGE and immunoblotting were performed as described (Smart and McIntosh, 1991). To resolve the PsaA/PsaB proteins, 10% SDS-PAGE gels were used; 17% gels were used to resolve PsaC, PsaD, PsaE and PsaF. D2 protein in Photosystem 11 (PS 11) was resolved with 17% gel to serve as a control. Protein assays were performed using the method of Lowry et al. (1951). Equal 52 amounts of protein (150 ug) was loaded in each lane for thylakoid preparations and equal amounts of chlorophyll (4 u g) were loaded in each lane for PS I complexes. Rabbit antiserum to PsaC or PsaD were raised using protein purified from strains of E. coli expressing, respectively, the psaC gene from Synechococcus sp. PCC 7002 or the psaD gene from Nostoc sp. PCC 8009 (Li et al., 1991b). Antibodies against PsaA/B proteins from Synechococcus were raised in rabbits as previously described (Henry et al., 1992). Antibodies against PsaE and PsaF (gifts from Dr. Parag Chitnis, Kansas State University) were raised in rabbits immunized with PsaE and PsaF purified from Synechocystis sp. PCC 6803. Rabbit antibodies to the D2 polypeptide from spinach (gifts from Dr. Wim Vermaas, Arizona State University) were raised as described (Vermaas et al., 1988). Chlorophyll was extracted with methanol and quantified using published extinction coefficients (Lichtenthaler, 1987). Oxygen Evolution LAHG-grown cells were washed once in 40 mM HEPES buffer, pH 7.0 and cells containing 10 pg Chl were resuspended in 1 ml of the same buffer and illuminated by saturating light at 25 °C. Rates of oxygen evolution were determined with a Rank-type oxygen electrode unit. Whole-chain electron transport was measured in the presence of 10 mM NaHCO,. PS 11 electron transport was measured in the presence of 1 mM 2,6-dichloro-p-benzoquinone. Membrane Isolation and Purification of PS I Complex Thylakoid membranes from Synechocystis sp. PCC 6803 were isolated using a modification of the procedure described in Yu et al. (1995). The cells were suspended in 0.8 M sucrose, 50 mM HEPES buffer, pH 7.8 and pelleted by centrifirgation. The cells were washed twice and suspended in the same buffer containing 0.8 M sucrose and protease inhibitors. The cells were broken in 10 cycles of a pre-chilled bead-beater 53 (Biospec Products, Bartelsville, OK); one cycle consisted of a 45 sec ‘on phase’ and a 10 min ‘off phase’. The cell solution was removed from the beads by vacuum suction and centrifirged at r,,, of 5,000 x g to remove unbroken cells. Thylakoid membranes in the supernatant was then pelleted by ultracentrifugation at 100,000 x g with repetitive washing. The thylakoid membranes were suspended in 50 mM Tris, pH 8.3, frozen in liquid nitrogen, and stored at -95°C. n-Dodecyl B-D-maltoside-PS I (DM-PS 1) complexes were isolated using the protocol described in Chitnis and Chitnis (1993) with minor modifications. The membranes were solubilized in 1% n-dodecyl B-D-maltoside (DM, Calbiochem, La Jolla, CA) at 4C for 1 h at the Chl concentration of 0.5 mg ml". DM-PS I complexes were isolated fi'om the lower green zone of PS I trimers which appeared after centrifugation of the solubilized membrane suspension loaded in a sucrose density gradient (0.1 M to 1.0 M sucrose) for 24 h at 4°C. In the alanine and double aspartate mutants, a mixture of PS I monomers and trimers was used because it was difficult to completely separate the lower and the upper bands. The problem is that PsaL is easily lost in the presence of 1% DM at 0.4 mg Chl/ml due to the absence of PsaD (Mannan et al., 1991). Even though the lower part of the broad band containing mostly PS I trimers was used (containing <10% of the total Chl), there was nonetheless some contamination by PS I monomers. Isolated PS I complexes were dialyzed in 50 mM Tris, pH 8.3, resuspended with the same buffer containing 15% glycerol and 0.03% DM, frozen as small aliquots in the liquid nitrogen, and stored at -95°C. Steady State Reductase Activity The steady state reductase activities of PS I complexes were measured according to Jung et al. (1995). The absorbance kinetics were measured using a Cary 219 spectrophotometer with the photomultiplier shielded by appropriate narrow band and interference filters. The sample was illuminated from both sides using two banks of high intensity, LEDs emitting at ca. 660 nm (LSl, Hansatech Ltd.). The light intensity was 54 saturating at the chlorophyll concentration used. Rates of flavodoxin photoreduction were measured in a 1.3-ml volume using 15 11M flavodoxin and DM-PS I at 5 ug ml'1 of Chl in 50 mM Tricine, pH 8.0, 50 mM MgC12, 15 12M cytochrome c6 from Spirulina maxima, 6 mM sodium ascorbate, 0.05% DM. The measurement was made by monitoring the rate of change in the absorption of flavodoxin at 467 nm. Rates of flavodoxin-mediated NADP+ photoreduction were measured in a 1.3-ml volume using 15 uM flavodoxin and 0.8 uM spinach ferredoxinzNADP+ oxidoreductase (FNR, Sigma), and DM-PS I at 5 ug ml'l of chlorophyll in 50 mM Tricine, pH 8.0, 10 mM MgC12, 15 12M cytochrome c6 from Spirulina maxima, 6 mM sodium ascorbate, 0.05% DM, 0.5 mM NADP+, 0.1% B—mercaptoethanol. Rates of ferredoxin-mediated NADP+ photoreduction were measured in a 1.3-ml volume using 5 uM spinach ferredoxin and 0.8 uM spinach ferredoxinzNADP“ oxidoreductase (FNR, Sigma), and DM-PS I at 5 ug ml"1 of chlorophyll in 50 mM Tricine, pH 8.0, 10 mM MgC12, 15 11M cytochrome c6 from Spirulina maxima, 6 mM sodium ascorbate, 0.05% DM, 0.5 mM NADP+, 0.1% B-mercaptoethanol. The flavodoxin was purified from the a strain of E. coli containing the gene by DEAE-Sepharose CL 6B and Sephadex G-75 (both, Sigma) (Muhlenhoff et al., 1996a and 1996b). NADP+ reduction were measured by monitoring the rate of change in the absorption of NADPH at 340 nm. Time-Resolved Optical Absorption Spectroscopy Transient absorbance changes of P700 at 820 nm (AAm) were measured from the microseconds to tens-of-seconds time domain with a laboratory-built double-beam spectrometer as described in Vassiliev et al. (1995) upon excitation with a frequency- doubled Nd-YAG laser with a flash energy of 250 m]. The decay transients were fitted to ‘sum of several exponentials with baseline’ using the Marquardt algorithm in Igor Pro. The user-defined fit firnction enabled a fit up to 7 exponentials with all amplitudes and rate 55 constants set free during the fit. In most cases the fit comprised a baseline component accounting for long-term phases and/or possible drift of signal zero during long time scale acquisition. The quality of the fit was estimated using standard techniques including analyses of the residuals plots and comparison of the chi-square values and standard errors of the fit parameters between different fits. Using this approach, the PS I backreactions can be compared visually on a log time scale, and the ratio between the different pathways that contribute to the backreactions with P700+ can be estimated on the right abscissa. Results Genetic Characteristics of the PS I Mutants Construction and Characterization of AC -RCPT To allow rapid segregation of mutations in the psaC gene, a recipient strain of Synechocystis sp. PCC 6803 was engineered with the psaC coding region deleted and replaced by a kanamycin resistant (KmR) cassette (Fig. 32A). PCR-amplified psaC upstream (454bp) and downstream (208bp) flanking regions, with linker sequences for BamHI and EcoRI at either end, were cloned into pUC118 (Vierra and Messing, 1982). A KmR cassette excised from pUC4K (Taylor and Rose, 1988) using EcoRI, was inserted into the EcoRI site that separated the upstream and downstream flanking regions to form the plasmid pUCl 18-AC. Glucose-tolerant but otherwise wild-type Synechocystis sp. PCC 6803 was transformed with pUCl 18-AC, and KmR colonies were selected under LAHG conditions and were genetically verified by Southern hybridization using a 1.5-kb EcoRI-Ncol fragment containing psaC as probe, and by PCR. Complete segregation of the deletion mutation was confirmed (data not shown). psaC Mutagenesis and Genetic Characterization of the Mutants Plasmids for site-directed mutagenesis were constructed in vitro and manipulated in E. coli. A 1.5-kb EcoRI-NcoI fragment containing psaC was cut out of the plasmid 56 E sepsaC N A wreaoa pUC118-AC X KmFl 1 1Kb . r ' ' (B) H (B) so Km“ (N P B AC—RCPT X pCG (E) so psaC (N P GmR :fzfzffizfzfzfszrfzfz325:}:33:}:§:§:f:§:§:§:§:§:f:f:§:lefzfzf:fzfzf:Ezf: (B) H (B) Figure 3.2. PsaC Mutagenesis strategy. (A) Creation of a AC-RCPT. (B) Introduction of psaC mutations. See text for details. Restriction sites: B, BamHI; E, EcoRI; G, Bgl 11; H, Hind III; N, NcoI; P, PstI; S, Bbs 1. Restriction sites in parenthesis on a plasmid have been inactivated. 57 p6. 183.51 (Steinmuller, 1992) and cloned into pUC119 (Vierra and Messing, 1982) at the SmaI site to create plasmid pC. A 2.0-kb gentarrricin resistance (GmR) cassette, cut with BamHI from pUC119-gen (Smart et al., 1994) was inserted at the BbsI site downstream of psaC gene on pC to create plasmid pCG. Single-stranded DNA of pC was used as template for site-directed mutagenesis. Oligonucleotides were designed to effect the desired changes in the coding sequence, while also destroying an endonuclease restriction site (RsaI for the C14 site and Bva for the C51 site). The resulting change in digestion pattern serves as an effective and simple means of screening for the desired mutation. After verification of the mutations on pC using restriction mapping and DNA sequencing, a 953-bp BglII-Pstl fragment containing the psaC mutation was excised from pC and ligated with a 5.7-kb partial digestion product from pCG devoid of the corresponding fragment to form pCG with a mutated psaC gene. The mutated pCG plasmids were checked by restriction mapping and DNA sequencing to verify the presence of the desired mutations and of the proper sequences. Taking advantage of an XbaI site between the C14 site and the C51 site, pCG with a double mutation C14D/C51D was created by ligating a 758-bp XbaI-XbaI fragment from pCG with a single mutation C14D and a 5.9-kb XbaI-Xbal fragment from pCG with a single mutation C51D. Plasmid pCG with wild type psaC and its mutated variants (shown as an asterisk on psaC) were then used to transform the strain AC-RCPT (Fig. 3.2B). GmR colonies were selected under LAHG conditions and were genetically characterized by three different methods: (i) Southern hybridization of genomic DNA using a 1.5-kb EcoRI-NcoI fragment containing psaC as probe; (ii) restriction mapping and direct DNA sequencing of PCR product amplified from single colonies; and (iii) growth in the presence of kanamycin. Gene replacement by double crossover and complete segregation of all the mutations was confirmed (data not shown). 58 Physiological Characterization of the PS I Mutants Growth Analysis of the Mutants As shown in Table 3.1, all of the PsaC mutants were unable to grow autotrophically under light intensities ranging from 2.2 to 22 umol rn'2 s". They were able to grow mixotrophically under 2.2 umol m'2 s'1 but not under 22 umol m'2 s". In addition, C51D and C148 were tested and found unable to grow photoautotrophically or mixotrophically under white light of 60 umol m'2 5". All PsaC mutants showed similar growth rates to wild-type when growing under LAHG or mixotrophically under 2.2 umol m“2 5". These results show that the mutants are deficient in photosynthesis, that they are light-sensitive and that their mixotrophic grth is inhibited by moderate light intensity of 22 umol rn'2 s". The likely source of light inhibition is the overreduction of the electron transfer chain between P811 and PSI by PSII activity. This hypothesis arises fiom the following results: (1) C51D and C148 were found to grow photoheterotrophically under 22 umol m'2 8'1 white light in the presence of 10 mM DCMU, a PS H inhibitor; (2) C51D and C 148 were found to grow mixotrophically when 22 or 60 umol m’2 s" of white light was filtered by a red plastic sheet; and (3) C51D grows photoautotrophically when 60 umol m'2 s’1 of white light was filtered by a red plastic sheet. The hypothesis is also supported by a previous study of cyanobacterial PS I-less mutants (Shen et al. , 1993), where light inhibition of mixotrophic growth is relieved by genetic inactivation of phycobilisomes. Transformation of C148 and C51D with wild-type psaC DNA restored photoautotrophic growth and relieved light inhibition under mixotrophic conditions, demonstrating that the only lesion in these mutants is in psaC (data not shown). Oxygen Evolution of the Mutant Cells Whole-chain electron transport (HzO-' C02) and PS 11 electron transport (HzO- 2,6-dichloro-p-benzoquinone) were measured using LAHG-grown cells. As shown in Table 3.1, there was essentially no 02 evolution in AC-RCPT indicating that consistent 59 Table 3.1. Physiological characterization of the PsaC mutants. WT AC- 0510 0518 651A 0140 C148 C14A c140 Gmll RCPT 051D Photoeutotrophlczgrowth + - - - - - - - - (2. 2-22 pmol m s ) Mixotrophic growth + + + + + + + + + (2. 2 pmol m 2 s ) Mixotrophic growth + - - - . - - - - (22 pmol m Whole-chain 0, evolution 4301 3 1 341: 1011 39 t 1181 1411 33 :t 30 t H20. 