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L' HESIS

M |CHIGAN SirATEI UiIIWEIIRISlTIiII III flIFI‘lII-IIIIEIII II

3 1293 00908 5741

This is to certify that the

dissertation entitled

EPR AND ENDOR CHARACTERIZATION OF TYROSINE
AND PHENOL RADICALS IN FROZEN MATRICES

presented by

MOHAMED KAMAL EL-DEEB

has been accepted towards fulfillment
of the requirements for

Ph.D. Chemistry

degree in

 

 

Major professor

l

Date/1M /5 . /7?/
/

MSU is an Affirmative Action/Equal Opportunity Institution 0- 12771

LIBRARY

Michigan State
University

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution
c:\clrc\dmsdue.pm3-o.1

EPR AND ENDOR CHARACTERIZATION OF MODEL TYROSINE
AND PHENOL RADICALS IN FROZEN MATRICES

By

Mohamed Kama] EI-Deeb

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1991

ABSTRACT

EPR AND ENDOR CHARACTERIZATION OF MODEL TYROSINE
AND PHENOL RADICALS IN FROZEN MATRICES

By

Mohamed Kama] El-Deeb

In recent years, tyrosine radicals have emerged as important cofactors in the

redox chemistry taking place in several proteins. In oxygen-evolving photosynthesis, for
example, two redox active tyrosine residues, YZ and YD, occur in Photosystem II. The

tyrosine radical Y2. acts as an electron transfer intermediate between the water oxidation

site and the reaction center chlorophyll, a function vital to the highly efficient
photosynthetic process. In ribonucleotidediphosphate reductase, a radical has been
identified in the active site, and it was suggested that the radical initializes the enzymatic
reaction by hydrogen atom abstraction from the substrate. Tyrosine radicals have also
been found in prostaglandin synthase and galactose oxidase; in the latter case, the
tyrosine being linked to a cysteine residue via a thio-ether bridge. An interesting feature
of these naturally-occurring tyrosine radicals is the wide variation of their EPR spectral
line shapes, despite the similarity of their chemical identity. In order to explore the
factors that influence the properties of tyrosine radicals in proteins, we have used a
combination of EPR and ENDOR spectroscopic techniques, isotopic labeling, and
spectral simulations, to characterize photochemically-generated model tyrosine radicals
in disordered solid matrices. Our results show that the spin density distribution in
tyrosine radicals follows an odd-altemant pattern, with spin density at the oxygen and at

the ortho- and para- positions of the phenol ring. The occurence of a significant spin

density at the oxygen is indicated by the broadening of the tyrosine EPR spectrum upon
170-enrichment at the phenolic oxygen. Comparing the results of our model compound
study to the results on the tyrosine radicals in Photosystem II and in ribonucleotide-
diphosphate reductase, we conclude that the spin density distribution is essentially similar
in all species. The spectral properties of the various tyrosine radical species are discussed
in terms of the conformation of the B-methylene group with respect to the aromatic ring.
To model the cysteinyl-substituted tyrosine radical in galactose oxidase, we investigated
radicals derived from phenol analogues, with and without thioalkyl substitution. We
show that the sulfur substitution does not severely perturb the radical, and that the spin
density is confined mainly to the phenol ring. The odd-altemant spin distribution pattern
is retained in the sulfur-substituted compounds, despite a 25 % decrease in the spin
density at the para-position. Based on the B-methylene proton hyperfine coupling and the
para-position spin density, we are able to predict the conformation of the methylene
group with respect to the ring, and we present models for the tyrosine radicals in

Photosystem II and the cysteinyl-substituted tyrosine radical in galactose oxidase.

To the memory of my father, Kama! Mukhtar El-Deeb,

whose teachings have been behind every success in my life

iv

 

ACKNOWLEDGMENTS

I am deeply indebted to Prof. Babcock for his continuous guidance and
enthusiastic encouragement. Financial support through a research assistantship
throughout the course of this work is also greatly appreciated. I would also like to
thank Prof. Harrison, Prof. Popov, and Prof. Wagner for taking the time to serve on
my guidance committee, and for their constructive criticism. Several fruitful and
stimulating discussions with Chris Bender, Matt Espe, 0th Hoganson, Ivan
Rodriguez, and Peter Sandusky are greatly acknowledged. Chris’s expert advice on
ENDOR coil construction was invaluable. The numerous discussions I had with
Curt about ENDOR, EPR simulations, and POWFUN were really fun! My
appreciation is extended to all present and former members of Professor Babcock’s
group for their cooperation, friendship and good will. The completion of this work
was made possible by the excellent technical support of the staff of the electronics
shop, machine shop, and glass shop at the Chemistry Department, and of our genius

electronics designer, Martin Rabb.