602; pmoloz mgChl" Hr“ 165 18 126 56 27 62 61 15 18 PSII 02 evolution 2921 312: 553: 326: 4841 368: 455: 409:1: 370:1: H20~ DCBQ; pmolO, mgChl" Hr‘1 75 63 253 39 124 80 44 117 68 PS I electron transport cyt c, flavodoxin 900 40 460 480 60 510 480 40 60 pmol flavodoxin mgChl Hr . PS I electron transport cyt c, flavodoxin- N1ADP: 930 70 700 670 170 700 730 200 170 pmolNADP mgChl‘ Hr PSI electron treneport cyt 0, ~ ferredoxin NADP 820 40 540 430 50 480 380 50 7O pmolNADP mgChl Hr ‘ Single turnover efficiency (96) 80 ‘ 0 28 21 0 41 32 0 O AAm , P700’ [FA/F3] ’ Growth tests: 3611 plates supplemented with appropriate antibiotic and glucose, when applicable, were incubated at 30°C. +: growth; -: no growth in 50 days. 02 evolution: LAHG-grown cells containing 10 microgram Chl were resuspended in lml 40 mM HEPES buffer, pH 7.0 and illuminated by saturating light at 25°C. Rates of oxygen evolution were determined with a Rank type oxygen electrode unit. Whole- chain electron transport was measured in the presence of 10 mM NaHC03. PS H electron transport was measured in the presence of 1 mM 2,6-dichloro-p-benzoquinone. PS I-mediated substrate reduction: See text for measurement conditions. Efficiency of electron donation from P700 to FA/FB is shown as the % backreaction with a lifetime 2 7 ms. 60 with previous observations (Yu et al. , 1995), PsaC is required for whole-chain photosynthetic electron transport. Compared to the wild type control, all the PsaC mutants had lowered whole-chainO2 evolution rates to varying degrees, with Asp and Ser mutants showing the highest rates, and Ala mutants and the double Asp mutant showing the lowest rates. However, all the mutants including AC-RCPT showed near- wild-type—levels of PS 11 02 evolution rates, indicating that electron transport in PS 11 was not affected in the short term in the mutants. The lower PSII rates compared to whole chain rates suggest the P811 capacity measurement conditions were not optimal, and DCBQ may not work in cyanobacterial cells as well as in chloroplasts. Rates of NADP+ Photoreduction Table 3.1 shows rates of flavodoxin reduction and ferredoxin- and flavodoxin- mediated NADP+ photoreduction for DM-PS I complexes isolated from the wild-type and mutant strains. The wild type PS I complex supports high rates of reductase activity: 800 to 900 umol mgChl'l Hr’l . The AC-RCPT mutant (which lacks PsaC, PsaD, and PsaE) photoreduces flavodoxin at a rate of 40 umol mgChl‘l Hr'l and photoreduces flavodoxin and ferredoxin-mediated NADP+ at rates of 70 and 40 umol mgChl'l Hr". The C14D-PS I and C51D-PS 1 complexes show lower levels of reductase activity compared to the wild type PS I complex: 460 to 510 umol mgChl'l Hr’l for flavodoxin reduction, 700 umol mgChl‘l Hr‘l for flavodoxin-mediated NADP+ photoreduction and 480 to 540 umol mgChl" Hr'l for ferredoxin-mediated NADP+ photoreduction. Both the C14S-PS I and CSIS-PS I complexes show nearly the same level of reductase activity as the aspartate mutants. However, the C14A-PS I, C51A-PS I and C14D/C51D-PS I complexes supported minimal rates of PS I reductase activities similar to those for the AC-RCPT complex. 6] Biochemical Characterization of the PS I Mutants PS I Subunit Composition in the Mutants Immunoblotting of thylakoid membrane proteins was performed on an equal protein basis, and PS I complexes on an equal chlorophyll basis, using antibodies against the proteins PsaA/B, PsaC, PsaD, PsaE, and PsaF. Antibody against the D2 protein in PS 11, which should not vary significantly in the PS I mutants, was used to make sure similar amout of protein was loaded in each lane. Compared to wild-type thylakoid membranes, the levels of PsaC, PsaD, and PsaE were lowered to varying degrees in all mutants (Fig. 3.3). The Asp and Ser mutants demonstrated moderately low levels of these proteins, while Ala mutants and the double Asp mutant did not contain detectable amounts of these three subunits. All mutants contained near-wild- type levels of PsaF and PsaA/PsaB in all samples (Fig. 3.3). It is difficult to quantitate PsaA/PsaB with immunoblots (Smart et al. , 1993) or by Elisa (Golbeck, unpublished); however, it is assumed that the levels of PsaA/PsaB in the PsaC mutant thylakoids are similar to that in wild-type since it has been demonstrated that assembly of the PsaA/PsaB dimer does not require PsaC, PsaD or PsaE (Chapter 2; Mannan et al. , 1991). In all mutants the levels of PsaC, PsaD, and PsaE appeared to be closely related, an observation that is consistent with the finding that the stable binding of PsaD and PsaE to PS I complex is dependent on the presence of PsaC (Yu et al. , 1995; Li et al. , 1991a). The levels of PsaA/B and PsaF are representative of the amounts of PS I in thylakoids (Golbeck and Bryant, 1991). The levels of PsaC, PsaD, and PsaE represent the amounts of these three subunits bound on PS I core to form a complete PS I complex. These results show that the amount of complete PS I complexes is lower to varying degrees in all the mutants compared to the wild type. In thylakoids from the Asp and Ser mutants, a minor portion of PS I contains PsaC, PsaD, and PsaE bound to form a complete PS I complex, while the majority of PS I cores are lacking these D2 PsaA/B PsaF PsaD PsaE PsaC 62 WT RCPT SID SIS 51A MD MS 14A l4D/SID f 3‘11 ‘.I l I all! "" ~ -l If..- - —.—. “ m N” . 0v . u— - Figure 3.3. Western blots of thylakoid membranes of PsaC mutants. Thylakoid membrane preparations containing 150 microgram protein was loaded in each lane. 63 subunits (it is difficult to specify the precise amount from the Western blots and efforts to do this by Elisa techniques were unsuccessful). In thylakoids from Ala mutants and the double Asp mutant, no complete PS I complexes are found. To our knowledge this is the first report that the p0pulation of PS I complexes is found to be heterogeneous in vivo as a result of mutagenesis. Heterogeneous PS I populations, assayed by immunoblot analysis, were also observed for purified PS I complexes from the Asp and Ser mutants after solubilizing the membranes with 1% DM at 1 mg/ml Chl for 20 min followed by a single sucrose density centrifugation step (data not shown). Heterogeneity complicates functional analysis of the mutant PS 1 complexes, making it is difficult to distinguish between (i) inefficient electron transfer from Al to Fx to F AIFB and (ii) a mixed population of P700-PX cores and P700-FA/FB complexes. To solve this problem, a purification procedure with sucrose density gradient centrifugation was adopted to separate the PS I core subpopulation (i.e. those devoid of PsaC, PsaD and PsaE proteins) from the integral PS I complex. The separation principle is based on the finding that at high ratios of DM to Chl, PsaL is readily lost in PS I complexes which lack PsaC, PsaD and PsaE (Mannan et al. , 1991). On the other hand, the PsaL-less P700-Fx core was shown to be present in the upper, monomeric band, while the intact PS I complex is present in the lower, trimeric band (Chitnis and Chitnis, 1993). As the result nearly homogeneous PS I holocomplexes, as analyzed by immunobloting, were isolated from the Asp and Ser mutants. Compared to wild-type PS I complexes, all the mutants had similar levels of PsaB and PsaF (data not shown); and the single Asp and Ser mutants had similar levels of PsaC, while the Ala mutants and the double Asp mutant had no detectable levels of PsaC (Fig 3.4). 64 WT SID SIS 51A 14D MS 14A l4D/SID PsaC m I I PsaB PsaF is, *5!- »'-m-;h briefly. rev-'1». “1;,“ m ,w Figure 3.4. Western blots of purified mutant PSI complexes. PS I particles containing 4 microgram chlorophyll was loaded in each lane. 65 Single Turnover Flash Studies of Mutant PS I Complexes The effects of different ligand substitutions on P700+ re-reduction kinetics were determined in homogeneous DM-PS I complexes after a single turnover, saturating flash. In wild-type PS 1 complexes, the vast majority of the backreaction is derived from [F A/FBI . As shown in Fig. 3.5A, 60% of the recombination kinetics are derived from the 25-ms and 112-ms components attributed to P700+ [FA/FBI recombination. An additional 26% are derived from slower phases with lifetimes of 221 ms and 2.2 s, leading to an 86% efficient electron transfer to FA/FB. The slowest kinetic phases are due to exogenous donors undergoing redox reactions with P700“ and come about in reaction centers where [F A/FBI has become oxidized by exogenous electron acceptors in the medium. The sum contribution of earlier acceptors, including Fx’ and Al‘, is 14% of the total absorption change. The major contribution to the absorbance change in the Synechocystis sp. PCC 6803 PS I complex with chemically reduced terminal iron-sulfur clusters and in the P700-Fx PS 1 core isolated by urea treatment of the PS I complex is brought about by the components with life times of ca. 400 as and 1.5 ms, which result from back reaction of P700+ and Fx' (Vassiliev et al., 1995). The faster components with life times of ca. 10 as and 100 as appearing in these preparations may result from recombination of P700* and A,‘ (Brettel and Golbeck, 1995) in a fraction of centers which either have a lower quantum efficiency of electron transfer between A1 and Fx due to some changes of Fx microenvironment or have have the Fx cluster missing or chemically reduced. There is also evidence that decay of the Chl triplets formed upon laser flash excitation in the antenna may contribute to AA decay in the tens-of-us time domain. The kinetics of the C14D/C51D-PS I (Fig. 3.5B), C14A-PS I (Fig. 3.5C), and C51A-PS I (Fig. 3.5D) complexes are similar to those for the P700-PX PS I core isolated by urea treatment of the PS I complex (data not shown; see Vassiliev et al. , 66 Figure 3.5. Kinetics of AA820 absorbance changes (I). (A) Wild-type PS I Complex, (B)C14D/C51D-PS I Complex, (C) Cl4A-PS I Complex, (D) C51A-PS I Complex. The wild-type and mutant PS I complexes were isolated from membranes with B-D- dodecyl maltoside. The reaction media is 25 mM Tris buffer, pH 8.3 with 0.03% DM, 10 mM sodium ascorbate and 4 uM DCPIP; the chlorophyll concentration is 50 ug ml'l . Each trace (dots) is an average of 16 measurements taken at 50 3 intervals. The multiexponential fit is overlaid as a solid line; residuals of the fit shown at the top of the graph. The major individual exponential components are shown as dashed lines, with the percent on the right ordinate, and with offsets equivalent to the relative contribution of the component. The vertical dotted bars separate the time domains in which most of the backreactions of Al (left), Fx (middle) and FA/FB (right) correspondingly occur. 67 1995). The majority of the backreaction kinetics for all three mutant complexes is derived from tens-of-us to ms decay phases, with little or no measurable backreaction in the tens-to-hundreds of ms timescale. Since the PsaC protein is missing in these mutants (Fig. 3.3), and therefore the F A and PB clusters, which govern the tens-of-ms decay kinetics are lacking, this is the expected result. The larger AA in these three mutants, which is due to a significant as component, may be derived from additional absorption changes from the decay of Chl triplets in the antenna bed. The optical kinetic data are in agreement with the steady-state rate data (Table 3.1) in which no significant N ADP+ reduction occurs in the absence of FA and F3. The kinetics of the C14D-PS I, C14S-PS I, C51D-PS I and C51S-PS I complexes are mixed 'core- and ‘complex-type, with each mutant showing a slightly different fractions of the backreaction derived from tens-of-us to ms decay phases. The C14D-PS 1 complex has the largest and the CSIS-PS I mutant has the smallest, percentage of electrons which arrive at FA/FB. The [F A/FBI backreaction in C14D-PS I (Fig. 3.6A) has lifetime components of 33 ms and 335 ms which contribute 26% to the total absorption change; an additional 15% is contributed by the slower donation to P700+ by external donors, leading to a 41% efficient transfer to FA/FB. The remainder of the backreaction occurs from components with lifetimes of 712 as (19%) and 2.4 ms (20%) derived from Fx, and 19% occurs from a component with a lifetime of 45 ,us. The [FA/FBI backreaction in C51D-PS I (Fig. 3.63) has lifetimes of 9 ms and 106 ms which contribute 15 % to the total absorption change; an additional 13% is contributed by the slower donation to P700+ by external donors, leading to a 28% efficient electron transfer to F A/F B. The remainder of the backreaction occurs from components with lifetimes of 206 as (20%) and 1.0 ms (12%) derived from Fx, and over 39% occurs from a component with a lifetime of 34 as. The C14S-PS 1 complex shows relatively similar kinetic behavior to the above two mutants. The [F A/FBI backreaction in C14S-PS I (Fig. 3.6C) has lifetime 68 Figure 3.6. Kinetics of AA820 absorbance changes (II). (A) C14D-PS I Complex, (B) C51D-PS I Complex, (C) C14S-PS 1 Complex, (D) C51S-PS 1 Complex. The wild- -type and mutant PS 1 complexes were isolated from membranes with B-D-dodecyl maltoside. The reaction media 13 25 mM Tris buffer, pH 8. 3 with 0. 03% DM, l10 mM sodium ascorbate and 4 ,uM DCPIP; the chlorophyll concentration is 50 ug m1". Each trace (dots) 15 an average of 16 measurements taken at 50 5 intervals. The multiexponential fit is overlaid as a solid line; residuals of the fit shown at the top of the graph. The major individual exponential components are shown as dashed lines, with the percent on the right ordinate, and with offsets equivalent to the relative contribution of the component. The vertical dotted bars separate the time domains in which most of the backreactions of A, (left), Fx (middle) and FA/FB (right) correspondingly occur. 69 components of 51 ms and 148 ms which contribute 26% to the total absorption change; an additional 6% is contributed by the slower donation to P700+ by external donors, leading to a 32% efficient transfer to F A/F,,. The remainder of the backreaction occurs from components with lifetimes of 1.13 as (25%) and 2.81 ms (13%) derived from Fx, and over 31% occurs from components with lifetimes of 32 and 198 as. The [FA/FBI backreaction in C51S-PS I (Fig. 3.6D) has lifetime components of 28 and 89 ms which contribute only 9% to the total absorption change; an additional 12% is contributed by the slower donation to P700+ by external donors, leading to a 21% efficient electron transfer to F A/FB. The remainder of the backreaction occurs from components with lifetimes of 580 as (41%) and 2.3 ms (22%) derived from Fx, and only 16% occurs from a component with a lifetime of 36 us. Discussion Multiple site-specific mutations to individual cysteine ligands for each of the two PS I [4Fe-4S] centers FA and FB were used to produce seven mutant strains of PsaC in Synechocystis sp. PCC 6803. Cysteines 14 and 51 were changed to alanine (C14A, C51A), aspartic acid (C14D, C51D) and serine (C148, C51S), and the results were compared with in vitro reconstitution studies (Yu et al., 1995a and 1995b; Jung et al. , 1996). In each instance, the PsaC mutant strains could not grow under standard photoautotrophic growth conditions, but could grow mixotrophically under weak light, indicating a light-sensitive lesion in PS I that had become limiting for growth. The characteristics of these separate lines were dependent on the specific ligand substitution for the cysteines in two separable ways. First, some of the mutations resulted in PS I reaction centers where stable incorporation of PsaC was precluded by the specific mutation. These strains included the substitutions C51A, C14A and the double aspartate substitution C14D/C51D. Second, some of the mutations resulted in PS I reaction centers incorporating lower-than-wild-type levels of PsaC. These strains included the 7O substitutions C51D, C518, C14D, and C148. In spite of the heterogeneity of PS I found in the membranes, near-homogeneous PS 1 complexes with bound PsaC, PsaD and PsaE as judged by protein blotting were isolated from these four mutant strains by detergent fractionation. These results vary from those recently published for two mutant strains constructed in Anabaena variabilis ATCC 29413 (Mannan et al. , 1996). In A. variabilis, the C13D and C50D mutants (the same functional cysteines in mutants C14D and C51D in Synechocystis sp. PCC 6803), grew photoautotrophically, and electron transport rates, measured using ascorbate/DCPIP as a donor, were similar to the wild-type. Although species-specific differences may be invoked to account for these differences, the discrepancy deserves further investigation. PsaC Mutations and PS I Biogenesis. The biogenesis and redox properties of membrane complexes containing bound iron-sulfur complexes is dependent upon the associated protein structure; however, protein structure can also be modified by incorporation of mutations to ligands of iron- sulfur clusters (Martin et al. , 1990). The in vivo experiments with Synechocystis sp. PCC 6803 demonstrate that some mutations give rise to altered forms of PsaC which are not stable in the PS I complex or not associated with thylakoids: C51A, C14A and the double mutant C14D/C51D. In these cases little PsaC is seen in thylakoid membranes and none is detected in purified PS I complexes (Fig. 3.3 and 3.4). The Ala mutants, which contain an -CH2 side group, are not capable of providing a ligand to an iron in the modified site of the cluster. These substitutions are only capable of supporting [3Fe-4S] clusters (Mehari et al., 1995). The implication is that PsaC proteins containing [3Fe-4S] clusters are unable to bind to P700-PX cores. We suspect that PsaC hosting a [4Fe-48] cluster and a [3Fe-48] cluster, as has been found in the unbound PsaC mutants C14D, C51D, C148 and C518 (Yu et al., 1993; 71 Mehari et al. , 1995) has a structure sufficiently different from that of wild type PsaC to preclude its incorporation into PS I. The single substitutions with either aspartic acid (C14D, C51D) or serine (C148, C518) lead to lower-than-wild-type levels of PsaC incorporation into PS 1 complexes in thylakoids (Fig. 3.3). The Asp and Ser mutants, which contain carboxylate and hydroxy side groups respectively, are potentially capable of providing an oxygen ligand to an iron in the modified site of the cluster. These substitutions are capable of supporting mixed-ligand [4Fe-4S] clusters. The implication is that two [4Fe-4S] clusters must be present in PsaC to be incorporated into PS I complexes. It is likely that the C51D, C518, C14D and C148 proteins also have altered structures, resulting in less-than-perfect interactions with PsaA/PsaB. Yet, size and charge considerations are only one possibility. If the iron-sulfur center insertion requires an efficient ligand exchange reaction at the C14 or C51 positions (this is not provided by the Ala mutations and may be altered to some extent in the Ser and Asp changes), then PsaC biogenesis could be suppressed with the resulting phenotype. Consequently, the mutant PsaC proteins may not bind tightly, or they may dissociate easily, or they may be degraded in the cell, leading to a decreased amount of fully- assembled PS 1 complex. PsaC Mutations and Electron Transport The C51D, C518, C14D and C148 mutant strains demonstrate whole chain oxygen evolution of modest but significant levels along with at least 50% or better wild-type PS I electron transport capacity. Characterization of isolated PS I complexes (Fig. 3.4) demonstrates that electron transport is less efficient when incorporating a modified PsaC subunit. Assuming that the levels of PsaC revealed by Western blots in the isolated PS 1 complexes of the above four mutants are similar to the wild type, the AA820 kinetics results imply that the lower rates of electron transport are due to a qualitative functional alteration of PS I. The single-tumover flash data are in an 72 agreement with the NADP+ reduction data and show that the inefficient electron transfer step occurs between Fx and F A/ FB regardless of whether the mutation is in cysteine 14, which is associated with the FB site, or in cysteine 51, which is associated with the FA site. Particular values of lifetimes and contributions of the AAszo decay phases vary slightly from preparation to preparation even in the wild type samples. However, analysis of decay kinetics over several orders of time scale provides a rationale to distinguish between the “integral-complex” and “core-type” kinetics signature and detect a decrease in efficiency of electron transport to F A and F8 (Vassiliev et al. , 1995). We have tabulated the overall amplitude of the AAszo kinetic components with lifetimes longer than 7 ms as an indicator of efficient photoreduction of F A and FE. As shown in Table 3.1, electron transfer to F A/FB in the serine and aspartate mutants roughly correlates with the rates of ferredoxin-mediated NADP+ photoreduction. Comparison of some of these mutants (C148, C518) with analogous in vitro PS I mutants (Jung et al., 1996) indicates that F A/FB photoreduction in the in vivo mutants samples occurs with a lower apparent quantum efficiency. One possible explanation is that the external thiolate of B-mercaptoethanol, used in the iron-sulfur insertion protocol, provides the ligand to the [4Fe-48] cluster. The PsaC conformation would be rendered closer to that of the wild type, which would then confer higher efficiency of forward electron transfer to the PsaC-bound clusters. On the other hand, the higher contribution of the fast kinetic phases (in the as to ms time domain) in the in vivo PS I mutants may occur due partially to a decrease of the amount of PsaC per reaction center, which would not be resolved in the western blots of the PsaC protein. In any event, one important question regarding the functional properties of PsaC in the in vivo mutants is whether only one or both of the PsaC-bound [4Fe-4S] clusters are functioning as the electron acceptors. The goal of the next chapter is to probe this question using low-temperature EPR spectroscopy and absorbance difference kinetics 73 measurements using repetitive flash excitation. A redox equilibrium between A, and F,( has been proposed (Sétif and Brettel, 1993). Based on this proposal, a reasonable rationale for the function of FA and F3 is to draw the equilibrium completely away from A, , thereby ensuring a high quantum yield in PS I . The single substitutions with either aspartic acid (C14D, C51D) or serine (C148, C518) are sufficiently similar to wild-type PsaC in structure and charge for the mutant PsaC subunit to draw the equilibrium away from A, . For the Ala substitutions, C14A and C51A, and the double Asp mutant, C14D/C51D, the modified PsaC was not stably incorporated into PS I thylakoids in vivo. These mutants also demonstrated greater impairment in whole chain oxygen evolution than the other mutants, with rates similar to the AC-RCPT recipient strain that entirely lacks a functional PsaC. PS I electron transport measured in three different assays also displayed baseline activity, indicating that no functional PS I was present. Decreased amounts of complete PS 1 complexes and reduced electron transport efficiencies in complete complexes could both lead to reduced PS I activity, making PS I the rate-limiting step in photosynthetic electron transport. The reduced PS I activity apparently causes dramatically different phenotypes in growth of the PsaC mutants by two different mechanisms. Under dim light the reduced PS I activity limits photosynthetic conversion of light energy to the extent that cells cannot grow photoautotrophically; under moderate white light the electron transfer chain between P811 and P81 is over-reduced, resulting in inhibition of growth. Acknowledgment The PSI activity measurement and optical spectrocopy were performed by Y.-S. Jung and I. Vassiliev in J. Golbeck lab. 74 References Anderson SL, McIntosh L: Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process. J Bacteriol 173: 2761- 2767 (1991a). Anderson SL, McIntosh L (1991b) Partial conservation of the S'ndhE-psaC-nth 3' gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts. Plant Mol Biol 16: 497-499. Brettel K and Golbeck JH (1995) Spectral and kinetic characterization of electron acceptor A, in a photosystem 1 core devoid of iron-sulfur centers F x, F ,3, and F A. Photosynth. Res. 45, 183-193 Chitnis PR, Xu Q, Chitnis VP, and Nechushtai R (1995) Function and organization of photosystem I polypeptides. Photosynth Res 44: 23-40 Chitnis VP and Chitnis PR (1993) PsaL is required for the formation of photosystem I trimers in the cyanobacterium Synechocystis sp. PCC 6803. FEBS Lett. 336, 330-334 Golbeck JH (1994) Photosystem I in cyanobacteria. In: The molecular biology of cyanobacteria. Bryant DA, ed. Kluwer Academic Publishers, pp. 319-360 Golbeck JH and Bryant (1991) Photosystem 1. Curr. Topics Bioenerg. 16: 83-177 Henry R, Takemoto L, Murphy J, Gallegos G, and Guikema J (1992) Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1. Plant Physiol. and Biochem. 30, 357-364 Jung Y-S, Yu L, Golbeck TH (1995) Reconstitution of iron-sulfur center F3 results in complete restoration of NADP+ photoreduction in Hg-treated photosystem I complexes from Synechococcus sp. PCC 6301. Photosynth Res 46: 249-255 Jung, Y-S., Vassiliev, I.R., Qiao, F., Yang, F., Bryant, D. and Golbeck, J .H. (1996) J. Biol. Chem. (In press) Jung Y8, Vassiliev I, Yu J, McIntosh L, and Golbeck JH (1996) Strains of Synechocystis sp. PCC 6803 with altered PsaC. II. EPR and optical spectroscopic properties of F A and FB in aspartate, serine and alanine replacements of cysteines l4 and 51. J. Biol. Chem. (In press) Li N, Warren PV, Golbeck JH, Frank G, Zuber H, and Bryant DA (1991a) Polypeptide composition of the photosystem I complex and the photosystem I core protein from Synechococcus sp. PCC 6301. Biochim Biophys Acta 1059: 215-225. Li N, Zhao J, Warren PV, Warden JT, Bryant DA, and Golbeck JH (1991b) PsaD is required for the stable binding of PsaC to the photosystem I core protein of Synechococcus sp. PCC 6301. Biochemistry 30: 7863-7872. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148: 350-3 82. 