My gratitude and appreciation for life goes to my Professors at Alexandria

University, who laid the foundation for the young scientist being born today.

My friends at the MSU Chapter of the Egyptian Student Association created
an atmosphere that I consider a home away from home. Keep up the good work,
folks. I wish to express my special gratitude to the president of the ESA, my good
friend and roommate, Gamal Khedr. Thanks for everything, Jimmy.

And last, but not least, my gratitude to my wife, J clan, and my children, Islam,
Israa, and Mustafa, for their unselfish love, encouragement and support, which
helped me through my long years in graduate school. THANK YOU for your love

and understanding, and to you I dedicate this work.

Thank GOD ....... I survived 2228 days at MS U !
v

 

TABLE OF CONTENTS

 

 

 

 

 

Page
LIST OF TABLES .......... - ....... _ ix
LIST OF FIGURES -- -- _ - - x
LIST OF ABBREVIATIONS ..................................... xvi
CHAPTER 1 GENERAL INTRODUCTION
1.1 Tyrosine Radicals In Biological Systems ............. 1
1.1.1 Tyrosine Radicals In The Photosynthetic
Oxygen Evolution Process ......................... 1
1.1.2 Tyrosine Radicals In Ribonucleotidediphosphate
Reductase - -- - - . ..... - - ..... 6
1.1.3 Other Biological Systems Containing Tyrosine
Radicals ................. . ......... 9
1.2 Tyrosine — Electronic Structure And Spin
Density Distribution - -- 11

 

1.2.1 Hilckel Molecular Orbital Treatment Of Benzene 11

 

1.2.2 Tyrosine And Simpler Phenolic Analogues 18
1.3 Photochemical Formation Of Tyrosine Radicals In Vitro 23
1.4 Rationale Of The Present Work ......................... 26
References ............................... 28
CHAPTER 2 THEORY OF EPR AND ENDOR SPECTROSCOPY

2.1 The Electron Paramagnetic Resonance (EPR) Experiment 33
2.2 Electron-Nuclear Hyperfine Interaction ............. 36

vi

 

CHAPTER 3

2.3

2.4
2.5

3.1
3.2

3.3

2.2.1 Dipolar Hyperfine Interaction .............
2.2.2 Isotrpic Hyperfine Interaction .............
Mechanism Of Isotropic Hyperfine Interaction In
w—Radicals ..............

 

2.3.1 Isotropic Hyperfine Coupling To a-Protons

 

— Spin Polarization .....
2.3.2 Isotropic Hyperfine Coupling To B—Protons

— Hyperconjugation .....................................
The Spin Hamiltonian - -- -
The Electron-Nuclear Double Resonance (ENDOR)

 

Experiment ..................................................

References ..................................................

EPR CHARACTERIZATION OF IMMOBILIZED
TYROSINE RADICALS: A MODEL FOR THE
TYROSINE RADICALS IN PHOTOSYSTEM H

Introduction .....................................................

Materials .....................................................
Tyrosine Radical Generation .............................
17O-Enriched Tyrosine Radicals .............................
EPR Measurements .........................................

Results .....................................................

vii

Page

36
39

39

4O

44
47

60
65

67
69
69
69
71
71
75

 

 

 

 

CHAPTER 4

CHAPTER 5

3.4

4.1
4.2

4.3
4.4

Specific Deuteration Effects on the EPR Spectra

of Tyrosine Radicals

 

The EPR Spectrum of 17O-Enriched Tyrosine Radicals:

Spin Density at the Phenolic Oxygen .................
Discussion .....................................................

References .....................................................

EPR AND ENDOR CHARACTERIZATION OF
FROZEN PHENOL AN ALOGUES: A MODEL FOR
THE DERIVATIZED TYROSINE RADICAL IN
GALACTOSE OXIDASE

Introduction .....................................................
Materials And Methods
Materials .....................................................

 

Phenol Radical Generation .........................................
EPR And ENDOR Measurements .............................
Results ........................................................
Discussion ........................................................

References ........................................................