75 Lowry OH, Rosebrough NJ, Farr AL, and Randall R] (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-275. Mannan RM, He W-Z, Metzger S, Whitmarsh J, Malkin R, and Pakrasi HB (1996) Active photosynthesis in cyanobacterial mutants with directed modifications in the ligands for two iron-sulfur clusters in the PsaC protein of photosystem I. EMBO J 15: 1826-1833 Mannan RM, Pakrasi HB, and Sonoike K (1994) The PsaC protein is necessary for the stable association of the PsaD, PsaE, and PsaL proteins in the photosystem I complex: analysis of a cyanobacterial mutant strain. Arch Biochem Biophys 315: 68-73. Martin AE, Burgess BK, Stout CD, Cash VL, Dean DR, Jensen GM, and Stephens PJ (1990) Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-8] cluster- dn'ven protein rearrangement. Proc. Natl. Acad. Sci. USA 87, 598-602 Mehari T, Qiao F, Scott PM, Nellis DF, Zhao J, Bryant DA, and Golbeck JR (1995) Modified ligands to FA and PE in photosystem I. 1. Structural constraints for the formation of iron-sulfur clusters in free and rebound PsaC. J. Biol. Chem. 270, 2108- 281 17 Muhlenhoff U, Kruip J, Bryant DA, Rogner M, Sétif P, and Boekema E (1996a) Characterization of a redox-active cross-linked complex between cyanobacterial photosystem I and its physiological acceptor flavodoxin. EMBO J. 15, 488-497 Muhlenhoff U, Zhao J, and Bryant DA (1996b) Interaction between photosystem I and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 as revealed by chemical cross-linking. Eur. J. Biochem. 235, 324-331 Ohad N, and Hirschberg J (1992) Mutations in the D1 subunit of photosystem II distinguish between quinone and herbicide binding sites. Plant Cell 4: 272-283 Sambrook J, F ritsch EF, and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Sétif P and Brettel K (1993) Forward electron transfer from phylloquinone-A, to iron sulfur centers in spinach photosystem 1. Biochemistry 31, 7 846-7854 Shen G, Boussiba S, and Vermaas WFJ (1993) Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome fiinction. Plant Cell 5: 1853-1863. Smart LB, Bowlby NR, Anderson SL, Sithole I, and McIntosh L (1994) Genetic manipulation of the cyanobacterium Synechocystis sp. PCC 6803: development of strains lacking PSI for the analysis of mutations in PSII. Plant Physiol. 104, 349-3 54 Smart LB and McIntosh L (1991) Expression of photosysthesis genes in the cyanobacterium Synechocystis sp. PCC 6803: psaA-psaB and psbA transcripts accumulate in dark-grown cells. Plant Mol Biol 17: 959-971 Smart LB, Warren PV, Golbeck JH, and McIntosh L (1993) Mutational analysis of the structure and biogenesis of the photosystem I reaction center in the cyanobacterium Synechocystis sp PCC 6803. Proc Natl Acad Sci USA 90: 1132-1136 Steinmuller K (1992) Identification of a second psaC gene in the cyanobacterium Synechocystis sp PCC 6803. Plant Mol Biol 20: 997-1001. 76 Taylor LA and Rose RE (1988) A correction in the nucleotide sequence of the Tn903 kanamycin resistance determinant in pUC4K. Nucl Acids Res 16: 358-358 Vassiliev IR, Jung Y8, Mamedov MD, Semonoc AY, McIntosh L, and Golbeck JH (1996) Absorbance changes and electrogenic reactions in the usec-to-sec time domain in photosystem I. Biophys J (In press) Vassiliev IR, Jung YS, Smart LB, Schulz R, McIntosh L, and Golbeck JH (1995) A mixed-ligand iron-sulfiir cluster (C556S,.,,,,3 or C5658”) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I. Biophys J 69: 1544- 1553 Vermaas WFJ, Ikeuchi M, and Inoue Y (1988) Protein composition of the photosystem 11 core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp PCC 6803. Photosynth Res 17: 97-113. Vieira J and Messing J (1982) The pUC plasmids, an Ml3mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259-268. Warren PV, Smart LB, McIntosh L, and Golbeck JH (1993) Site-directed conversion of cysteine-S65 to serine in PsaB of photosystem I results in the assembly of [3Fe-48] and [4Fe-4S] clusters in Fx. A mixed-ligand [4Fe-48] cluster is capable of electron transfer to F A and FE. Biochemistry 32: 4411-4419. Williams JGK (1988) Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Meth Enzymol 167: 766-7 78 Yu J, Smart LB, Jung Y-S, Golbeck JH, and McIntosh L (1995) Absence of PsaC subunit allows assembly of photosystem 1 core but prevents the binding of PsaD and PsaE in Synechocystis sp. PCC 6803. Plant Mol Biol 29: 331-342 Yu L, Bryant DA, and Golbeck JH (1995a) Evidence for a mixed-ligand [4Fe-4S] cluster in the C 14D mutant of PsaC. Altered reduction potentials and EPR spectral properties of the FA and F B clusters on rebinding to the P700-Fx core. Biochemistry 34, 7861-7868 Yu L, Vassiliev IR, Jung Y8, Bryant DA, and Golbeck JI-I (1995b)Modified ligands to FA and FE in photosystem I. II. Characterization of a mixed ligand [4Fe-4S] cluster in the C5 1D mutant of PsaC upon rebinding to P700-Fx cores. J. Biol. Chem. 270, 28118-28125 Yu L, Zhao J, Lu W, Bryant DA, and Golbeck JH (1993) Characterization of the [3F e-4S] and [4Fe-4S] clusters in unbound PsaC mutants C14D and C51D. The midpoint potentials of the single [4Fe-4S] clusters are identical of F A and F,3 in bound PsaC of photosystem 1. Biochemistry 32: 8251-8258 Chapter 4 STRAINS OF S I'NECHOC YSTIS SP. PCC 6803 WITH ALTERED PsaC. II. EPR AND OPTICAL SPECTROSCOPIC PROPERTIES OF FA AND F,3 WHEN CY STEINES 14 AND 51 ARE REPLACED BY ASPARTATE, SERINE AND ALAN INE Introduction There are several exceptions to the nearly universal occurrence of cysteine thiolate ligands to iron-sulfiir clusters in the ferredoxin class of electron transfer proteins (Moulis et al. , 1996). Examples include the N-ligands from histidine residues in the Rieske- subclass of iron-sulfur proteins (Davidson et al., 1992; Shergill and Cammack, 1994; Britt et al., 1991), O-ligands from (most likely) aspartate in Fd III from Desulfovibrio africanus (Telser et al., 1995), and a proposed O-ligand from serine to the pentacoordinated iron in the P- cluster of nitrogenase (Mouesca et al., 1994). Recently, oxygen ligands have been introduced in lieu of cysteine thiolates by site-directed mutagenesis in proteins which contain [2Fe-28], [3Fe-4S] and/or [4Fe-4S] clusters. Examples include the introduction of serine for cysteine in the interpolypeptide I"x cluster of PS I (Warren et al., 1993; Vassiliev et al., 1995) and the [4Fe-4S] cluster of Escherichia coli fiimarate reductase (Kowal et al., 1995). The consequence of these changes include a change in the EPR spectrum and a decreased electron transfer efficiency 77 78 in the case of serine- ligated Fx, and a change in the cluster midpoint potential and intercluster spin interaction in the case of E. coli fiimarate reductase. One instance of a functional [4Fe-4S] cluster derived from an aspartate-for- cysteine change is the modified PsaC subunit of PS I. In a previous study, mutant PsaC proteins expressed in Escherichia coli were reconstituted onto P700-FX cores, and the assignment of the ligands for the two terminal electron acceptors was determined by EPR spectroscopy (Zhao et al., 1992). The substitution of aspartate for cysteine in position 14 of PsaC led to the retention of an S = '/2, [4F e-4S] cluster-at the unmodified site with g-values characteristic of F A. Similarly, the substitution of aspartate for cysteine in position 51 of PsaC led to the retention of an S = V2, [4Fe-48] cluster at the unmodified site with g—values characteristic of F3. Since the pattern of cysteine ligation in PsaC is expected to be identical to that of ferredoxins with two [4Fe-4S] clusters whose structures have been determined, it follows that F,3 is ligated by cysteines 11, l4, l7, and 58 and F A is ligated by cysteines 21, 48, 51, and 54 (Zhao et al., 1992). The in vitro reconstitution experiments led to several hypotheses regarding alternative ligands to iron-sulfur clusters: 1) unbound PsaC refolds only in the presence of one [3Fe-4S] and one [4Fe-4S], or two [4Fe—4S] clusters when cysteine is replaced in positions 14 and 51 by aspartate, serine and alanine (Yu et al., 1993; Mehari et al., 1995). According to these results, a stable 3-dimensional structure requires the presence of two iron-sulfirr clusters, one of which must be a [4Fe-4S] cluster. 2) The failure to observe a [3F e-48] cluster in the in vitro reconstituted C14X-PS I or C5 lX-PS I complexes (where X = D, A or 8) indicates that the binding of PsaC onto P700-Fx cores requires the presence of two [4Fe-48] clusters. If rigorously true, it may be difficult to introduce a 79 [3F e-48] cluster motif into PsaC in vivo. 3) An oxygen ligand to the [4Fe-4S] cluster in the mutant site of the C14D-PS I and the C51D- PS I complexes leads to a high-spin state (likely S = 3/2). In this work, these hypotheses were tested through the generation of a series of in vivo mutations in F A and F3. The choices to replace cysteine residues with aspartic acid, serine and alanine were made because aspartate is known to support [3Fe-48] and [4Fe-48] clusters in naturally-occurring ferredoxins (Armstrong et al., 1989; Conover et al., 1990), serine can support [3Fe-4S] and [4Fe-4S] clusters in the Fx binding site of PS I (Warren et al. , 1993) and in the Azotobacter vinelandii hydrogenase small subunit (McTavish et al., 1995), and alanine, with the absence of a suitable ligand, should support only [3F e-4S] clusters. This chapter summarizes the EPR spectral characteristics and electron transfer properties of aspartate (C14D, C51D, C14D/C51D) alanine (C14A, C51A) and serine (C148, C518) mutations in PsaC in Synechocystis sp. PCC 6803. The premise tested is that [4Fe-4S] clusters will only be assembled in those mutants where oxygen ligands are available from the side chains of the replacement amino acids. Results and Discussion Summary of results A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues which ligate the F A and PB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14D, C51D, C14D/C51D), serine (C 148, C518) and alanine (C 14A, C51A) and the properties of F A and F,3 were 80 characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14D-PS I and C148-PS I complexes showed high levels of photoreduction of F A with g values of 2.045, 1.944 and 1.852 after illumination at 15 K, but there was no evidence of reduced F3 in the g = 2 region. The C51D-PS I and C518- PS I complexes showed low levels of photoreduction of F,3 with g values of 2.067, 1.931 and 1.881 after illumination at 15 K, but there was no evidence of reduced F A in the g = 2 region. The presence of F,3 was inferred in C14D-PS I and C148-PS I and the presence of F A was inferred in C51D-P8 l and C518-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700+ cation generated by illumination at 77 K. Successive-flash experiments at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after F,( in the four mutant PS 1 complexes at room temperature. Number of Electron Acceptors Present in the C14X-PS I and C51X-PS [Mutants The following generalizations can be made about the Cl4X-PS I and C51X-P8 I mutants (where X = D, S, and A). First, only [4Fe-48] clusters are found in the in vivo mutant PS I complexes, even though the unbound mutant PsaC expressed in E. coli contain [3Fe-4S] and mixed-ligand [4Fe-48] clusters in the altered site (Yu L et al., 1993; Mehari et al., 1995). The PsaC protein does not assemble in the in vivo mutants for which cysteines l4 and 51 are substituted with alanine, C14A and C51A, most likely because a [4F e-48] cluster cannot assemble in the absence of a suitable ligand. The absence of a suitable oxygen or sulfiir ligand fi'om the replacement amino acid, alanine, and the resulting destabilizing effect of a [3Fe-4S] cluster on PsaC binding to the P700-Fx 81 heterodimer, is apparently the reason for the inability of the C14A and C51A PsaC to bind to PS 1. Second, all mutant PS I complexes containing mixed-ligand [4Fe-48] clusters are capable of electron transfer to F A and F,3 at 15 K. These mutant complexes are also capable of supporting electron throughput to NADP+ at room temperature. This is also true for PS I complexes reconstituted in vitro with C14D and C51D (Yu et al., 19953). Third, the results of the in vivo experiments agree with the cysteine ligand assignments to PA and FB made using in vitro reconstitution of E. coli expressed proteins onto P700-Fx cores (Zhao et al., 1992). It is clear from the optical results that both acceptors are photochemically active in C14D-PS I, C51D-PS I, C148-PS I and C518-PS Icomplexes and that they may operate with lower quantum efficiencies than in the wild type. However, heterogeneity due to incomplete binding of PsaC would give rise to the same kinetics in single turnover experiments as does forward electron transfer inefficiency. To this end, we have taken care to isolate near-homogeneous PS I complexes from the thylakoids based on the different densities of PS I reaction centers with and without PsaC, PsaD, PsaE and PsaL (Chapter 3). Comparison with in vitro Reconstituted C14D-PS I and C5ID-PS I Mutant Complexes The similarities and the differences between the in vivo engineered and in vitro reconstituted C14D-PSI and C51D-PS I complexes depend on whether the mutation is in the F A or the FB site. Under conditions of photoaccumulation, the in vivo C51D-PS I complex is similar in spectral appearance to Escherichia coli-expressed C51D-PsaC reconstituted in vitro onto P700-Fx cores (Yu et al., 1995b). These similarities indicate 82 that oxygen from aspartate (alternatively, water or OH“) provide the ligands to F A in the modified site. The slight differences in the g-values between the in vivo and in vitro PS I complexes may be related to species differences, since the in vivo C51D-PS I mutant PS I complexes were derived from Synechocystis sp. PCC 6803, whereas the in vitro reconstituted PS I complexes were hybrids, composed of a Synechococcus sp. PCC 6301 P700-Fx core, PsaC and PsaE derived from Synechococcus sp. PCC 7002, and a PsaD protein derived from Nostoc sp. PCC 8009 (Li et al., 1991). Under conditions where only one electron was promoted to the acceptor side, only a low spin concentration of the FB cluster was observed in the in vivo mutants and their in vitro counterparts; yet the size of the P700+ radical indicates that the majority of the electrons were promoted to the proposed high-spin F A cluster. However, a very low spin concentration of F A was also observed after the in vitro reconstitution, which may have been derived from a minority population of a S = '/2 cluster. One candidate is a sulfur thiolate ligand derived from carryover of the B-mercaptoethanol used in the reconstitution protocol as suggested in Jung et al.(l995). In contrast, the in vivo C14D-P8 1 complex differs substantially from the in vitro reconstituted C14D-PS I complex. In the in vivo mutant, the F,3 cluster is not observed in the g = 2 region, but is inferred from the size of the P700+ radical and the presence of new EPR resonances at very low temperatures which may be derived from intercluster spin interaction between FA and F8. The proposal is that the F,3 cluster is present as a high-spin system (likely 8 = 3/2). After the in vitro reconstitution, the F,3 cluster was observed as a ground state S = '/2 spin system with g-values of 2.1 18, 1.911, 1.883. The difference in the in vivo engineered and in vitro assembled C14D-PS I complexes is that only oxygen 83 ligands are available in the former whereas sulfur thiolate ligands may also be available in the latter. The B-mercaptoethanol used in the in vitro iron-sulfur reinsertion protocol may have been recruited as a ligand at the mutated site of the in vitro complex (Jung et al., 1995). It is likely that SUIlel' provides a better ligand to an iron-sulfur cluster, replacing some or all of the oxygen- ligated cluster in the in vitro experiments. In this in vitro reconstituted PSI complex, F B may be present as a mixed population of sulfur-ligated 8= ‘/2 clusters visible in the g = 2 region, and oxygen-ligated Si= 3/2 clusters invisible in the g = 2 region. The fraction of sulfur- and oxygen-ligated clusters may have more to do with steric hindrance or accessibility to solvent than with inherent differences in the C14D and C51D sites of PsaC. The inability to engineer a PsaC protein with either a missing cluster or a [3Fe—48] cluster in vitro in PsaC from Synechocystis sp. PCC 7002 (Mehari et al., 1995) or in vivo in Synechocystis sp. PCC 6803 (this work) is further evidence that two [4F e-4S] clusters must be present for PsaC to bind to the PS I core. This result is consistent with in vitro reconstitutions of E. coli-expressed mutant PsaC proteins, where only those proteins with two intact [4F e-4S] clusters could be rebound onto P700-Fx cores (Jung et al., 1995). In conclusion, the studies presented here demonstrate that mixed-ligand [4F e-4S] clusters can assemble in the PsaC protein of PS I in vivo, and that PsaC protein containing such mixed-ligand clusters can bind to PS I core complexes and fimction in electron transfer reactions from P700 to ferredoxin or flavodoxin. The efliciency of iron-sulfur center insertion into the mutant proteins, or the stability of mutant proteins after cluster insertion, varies depending upon the chemical nature of the side group on the replacement amino acid. Differences observed between the spectroscopic properties of PS I complexes 84 containing mutant PsaC proteins formed by in vitro or in vivo methods are most likely due to the chemical nature of the ligands to the [4Fe-4S] clusters. The common denominator is that only those PsaC proteins containing two [4Fe-48] clusters are capable of assembling onto P700-Fx cores either in vivo or in vitro. Note: This chapter is condensed from a manuscript in press in J. Biol. Chem. by Yean-Sung Jung, Ilya R. Vassiliev, Jianping Yu, Lee McIntosh, and John H. Golbeck. 85 References Armstrong FA, George SJ, Cammack R Hatchikian EC, and Thomson AJ (1989) Electrochemical and spectroscopic characterization of the 7Fe form of ferredoxin 111 from Desulfovibrio africanus. Biochem J 264: 265-273 Britt RD, Sauer K, Klein MP, Knaff DB, Kriauciunas A, Yu CA, Yu L, and Malkin R (1991) Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske Fe-S centers of the cytochrome b6f complex of spinach and the cytochrome bcl complexes of Rhodospirillun rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria. Biochemistry 30: 1892- 1901 Conover PR, Kowal AT, Fu W, Park JB, Aono S, Adams MWW, and Johnson MK (1990) Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin. J Biol Chem 265: 8533-8541 Davidson E, Ohnishi T, Attaasafoadjei E, and Daldal F (1992) Potential ligands to the [2Fe-28] Rieske cluster of the cytochrome bcl complex of Rhodobactter capsulatus probed by site-directed mutagenesis. Biochemistry 31: 3342-3351 Jung Y-S, Yu J, Yu L, Zhao J, Bryant DA, McIntosh L, and Golbeck JH (1995) In vivo site-directed mutations of the cysteine ligands to PA and F,, in Synechocystis sp PCC 6803: a comparison with in vitro reconstituted PSI complexes. in Photosynthesis: From Light to Biosphere (Mathis, PA ed) Vol II, pp. 127-130. Kluwer Academic Publishers, Dordrecht Kowal AT, Werth MT, Mandori A, Cecchini G, Schroder I, Gunsalus RP, and Johnson MK (1995) Effect of cysteine to serine mutations on the properties of the [4Fe-48] center in Escherichia coli fumarate reductase. Biochemistry 34: 12284-12293 Li N, Warren PV, Golbeck JH, Frank G, Zuber H, and Bryant BA (1991) Polypeptide composition of the photosystem I complex and the photosystem I core protein from Synechococcus sp. PCC 6301. Biochim Biophys Acta 1059: 215-225 McTavish H, Sayavedrasoto LA, and Arp DJ (1995) Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by 02 and preferentially damages H2 oxidation over H2 evolution. J Bacteriol 177: 3960-3964 Mehari T, Parrett KG, Warren PV, and Golbeck JH (1991) Reconstitution of the iron- sulfur clusters in the isolated F ,,/F,3 protein: ESR characterization of the same-species and cross-species photosystem I complexes. Biochim Biophs Acta 1056: 139-148 Mehari T, Qiao F, Scott PM, Nellis DF, Zhao J, Bryant DA, and Golbeck JH (1995) Modified ligands to F A and F B in photosystem I. 1. Structural constraints for the formation 86 of iron-sulfur clusters in free and rebound PsaC. J Biol Chem 270: 28108-28117 Mouesca JM, Noodleman L, and Case DA (1994) Density firnctional/Poisson-Boltzmann calculations of redox potentials for iron-sulfilr clusters. Inorg Chem 33: 4819-4830 Moulis JM, Davasse V, Golinelli MP, Meyer J, and Quinkal I (1996) The coordination sphere of iron-sulfur clusters: lessons from site-directed mutagenesis experiments. J Inorg Biol Chem 1: 2-14 Shergill JK and Cammack R (1994) ESEEM studies of the iron-sulphur clusters of succinate dehydrogenase in Arum maculatum spadix mitochondrial membranes. Biochim Biophys Acta 1185: 35-42 Telser J, Smith ET, Adams MWW, Conover R, Johnson MK, and Hoffman BM (1995) Cyanide binding to the novel 4F e ferredoxin from Pyrococcusfilriosus: Investigation by EPR and ENDOR spectroscopy. J Am Chem Soc 117: 5133-5140 Vassiliev I, Jung Y-S, Smart LB, Schulz R, McIntosh L, and Golbeck JH (1995) A mixed- ligand iron-sulfur cluster (C556S,.,,,B or C565 SM) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I. Biophst 69: 1544- 1553 Warren PV, Smart LB, McIntosh L, and Golbeck III (1993) Site-directed conversion of cysteine 565 to serine in PsaB of photosystem I results in the assembly of [3F e-48] cluster and [4F e-4S] clusters in Ex. A mixed-ligand [4Fe-48] cluster is capable of electron transfer to F A and F3. Biochemistry 32: 4411-4419 Yu L, Bryant DA, and Golbeck JH (1995a) Evidence for a mixed-ligand [4F e-4S] cluster in the C 14D mutant of PsaC. Altered reduction potentials and EPR spectral properties of the F A and FB clusters on rebinding to the P700-Fx core. Biochemistry 34, 7861-7868 Yu L, Vassiliev IR, Jung Y8, Bryant DA, and Golbeck JH (1995b)Modified ligands to F A and F,3 in photosystem I. II. Characterization of a mixed ligand [4Fe-48] cluster in the C51D mutant of PsaC upon rebinding to P700-Fx cores. J. Biol. Chem. 270, 28118-28125 Yu L, Zhao J, Lu W, Bryant DA, and Golbeck JH (1993) Characterization of the [3Fe-48] and [4Fe-4S] clusters in unbound PsaC mutants C 14D and C51D. The midpoint potentials of the single [4Fe-48] clusters are identical to F A abd F,3 in bound PsaC of photosystem 1. Biochemistry 32: 8251-8258 Zhao J, Li N, Warren P, Golbeck JH, and Bryant DA (1992) Site-directed conversion of a cysteine to an aspartate leads to the assembly of a [3Fe-48] cluster in PsaC of photosystem I. Photoreduction of F A is independent of F8. Biochemistry 31: 5093-5099 Chapter 5 ISOLATION AND GENETIC CHARACTERIZATION OF PSEUDOREVERTANTS FROM SITE-DIRECTED PSI MUTANTS IN S IWE CHOC YS T IS 6803 Introduction Reversion of mutation results fi'om a change in DNA that either exactly reverses the original alteration (a true reversion), or compensates for it (a suppressor mutation). Suppressor mutations yield valuable information about other sites and/or other polypeptides that interact with the site altered by the original (primary) mutation. An organism with a suppressor mutation can be called a pseudorevertant. Selection of pseudorevertants can yield an array of suppressor mutations. Suppressor selection has been applied to Synechocystis sp. PCC 6803 for the molecular analysis of the structure, function, and assembly of photosystem I (PSI). This methodology involves the isolation of spontaneous pseudorevertants from site-directed PSI mutants. Suppressor mutations can then be located to a specific gene or operon by complementation and/or identified by DNA sequencing. In this way, specific amino acids and electron carriers may be assigned to a PSI structural model, and insights may be gained into the roles of specific amino acids on the assembly/stability of the complex and on modulating electron transport. 87 88 Synechocystis 6803 is a model organism for the study of oxygenic photosynthesis. Its genome (3.57Mb) has recently been sequenced (Kaneko et al., 1996) and it is naturally competent, can be easily transformed. It also has an active homologous recombination mechanism (Williams, 1988). Furthermore, it can grow photoheterotrophically on glucose, which has allowed propagation of PSII mutants that are incapable of photosynthesis (Williams, 1988), and it can grow under light-activated-heterotrophic-growth (LAHG) conditions, permitting isolation of P81 mutants in the absence of P81 function (Anderson et al., 1991). Site-directed mutagenesis has been successfirlly used in this organism to study structure-function relationships both in PSH (Williams, 1988) and in PSI (Smart et al., 1993; Schulz et al., 1995; Yu et al., submitted). PSI consists of at least 11 different proteins in cyanobacteria (Chitnis, 1996). All PSI proteins are believed to be present as one copy per P700 reaction center. They vary considerably in their molecular weights, hydrophobicities, and locations with respect to the lipid bilayer. In addition to proteins, the PSI complex contains approximately 100 Chl a molecules, two B-carotenes, two phyloquinone molecules, and three [4F e-48] clusters. The cofactors of P81 are bound to the PsaA, PsaB, and PsaC proteins. Trimers of P81 have been observed by electron microscopy in the photosynthetic membranes of cyanobacteria and are considered to be in vivo functional units (Boekema et al., 1994). On the basis of the electron density maps, a structural model for trimeric PSI from Synechococcus elongatus was initially proposed at a resolution of 6 A (Krauss et al., 1993). From new data, many aspects of this model have been later refined to a resolution of 4.5 A (Schubert et al., 1995; Fromme et al., 1996). Resolution of P81 structure at the atomic level will require further x-ray difii’action analyses of better crystals of P81. These 89 fine structure determinations are being complemented by topological explorations, electron microscopy, and genetic analysis. Genetic inactivation of the psaA, psaB, or psaC genes in Synechocystis 6803 produced mutants that lack PSI function and depend on glucose for grth (Smart et al., 1991; Chapter 2). Photosynthesis-deficient PSI mutants have also been created by site- directed mutagenesis of these genes (Smart et al., 1993; Schulz et al, 1995; Chapter 3). An additional phenotype, inhibition of mixotrophic growth by moderate light intensities, has also been observed for some PSI mutants (Smart et al., 1993; Chapter 3). Characterization of these site-directed mutations has yielded valuable information on biogenesis of P81 and on functions of electron carriers. One limitation of the site-directed mutagenesis approach is that it relies on proposed models or predictions. Studies using pseudorevertants will add additional power to the mutagenesis approach and may potentially reveal information about PSI biogenesis and firnction that may not have been predicted. This chapter describes a procedure for isolating spontaneous pseudorevertants fiom PSI mutants of Synechocystis 6803, localization and identification of the suppressor mutations, and some of the properties of the genetic transformation system in this cyanobacterium. Isolation and genetic characterization of pseudorevertants from PSI mutants A flow chart for isolation and genetic characterization of a ‘typical’ pseudorevertant from a primary PSI mutant in Synechocystis 6803 is shown in figure 5. 