CONCLUSIONS AND FUTURE WORK

viii

 

Page

75

82
89
110

115
116
116
117
124
125
140
147

151

Table 3.1:

Table 3.2:

Table 4.1:

Table 4.2:

LIST OF TABLES

 

 

 

Page
Isotropic hyperfine couplings and corresponding dihedral
angles for the methylene protons in different tyrosine
radical species. ....... -- - - 102
Parameters used to simulate the EPR spectra of the 3,5-
deuterated and the methylene-deuterated model tyrosine
radicals. .......... - - - - 108
Proton hyperfine coupling constants (G) in 2,6-R1-4-RH-phenol
radicals. ............................................................. 131

Parameters used in the simulation of the EPR spectra of 4-methyl

phenol and 2-methylmercapto-4-methyl phenol radicals. 139

 

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

LIST OF FIGURES

Electron transfer pathway in Photosystem H, with electron
transfer times according to references 6 and 11. Variations

in electron transfer rates depend on the S-state of the

 

manganese cluster. ............

The EPR spectrum of the Photosystem II tyrosine radical YD.

in the cyanobacterium Synechocystis 6803 (Reference 15).

The EPR spectrum of the tyrosine radical in RDPR from

the bacterium E. Coli (Reference 5). .................................

Hfickel molecular orbitals of benzene, where the numbers
shown indicate the orbital coefficients, and the dashed lines
represent nodal planes. S and A designate symmetric and

antisymmetric orbitals with respect to the plane of symmetry,

 

respectively (Reference 30).

Structures and antioxidation activities (kinh x 10'6 M'1 s'l)

of a-tocopherol (I) and related phenolic compounds

(References 43-48).

 

Molecular orbitals of the benzyl cation, radical, and anion (32).
The electronic structure of tyrosine resembles that of the

benzyl anion; the tyrosine radical resembles the benzyl radical.
x

 

Page

13

21

24

Page

Figure 2.1 Illustration of the electron-nuclear dipolar interaction (after
Bolton, J. , in Biological Applications of Electron Spin
Resonance; H. M. Schwartz et aL, eds.; 1972). ............ 37

Figure 2.2 Spin polarization of a C—H bond, leading to negative spin

density at the oz-proton (Reference 1). ........................ 41

Figure 2.3 Anisotropic hyperfine interaction with a-protons; the dotted

lines represent the region where 3 cos2 0 =-'- 1 (Reference 6). 43

Figure 2.4 Hyperfine interaction of B-methyl protons, arising from hyper-

conjugation with the aromatic ring (reference 1). ........... 45

Figure 2.5 Energy level diagram for a system of one electron interacting
with one proton (S = 1/2, I = 1/2) in a static magnetic field. v1

and v2 are the frequencies of the two allowed EPR transitions. 49

Figure 2.6 The variation of the direction of the nuclear spin quantization

(Heff) with respect to the applied field (H), depending on the
magnitude of the hyperfine field at the nucleus (HHF)

(Reference 1). ...... 52

Figure 2.7 Energy level diagram for a one electron - one proton system
where the hyperfine field is much smaller than the external

field, when the orbital containing the unpaired electron is

Figure 2.8

Figure 2.9

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Page

a) parallel to the external field; b) perpendicular to

the external field. ........................ - - -- - 54

 

Energy level diagram for a one electron - one proton system
where the hyperfine field is comparable to the external field
(Reference 1). ............................................................ 58

Energy levels (Hz) for S = 1/2 and I = 1/2 system, for
a) vN > a/2; b) vN < a/2 (Reference 13). - ..... - ......... 62

 

FAB-MS spectrum of 17O-enriched tyrosine. ...................... 70

X-band EPR spectra of photochemically-generated tyrosine
radicals in a) borate buffer, pH 10.0; b) saturated KOH;

c,d) 12 M H2804. Temperature: 120 K; modulation amplitude:
0.5 G; microwave power: 0.5 mW; scan width: 100 G (a-c),

600 G ((1). Spectrum e is an expansion of the feature at the

low field wing of spectrum (1 (see text). ........................ 72
Tyrosine numbering convention used throughout this work. 76

X-band EPR spectra of photochemically-generated model

tyrosine radicals, specifically deuterated as indicated, in borate

buffer (pH 10.0). Temperature: 120 K; modulation amplitude:

0.5 G; microwave power: 0.5 mW; scan width: 100 G. ....... 77

xii

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

X-band EPR spectra of specifically deuterated YD' radicals

in Photosystem II (7). ...........