1. 90 Primary mutant 1 Photoautotrophic grth or mixotrophic grth under inhibitory light intensity 1 Colonies are subject to three or more serial streakings under the selective conditions 1 PCR amplification of a fragment containing the primary mutation to confirm presence of the primary mutation by restriction analysis and/or sequencing l Transform the primary mutant with total DNA isolated fiom the revertants to demonstrate presence of suppressor mutations 1 Locate suppressor mutations by functional complementation 1 Identify the suppressor mutations by sequencing the cloned or amplified fi'agments 1 If necessary, the identified mutation may be introduced into the primary mutation background by site-directed mutagenesis to confirm its firnction. Figure 5.1. A flow chart for isolation and genetic characterization of a ‘typical’ pseudorevertant fi'om a primary PSI mutant. 91 Isolation of pseudorevertants from PsaC mutants The PsaC subunit, encoded by psaC , provides the ligands for two [4Fe-4S] clusters, F A and F8. The proposed cysteine ligands have been studied by a combination of site-directed mutagenesis and in vitro reconstitution. Mutant PsaC derived from overexpression in E. coli was reconstituted with a biochemical preparation of P81 cores. These experiments showed that Cys 51 in PsaC is a ligand to the F A cluster, whereas Cysl4 is a ligand to the F,3 cluster (Zhao et al., 1992). PsaC mutants C51D, C518, C51A, C 14D, C 148, and C14A were introduced into Synechocystis 6803, in which a cysteine ligand to PA or F ,,, respectively, is changed to aspartate, serine or alanine by site-directed mutagenesis. Physiological characterization has shown that these mutations have decreased PSI capacity and efficiency, making PSI limiting for whole chain photosynthesis. As a result, these mutants cannot grow photoautotrophically under white light and their mixotrophic growth (in the presence of glucose) is inhibited by white light (22-6O umol m‘2 5"; Chapter 3). To isolate pseudorevertants, 108 cells from a mid-log phase culture grown under LAHG (Anderson and McIntosh, 1991) conditions were spread onto 90-cm plates with solid BG-ll medium supplemented with gentamycin (glucose is added for mixotrophic growth). The plates were placed at 30°C under 10 umol m'2 8'1 white light for two days then transferred to 22 umol m‘2 3‘1 white light for up to forty days. Colonies have been found to appear spontaneously under these selective conditions at frequencies between 10‘5-10" in several tests. Hundreds of the isolates were serially streaked three to four times, with about half of them able to grow on the fourth and successive plates. Why other isolates could appear on the first selective plates but could not grow on the successive plates is not clear. Following the fourth serial plating, 92 selected isolates were characterized physiologically and genetically, as discussed below. Photoautotrophic and mixotrophic grth under a light intensity of 22 umol rn'2 s'1 was used to isolate and characterize pseudorevertants. Isolates obtained from one selective condition were tested for their ability to grow in other conditions by streaking on plates. Three classes of pseudorevertants have been found: those capable of growth under: (1) photoautotrophic conditions, but not mixotrophic conditions; (2) mixotrophic conditions, but not photoautotrophic conditions; (3) both conditions. Genetic conformation for suppressor mutations Since there are multiple copies of the genome in each Synechocystis cell (Williams, 1988), revertant colonies are serially streaked at least four times on selective medium in order to achieve genetic homogeneity. The presence of the primary mutations was checked using a rapid genetic screening technique developed based on polymerase chain reaction (PCR). Target fragments were directly amplified from cyanobacterial cells using a pair of specific primers (see Materials and Methods for details), then used for complementation tests, restriction analysis, and direct sequencing. Although true reversions have been found in C 148 revertants (codon TCT for serine was reverted to TGT for cysteine), most of the revertants of PsaC mutants contained the primary mutations, and no second-site mutations were found elsewhere in psaC. These results suggest that suppressor mutations in other genes may be responsible for growth under the selective conditions. However, the metabolism of cyanobacteria is very flexible. For example, flavodoxin can substitute for ferredoxin under conditions of iron-limitation (Ho and Krogmann, 1982). It is important therefore to demonstrate that the revertants have 93 come from genetic mutations rather than from physiological acclimation. DNA applied directly to the surface of a lawn of Synechocystis 6803 embedded in agar can result in the transformation of these cells (Dzelzkalns and Bogorad, 1988). This observation provides a simple procedure named “dot transformation” for the rapid assay of large numbers of DNA samples that may functionally complement a primary mutation. Dot transformation was used in characterization of revertants in this study. Total DNA was isolated from revertant colonies and was used to transform the primary PSI mutants. An example of such transformation assay is shown in figure 5.2. All the isolates tested so far demonstrated specific DNA modifications. Suppressor mutations can be localized to other proteins Mutations in PsaC such as C51D result in unstable assembly of the complex and lower electron transfer efficiency in assembled complexes. Conceivably the defects could be complemented by suppressor mutations in PsaA/PsaB dimer which may restore complex stability and/or electron transfer efficiency. Dot transformation was used to locate suppressor mutations to a particular DNA fragment by functionally complementing primary mutant cells using specific fi'agments amplified from the revertants. Suppressor mutations in ten pseudorevertant isolates from C5 1D have been localized to PsaA/PsaB in this way. These pseudorevertants show consistently different grth rates, indicating that the mutant phenotype that results from C51D can be suppressed by several different mutations on PsaA/PsaB. In contrast, no suppressor mutation for C148 has been localized to any tested genes for PSI subunits (psaA-psaB, psaC, psaD, and psaE). 94 10ng WT 100ng 10ng C14S-18 100ng Figure 5.2 In situ “dot transformation” demonstrates suppressor mutation. Log phase culture 0.1 ml (OD730 0.3) ofa primary PSI mutant C148 was added to 3 ml melted 0.7% Bacto-agar at 50°C, then the mixture was poured onto a 90cm petri dish containing 50 ml BG-l 1 solid medium supplemented with gentamycin. Total DNA (10 ng and 100 ng) isolated from wild-type and a pseudorevertant C148-18 was dropped in 5 ul on the lawn at marked areas. The plates were placed under 10 umol m'2 5" white light for four days before transfer to 22 umol m‘2 5" white light. The plate was photographed 40 days after transformation. In a control experiment total DNA isolated from C148 was dropped on a lawn of C148 cells, and no cluster of colonies appeared at the marked areas in 40 days. Note that some colonies appeared spontaneously outside of transformed areas. The protocol was modified from Dzelzkalns et al. (1988). 95 Implication for position and fimction of F A and F 3 The path of electrons through F A and F,3 is an unresolved area in the electron transfer pathway in PSI (Jung et al., 1995). The electrons may travel in a series: Fx to FB to F A to Fd, or Fx to F A to F3 to Fd . Alternatively, the electrons fi'om Fx may be transferred to F A or F8 and then one or both of these reduced clusters can donate electrons to Fd. The proposed locations of these redox centers in the x-ray crystallographic structure are at different distances from Fx, thus implying a serial flow of electrons between F,3 and F A (Krauss et al., 1993). As shown in figure 5.3, the most striking feature revealed by the PSI crystal structure is the arrangement of the three F e-S clusters in the form of an irregular triangle, with one cluster 15.4 A and the other, 22.2 A fiom Fx (Schubert et al., 1995). The crystal structure gives no hint as to which cluster represents F A and F3. Selective, specific inactivation of F B with mercurials such as HgCl2 indicates that photoreduction of F A is independent of F,3 (Jung et al., 1995). In contrast, Fd- mediated electron transfer from PSI requires functional FB (Jung et al., 1995). Our results with pseudorevertants show that only a mutation at F A site can be suppressed by mutations in PsaA/PsaB. The results imply that the F A site has extensive interaction with PsaA/PsaB, while the FB site is exposed on the stroma side. The combined data are not conclusive but argue that F A is the proximal cluster to Fx and F,3 is the distal cluster, and that electrons travel serially from Fx to F A to F3. Secondary mutation can be at the same site as the primary mutation The primary photochemical reactions catalyzed by the PSI reaction center occur on a core heterodimer composed of the subunits PsaA and PsaB, and a small subunit 96 It?" 12.5 I‘. .Ql‘ 22.2 Figure 5.3. FA/FB FBIFA (fifi) (A1) Arrangement of the geometrical centers of PSI electron carriers. Distances are in A. A/B, PsaA/PsaB dimer. C, PsaC. Modified from Schubert et al. (1995) 97 PsaC. The interaction between the PsaC subunit and the core heterodimer has been studied by molecular modeling and it was proposed that the two conserved regions between the cysteine ligands of the Fx cluster on PsaA and PsaB (CDGPRGGTC) are two flexible loops that form a surface-exposed cavity to accommodate the PsaC subunit (Rodday et al., 1993). To test the proposal an arginine residue R5619”, which may interact with a negatively charged residue on the PsaC subunit in Synechocystis 6803, was changed to glutamate by site-directed mutagenesis. The mutant R561E can not grow photoautotrophically due to reduced level of P81 (Schulz et al., 1995). To firrther understand the role of R561PsaB in assembly/stability of P81 complex, spontaneous revertants have been isolated from R561E at a rate of 4 x 10’7 under photoautotrophic growth conditions. The spontaneous mutations have been localized to the psaA-psaB operon in many revertants by functional complementation. Sequencing of PCR fragments amplified fi'om one of the revertants revealed a secondary mutation at 561M which converts codon GAA for glutamate to AAA for lysine. The reversion restores photoautotrophic growth to a rate comparable to wild type cells. This result strongly suggests a role for a positively charged residue at this site in stable assembly of the PSI complex. Genetic transformation in Synechocystis 6803 Some characteristics of genetic transformation in Synechocystis 6803 have been studied previously (Williams, 1988). When dot transformation is used in characterization of pseudorevertants, some colonies grow under selective conditions as a result of transformation, and other colonies appear on the plates spontaneously (see figure 5.2 for 98 an example). Therefore the success is dependent on a higher transformation frequency over the proportion of cells containing spontaneous mutations. Spontaneous mutations have been found to accumulate in cultures of the primary mutants, making aged cultures unusable for suppression test. To avoid such a problem, a fresh liquid culture should be used for suppression tests. In order to achieve a high transformation frequency, several parameters have been studied: growth conditions of the primary mutants, methods for preparation of transforming DNA, competency of primary PSI mutants, and transformation techniques. Comparison of growth conditions for primary PSI mutants Isolation of Synechocystis 6803 mutants deficient in PSI firnction was initially made possible by growing cells under light-activated-heterotrophic-growth (LAHG) conditions (Anderson et al., 1991). The PsaC mutants, including C51D, can grow under LAHG or mixotrophic conditions with dim light (5 umol m’2 8"; Chapter 3). The frequencies of transformation of C5 1D cells grown under these conditions was tested using plasmid pCG which contains a 3.5kb insert including a wild-type psaC gene (Chapter 3). As shown in table 5.1, mixotrophically grown cells were transformed with frequencies about three times that of LAHG cells. Although the reason for this differential is not known, the LAHG grown cells may have to go through an additional physiological acclimation to continuous light. It should be noted that the transformation frequency for both LAHG and mixotrophically grown PSI mutant cells varies considerably with different transforming DNA molecules, in agreement with a previous report with autotrophically grown wild—type cells (Williams, 1988). 99 Table 5.1. Comparison of transformation frequencies for LAHG and mixotrophically grown C51D cells. Grth conditions pCG amount (ng) Transformation frequency (10" cell) LAHG 2 6 Mixotrophic 2 20 LAHG 20 15 Mixotrophic 20 4O C5 1D cells (107 ) from LAHG culture (OD730 0.33) and mixotrophic culture (ODm 0.50) were harvested (OD73o = 0.25 corresponds to 1 x 10‘ cells ml") by centrifugation at 4500 g for 6 min at room temperature. The cell pellet was suspended in 20 111 BG-ll medium with 22 ng or 200 ng of plasmid pCG. The mixture of cells and DNA was incubated for 2 hr in a sterile test tube under 30°C, 12 umol m’2 s". Fresh medium was added to final volume of 500 111 then 50 ul diluted mixture (containing 10‘ cells, 2 ng or 20 ng pCG) was spread onto BG-ll agar plates supplemented with 1 ug ml‘l gentamycin. The plates were placed under 30°C, 12 umol m'2 s‘1 for 3 days before transferred to 22 umol m'2 s'1 . Colonies were counted 10 days after transformation. No colonies appeared in 10 days in a control experiment from which pCG DNA was omitted. 100 Comparison of methods for preparation of transforming DNA To study whether transformation is sensitive to impurities in transforming DNA, wild-type cells were transformed to kanamycin resistance using plasmid pKW1194 (Williams, 1988) prepared in four different ways: (1) direct use of a crude preparation made by ethanol precipitation of E. coli cell lysate (Wizard midiprep system, Promega); or further treatment of the crude preparation by the following means: (2) purification using Wizard miniprep system (Promega); (3) purification using QiaExH system (Qiagen); (4) purification by phenol-chloroform extraction. WT-GmR is a strain carrying a Gm resistance gene downstream of the psaC2 but otherwise wild-type (Chapter 3). Cells (3 ml) of this strain grown in BG-ll liquid medium supplemented with 1 pg ml" gentamycin were pelleted when OD730 was 0.41. The cells were resuspended in 3 ml of fresh BG-l 1. A 300 111 portion was transferred to a 15-ml Corning tube, and was mixed with 30 or 300 ng of plasmid. The mixture was then incubated for 20 hr at 30°C under 22 umol m'2 5'1 white light, and was agitated several times to keep the cells suspended. This mixture (100 til/plate) was then spread onto 9-cm BG-ll plates supplemented with 5 ug ml" kanamycin and was incubated as before. Colonies were counted nine days afier transformation. Transformation frequencies between 3 x 10'3 to 5 x 10'3 were observed for all four DNA preparations when 10 ng plasmid was mixed with 106 cells, and transformation frequencies between 5 x 10'3 to l x 10‘2 was observed for all four DNA preparations when 100 11g plasmid was mixed with 106 cells. No colonies appeared in a control experiment from which the plasmid was omitted. 101 Competency of the PSI mutant cultures at various stages of log phase Mixotrophically grown C51D cells in early log phase (ODm 0.15), mid log phase (OD-,3, 0.30), and late log phase (OD730 0.65) were plated onto BGll medium. Total DNA (10 ng in 5 111) isolated from the wild-type strain was dropped on cell lawns at marked areas, and the plates were put under 5 umol m’2 8'1 light for four days before transfer to 22 umol m'2 5'1 white light. Some clusters of colonies were observed at the areas on all the plates ten days following transformation. This shows that the PSI mutant cells were competent throughout the log phase. In addition, total DNA (10 ng in 5 111) isolated from the wild-type strain was dropped on plates containing the mid-log phase cells two days and five hours, respectively, before the plates were transferred to 22 umol m‘2 s'I white light. Transformation was observed in ten days, showing the PSI mutant cells stayed competent for at least four days under dim light on plates. Materials and Methods Strains and Growth Conditions Experiments were performed using a glucose-tolerant strain of Synechocystis sp. PCC 6803, which was acclimated for growth on solid medium in the dark. PSI mutants were maintained under LAHG conditions (Anderson, 1991). Cultures were grown in BG-ll medium prepared according to Williams (1988) except lab-purified Bacto-agar was used for LAHG (Anderson, 1991) Antibiotics were added in the following concentrations: kanamycin (Km), 5 mg/liter; gentamicin (Gm), 1 mg/liter. Photoautotrophic and rrrixotrophic growth were on solid media with or without supplemental glucose in a chamber providing continuous light. Cool white fluorescent 102 bulbs made by General Electric were used. The light intensity was varied by covering plates with layers of cheese cloth and was monitored using a L1-185A photometer (LICOR, Lincoln, NE). Fully grown plates may be wrapped with parafilm and stored in the dark at 4°C for up to six months. DNA Manipulations Polymerase Chain Reaction (PCR) was performed using a PTC 200 thermal cycler (MJ research Inc., Watertown, MA) and Taq polymerase (Boehringer Mannheim, Indianapolis, IN). Cells picked fi'om a medium size colony or an equivalent amount of cells collected from liquid culture were washed once with water and used as template. For amplification of long fragments (2 3 Kb), total DNA was used as template, and Pwo polymerase (Boehringer Mannheim) was used at 1/5 total polymerase activity in addition to Taq polymerase. Amplification products were checked in agarose gel and may be directly used for dot-transforrnation. For restriction analysis or sequencing, PCR products were purified using Wizard PCR purification kit (Promega Corporation, Madison, WI) or Ultrafiee-MC 30,000 NMWL filter unit (Millipore Corporation, Bedford, MA) which is recommended for long fragments. DNA sequencing was performed using dye-terminator chemistry for fluorescence sequencing at the Plant Biochemistry Facility, Michigan State University. The procedure for preparation of cyanobacterial DNA was adapted fi'om (Ohad, 1992), with two “loopfuls” of cells scraped from plates or cells from 10 ml liquid culture being used to extract DNA. RNA was removed from the preparation by incubation with RNase A, and DNA was recovered by ethanol precipitation. 103 Acknowledgment I thank Yong Wang for her contribution in characterization of R56lE-derived revertant S. l 04 References Anderson SL, McIntosh L: Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process. J Bacteriol 173: 2761- 2767 (1991). Boekema EJ, Boonstra AF, Dekker JP, and Rogner M (1994) Electron microscopic structural analysis of photosystem I, photosystem H, and the cytochrome b6/f complex from green plants and cyanobacteria. J Bioenerg Biomembr 26: 17-29 Chitnis PR, Xu Q, Chitnis VP, and Nechushtai R (1995) Function and organization of photosystem I polypeptides. Photosynth Res 44: 23 -40 Chitnis PR (1996) Photosystem 1. Plant Physiology 111: 661-669 Dzelzkalns VA, and Bogorad L (1988) Molecular analysis of a mutant defective in photosynthetic oxygen evolution and isolation of a complementing clone by a novel screening procedure. EMBO J 7: 333-338 Fromme P, Witt HT, Schubert W-D, Klukas O, Saenger W, and KrauB N (1996) Structure of photosystem I at 4.5 A resolution : a short review including evolutionary aspects. Biochim Biophys Acta 1275: 76-83 Ho K and Krogmann DW (1982) Photosynthesis. In The Biology of Cyanobacteria, Carr NG and Whitton BA, eds. Los Angeles: University of California Press, pp. 191-214 Jung Y-S, Yu L, Golbeck JH (1995) Reconstitution of iron-sulfirr center F,3 results in complete restoration of NADP+ photoreduction in Hg-treated photosystem I complexes from Synechococcus sp. PCC 6301. Photosynth Res 46: 249-255 Kaneko T, Sato S, Kotani H, Tanaka A, Asarrrizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouch T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo 8, Takeuchi C, Wada T, Watanabe A, Yamada M, Tasuda M, and Tabata S (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. Strain PCC 6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA research 3: 109- 136 Kless H, Vermaas WFJ, and Edelman M (1992) Photosystem 11 function and integrity in spite of drastic protein changes in a conserved region of the D2 protein. Biochemistry 31: 11065-11071 KrauB N, Hinrichs W, Witt I, Fromme P, Pritzkow W, Dauter Z, Betzel C, Wilson KS, Witt HT, and Saenger W (1993) Three-dimensional structure of system I of photosynthesis at 6 A resolution. Nature 361: 326-331 105 Ohad N, and Hirschberg J (1992) Mutations in the D1 subunit of photosystem II distinguish between quinone and herbicide binding sites. Plant Cell 4: 272-283 Rodday SM, Jun S-8, and Biggins J (1993) Interaction of the F A-FB binding subunit with the photosystem I core heterodimer. Photosynth Res 36: 1-9 Schubert WD, Klukas O, Krauss N, Saenger W, Fromme P, Witt HT (1995) Present state of the crystal structure analysis of photosystem I at 4.5 A resolution. In Mathis P, ed, Photosynthesis: From Light to Biosphere, Vol II. Kluwer Academic, Dordrecht, The Netherlands, pp 3-10 Schulz R, Smart LB, Yu J, and McIntosh L (1995) A new set of site-directed mutations in photosystem I core reaction center from Synechocystis sp. PCC 6803. In Mathis P, ed, Photosynthesis: from light to biosphere, vol II. pp. 119-122 Smart LB, Anderson SL, and McIntosh L (1991) Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp PCC 6803. EMBO J 10:3289-3296 Smart LB, Warren PV, Golbeck JH, and McIntosh L (1993) Mutational analysis of the structure and biogenesis of the photosystem I reaction center in the cyanobacterium Synechocystis sp PCC 6803. Proc Natl Acad Sci USA 90: 1132-1136 Williams J GK (1988) Construction of specific mutations in photosystem II photOsynthetic reaction center by genetic engineering methods in Synechocystis 6803. Meth Enzymol 167: 766-778 Zhao J, Li N, Warren PV, Golbeck JH, and Bryant DA (1992) Site-directed conversion of a cysteine to aspartate leads to the assembly of a [3Fe-48] cluster in PsaC of photosystem I. The photoreduction of F A is independent of F3. Biochemistry 31:5093-5099 Chapter 6 DISCUSSION AND POTENTIAL FUTURE EXPERIMENTS My thesis research has accomplished many of the goals established at its inception: identification of the gene for the PsaC subunit in PSI by gene inactivation, elucidating the function of PsaC in PSI biogenesis, and the site-directed mutagenesis of PsaC to study structure-firnction relationships. An unexpected finding was the isolation of pseudorevertants from the primary PSI mutants. The development of genetic techniques for characterizing PSI pseudorevertants has begun to show potential as a new approach in the study of P81. However, this new tool brings to the surface new questions that warrant future experimentation. The following is a summary on the study of PsaC mutants, and a brief discussion of some of the new questions and possible experimental approaches to address them. Summary on the study of the PsaC mutants Insertional inactivation of the psaC gene (Chapter 2) resulted in a photosynthesis- deficient strain. The mutant lacks the PsaC, PsaD and PsaE polypeptides in isolated thylakoid membranes, while the PsaA/PsaB and PsaF subunits were found. The EPR signals attributed to F A and F,3 were absent in the mutant strain, but a reversible FX signal was present. Addition of PsaC and PsaD proteins to the thylakoids from the PsaC minus 106 107 strain gave rise to resonances resembling that of wild-type PSI complex. In room- temperature optical spectroscopic analysis, addition of PsaC and PsaD to the thylakoids also restored a 30-ms kinetic transient which is characteristic of the P700*[FA/FB]' backreaction. These results demonstrate that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PSI complex, and that PsaC, PsaD and PsaE are not needed for assembly of the PsaA-PsaB dimer or for electron transport from P700 to Fx. PsaC provides ligands for two [4Fe-48] clusters, F A and F3, the terminal electron carriers in PS I. The cysteine ligands in positions 14 and 51 to F,3 and F A, respectively, were replaced with aspartate, serine, or alanine in order to probe the firnction of these two clusters in photosynthetic electron transport (Chapter 3). All mutant strains were unable to grow photoautotrophically and, compared to wild type, mixotrophic growth was inhibited under moderate light intensity. The mutant cell lines supported lower rates of whole-chain photosynthetic electron transport. Thylakoids isolated from the aspartate and serine mutants have lower levels of PsaC, PsaD, and PsaE and lower PS I capacity compared to the wild-type. The alanine and double aspartate mutants have no detectable levels of PsaC, PsaD, and PsaE. In addition, Asp and Ser substitutions decrease PSI quantum efficiency at the Fx -> F A/F B electron transfer step. The Ala and double Asp mutants failed to show any F A/F ,3 activity. These results indicate that the various mutations of the cysteine l4 and 51 ligands to F,3 and F A affect biogenesis and electron transfer differently depending on the type of substitution, and that the effects of mutations on biogenesis and firnction can be biochemically separated and analyzed. Further characterization (Chapter 4) of the mutant PSI complexes shows that a “mixed-ligand” (sulfirr ligand and oxygen ligand) [4Fe-48] cluster is present in the mutant 108 sites with Asp or Ser, and that it is capable of accepting electrons alter F x. The fourth ligand may be an oxygen from aspartate or serine residues, or alternatively, from water or CH . The PSI complexes from Ala mutants and the double Asp mutant show only the photoreduction of F x, consistent with the absence of PsaC. These results show that only those PsaC proteins containing two [4Fe-48] clusters are capable of assembling into PSI complexes. However, since neither F A or F B was specifically inactivated in the mutants, it could not be determined from these characterizations which of two clusters accepts electrons from Fx, or which one donates electrons to ferredoxin. Genetic characterization of pseudorevertants has been applied to the study of PSI structure and function in Synechocystis 6803 (Chapter 5). Photoautotrophic and mixotrophic growth under moderate light intensity were used to isolate and characterize pseudorevertants from the site-directed PsaC mutants. Three classes of pseudorevertants were found: those capable of grth under: (1) photoautotrophic conditions, but not mixotrophic conditions; (2) mixotrophic conditions, but not photoautotrophic conditions; (3) both conditions. Suppressor mutations in some pseudorevertants have been localized to the PsaA-PsaB dimer by firnctional complementation. In combination with a previous study on chemical inactivation of F,3 (Jung et al., 1995), the current data indicate that F A is the “proximal” cluster in the X-ray PSI crystal structural model, and that electron transfer proceeds serially from Fx to F A to F3. The mechanisms of suppressor mutations restoring PSI activity Since the primary PsaC mutations affected both complex assembly and quantum efficiency, suppressor mutations may be expected to restore either one or both, resulting 109 in partial or complete PSI activity. If a suppressor mutation stabilizes PSI assembly, its effects may be shown by PSI activity measurements and/or western blot analysis of thylakoid membranes isolated from the primary mutant and the pseudorevertant. It is expected that the levels of PsaC, PsaD, and PsaE subunits, and/or the PSI capacity in the pseudorevertant would be higher than that in the primary mutant. Ifa suppressor mutation restores quantum efficiency, its effects may be shown by kinetic studies using optical spectroscopy. If properties of the Fe-S clusters are changed by a suppressor mutation, the effects may be found by EPR spectroscopy and redox titration. Integration of the findings from physiological, biochemical and biophysical characterizations should help build a better structural model for PSI and firrther our understanding of its assembly and firnction. Light inhibition of PSI mutants Mutants of Synechocystis 6803 that lack PSI are sensitive to continuous white light greater than 3 umol m‘2 3'1 (Smart et al., 1993). The PsaC mutants with reduced PSI activity are less sensitive yet still cannot grow under white light greater than 22 umol m"2 s“. Clearly, PSI allows cells to grow mixotrophically under moderate light. How is this accomplished? A possible mechanism for the light inhibition is shown in figure 6.1. In cyanobacteria, part of the respiratory electron transport chain is shared with the photosynthetic electron transport chain (Kallas, 1994). Cytochrome oxidase has been shown to be the rate-limiting step in cyanobacterial respiratory electron transfer chain (Schmetterer, 1994). In PSI mutants, respiratory reactions may be feedback inhibited when the shared components are highly reduced by PSII activity under moderate light. 110 Figure 6.1. Over-reduction of the shared components inhibits growth. In cyanobacteria, part of the respiratory electron transport chain is shared with the photosynthetic electron transport chain. The shared components are shown in the dashed box. In PSI mutants, respiratory electron transfer may be inhibited when the shared components are highly reduced by PSII under white light. PBS, phycobilisome; COX, cytochrome oxidase. 111 The hypothesis that over-reduction of the electron transfer chain between P811 and P81 inhibits growth is supported by the following observations (Chapter 3): (1) C51D and C 148 were found to grow photoheterotrophically under 22 umol m’2 5'1 white light in the presence of 5 umol m‘2 s'1 DCMU, a P8 H inhibitor; (2) C51D and C148 were found to grow mixotrophically when 22 or 60 umol m'2 s‘1 of white light was filtered by a red plastic sheet; and (3) C51D grows photoautotrophically when 60 umol m’2 s’1 of white light was filtered by a red plastic sheet. Red light is primarily absorbed by PSI over PSII (F ujita et al., 1994), therefore it does not cause over-reduction of the shared components. In a study with isolated thylakoid membranes from spinach, it was found that DCMU could stimulate cyclic photophosphorylation as measured by ferredoxin-mediated 02 reduction, and the stimulation was eliminated by the addition of NADP+ (I-Iosler and Yocum, 1987). Since NADP+ should be present in the PsaC mutants, the presence of DCMU is not likely to affect cyclic phosphorylation. It has also been reported that light inhibition in a PSI-minus mutant is relieved when the assembly of phycobilisomes, which serve preferentially as antennae for PSH, is genetically inactivated (Shen, 1993). The available data indicate that PSI allows mixotrophic grth under moderate light by lowering redox poise of the shared components, thereby allowing respiratory reactions which are essential for growth. The available data also indicate that some suppressor mutations may reduce the activity of PSII, thereby restoring the balance between activities of P81 and P811 at a lower level, or they may alter the properties of certain key enzymes in respiration to relieve feedback inhibition. Identification of such suppressor mutations should broaden our knowledge of metabolism in this cyanobacterium. 112 Identification of suppressor mutations in the genome of Synechocystis 6803 Suppressor mutations have been localized to PSI subunits by genetic complementation using PCR fragments containing genes for these subunits (chapter 5). However, as discussed above, suppressor mutations could also occur outside of P81. This notion is supported by preliminary results from some of the pseudorevertants. For example, despite demonstration of the presence of a suppressor mutation in C148-18 (Fig 5.2), the mutation that restored photoautotrophy is not present in psaA, psaB, or psaC, as determined by DNA sequencing. Furthermore, transformation with total DNA and PCR fragments (containing genes for PsaA, PsaB, PsaC, PsaD, PsaE, and petF for ferredoxin) . from other C 148-derived pseudorevertants also imply there are suppressor mutations on other sites of the genome that eliminate light inhibition. Shotgun cloning strategies, such as the one shown in figure 6.2, will be developed to identify those mutations. An integration vector pJY 4 (figure 6.2) has been constructed in which a 3.3-Kb Synechocystis 6803 sequence cloned in pUC119 was interrupted by a KmR cassette. The plasmid transforms wild-type Synechocystis cells to kanamycin resistance at frequencies up to 10‘2 (Yu and McIntosh, unpublished). To clone a DNA fragment containing a suppressor mutation using this vector, total DNA from the pseudorevertant will be partially digested with Sau3A and ligated to pJY 4 at the unique BamHI site. Ligation products of appropriate sizes (9-15 Kb) will be purified from an agarose gel and directly used to transform primary mutant cells (cloning in E. coli is omitted to avoid potential problem in cloning genes for hydrophobic proteins). The mixture of DNA and cells will be spread onto plates containing Km, in the presence (for mixotrophic growth under moderate light) or absence (for photoautotrophic growth) of glucose. Colonies growing under the 113 Figure 6.2. A shotgun cloning vector, pJY 4. An integration vector pJY 4 is shown in which a 3.3-Kb Synechocystis 6803 sequence cloned on pUCl 19 was interrupted by a KmR cassette. See text for the detailed cloning strategy. Total DNA from a pseudorevertant will be partially digested with Sau3A and ligated to pJY 4 at the unique BamHI site. Ligation products will be used to transform the primary mutant cells. A and B, PCR primers near the BamHI site. The asterisk on the insert indicates a suppressor mutation. 114 selective conditions will serve as templates for PCR. The inserts ligated onto pJY 4 will be amplified using primers near the BamHI site (Figure 6.2), and will be tested for ability to transform primary mutants using methods described in chapter 5. Other cloning strategies may also be considered. Finally, the recent availability of the complete genome sequence of Synechocystis 6803 will undoubtedly simplify genetic studies using this model organism. The development of an efficient complementation-based cloning vector will be critical for the firll use of the sequence information. Combinatorial mutagenesis: Determination of properties and function of Fe-S clusters by protein environment A major question in protein biochemistry is how primary amino acid sequence leads to specific folding, assembly, stability and firnction. This question is particularly difficult to address with membrane proteins. For example, the three [4Fe-48] clusters in PSI have different thermodynamic and electronic properties allowing vectorial electron transfer from FX to F A/F B . Some of the ligands which bind these clusters have been identified by site-directed mutagenesis, and the regions surrounding these ligands are highly conserved. However, until recently (Jung et al., in press), little was known concerning how these sequences determined the properties of the iron-sulfur clusters and facilitated efficient electron transfer. The functional requirements in a short region of a protein can be studied by means of directed molecular evolution, in which a desired or novel function is selected from a pool of randomly arranged sequences of nucleic acids or amino acids. This approach, termed combinatorial mutagenesis, has been used in study of PSII (Kless and Vermaas, 1995), and could be used to further determine fimctional 115 requirements for the three F e-S clusters in PSI. For example, the sequences surrounding a F A ligand C51 (G50, C51, K52, R53) are conserved in all known PsaC proteins. Using site-directed mutagenesis, C51 has been changed to Asp, Ser, or Ala, and all the mutants lost photoautotrophy. Combinatorial mutagenesis may be applied by two-step PCR (Kless and Vermaas, 1995) to randomize these four amino acids, followed by transformation into a primary PsaC mutant such as C51D. Following positive selection for photoautotrophy, the combinations at these four sites that can support PSI activity will be identified. The contribution of each residue will then be evaluated in relation to assembly of the complex, properties of F A and efficiency of electron transfer. An appealing possibility for this approach is to generate a series of F A mutants with a range of mid-point potentials, which would define the relationship between this parameter and electron transfer efficiency, and further our understanding of electron transfer between Fe-S clusters. 116 References Fujita Y, Murakami A, Aizawa K, and Ohki K (1994) Short-terrn and long-term adaptation of the photosynthetic apparatus: homeostatic properties of thylakoids. In: The molecular biology of cyanobacteria. Bryant DA, ed. Kluwer Academic Publishers, pp.677-692 Hosler JP and Yocum CF (1987) Regulation of cyclic photophosphorylation during ferredoxin-mediated electron transport. Effect of DCMU and the NADPH/NADP+ ratio. Plant Physiol 83: 965-969 Jung Y-S, Yu L, and Golbeck JH (1995) Reconstitution of iron-sulfilr center FB results in complete restoration of NADP+ photoreduction in Hg-treated photosystem I complexes from Synechococcus sp. PCC 6301. Photosynth Res 46: 249-255 Kallas T (1994) The cytochrome bf complex. In: The molecular biology of cyanobacteria. Bryant DA, ed. Kluwer Academic Publishers, pp. 259-317 Kless H, and Vermaas WFJ (1995) Many combinations of amino acid sequences in a conserved region of the D1 protein satisfy photosystem II function. J Mol Biol 246: 120- 131 Schmetterer G (1994) Cyanobacterial respiration. In Bryant DA ed, The molecular biology of cyanobacteria. Kluwer Academic Publishers. pp. 409-435 Shen G, Boussiba S, and Vermaas WFJ (1993) Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome firnction. Plant Cell 5: 1853-1863. Smart LB, Warren PV, Golbeck JH, and McIntosh L (1993) Mutational analysis of the structure and biogenesis of the photosystem I reaction center in the cyanobacterium Synechocystis sp PCC 6803. Proc Natl Acad Sci USA 90: 1132-1136 nrcuran sran UNIV. LIBRARIES lllWimill”IWilliillHWill”lllllllllllllll 31293015795960