 

The broadening of the EPR spectrum of l7O-enriched tyrosine
radical ( — ) relative to the unlabeled compound ( - - - - ).
Temperature: 120 K; modulation amplitude: 5.0 G; microwave

power: 0.5 mW; scan width: 100 G.- ......

 

X—band EPR spectra of a) the fully protonated YD. tyrosine

radical in Photosystem II (7), b) the model tyrosine radical,
and c) the tyrosine radical in RDPR (10). .......................

X-band EPR spectra of the methylene-deuterated tyrosine
radicals: a) the Photosystem II YD. radical (7); b) the model

tyrosine radical; and c) the tyrosine radical in RDPR (10).

X-band EPR spectra of the Photosystem H YD. tyrosine

radical (a), the model tyrosine radical (b), and the tyrosine
radical in RDPR (c), all deuterated at the ring 3,5 positions.

A model for the methylene group conformation in the model

tyrosine radical and the Photosystem II YD. radical. a) side
view; b) end view, looking along the C3—C1 bond. ............

xiii

Page

80

85

91

96

99

103

Figure 3.11

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Page

Computer simulations of the EPR spectra of a) 3,5-deuterated,

and b) methylene-deuterated model tyrosine radicals, with the

 

parameters listed in Table 3.2. -- - 106

X-band EPR spectra of the radical derived from 2-methyl-
mercapto-4-methyl phenol by UV illumination in a) chloroform;
b) lmM KOH; c) 0.1 M KOH; d) 0.1 M KOH + 4 M KCL;

e) 0.1 M KOH + 12 M LiCl; and f) 10 M KOH.Temperature:

120 K; modulation amplitude: 0.5 G; microwave power: 0.5 mW. 118

300 MHz NMR spectra of 2-methy1 mercapto-4-methyl
phenol in CDC13 (a) and in 0.1 M NaOD/DZO (b). ............ 121

X-band EPR spectra of the radicals derived from phenol (a)
and 2-methylmercapto phenol (b). Temperature: 120 K;

modulation amplitude: 0.5 G; microwave power: 0.5 mW. 127

Experimental and simulated EPR spectra of 4-methyl phenol
(a,b) and 2-methylmercapto-4-methyl phenol (c,d). Experimental
conditions are the same as in Figure 4.3. The simulation

parameters are listed in Table 4.2. .................................... 129

EN DOR spectra of the radicals of 4-methyl phenol (a) and 2-
methylmercapto-4-methyl phenol (b), showing the 4-methyl

xiv

 

Figure 4.6

Figure 4.7

Page

proton axial features. Temperature: 120 K; microwave power:

10 mW; RF power: 40 W; RF modulation: 150 kHz. .............. 134

X-band EPR spectrum of the radical derived from 2,6-di-t.butyl-
4-methyl phenol. Temperature: 120 K; modulation amplitude:

0.5 G; microwave power: 0.5 mW. ...................................... 137

A model for the o-cysteinyl-substituted tyrosine radical in
apogalactose oxidase; a) side view; b) end view, looking along

the C5-C1 bond. The dihedral angle for the more strongly

coupled proton, 01, is 34°. ..... - .. 145

 

ENDOR
EPR

GO

HMO
HOMO
LCAO-MO
LUMO
PGHS

PS 11
RDPR

YD

Yz

LIST OF ABBREVIATIONS

Electron-nuclear double resonance

Electron paramagnetic resonance

Galactose oxidase

Htlckel molecular orbital

Highest occupied molecular orbital

Linear combination of atomic orbitals - molecular orbital
Lowest unoccupied molecular orbital

Prostaglandin H synthase

Photosystem II of the oxygen-evolving photosynthetic plants

Ribonucleotidediphosphate reductase

The redox-active tyrosine D in Photosystem II

The redox-active tyrosine Z in Photosystem II

CHAPTER 1

GENERAL INTRODUCTION

1.1 TYROSINE RADICALS IN BIOLOGICAL SYSTEMS

Tyrosine (2-amino-3-(4-hydroxyphenyl)propanoic acid) is one of twenty amino
acids that are of general occurrence in all proteins (1). During the last two decades,
tyrosine radicals have been found to play a vital role as intermediates in the redox
chemistry taking place in several biologically relevant systems (2-4). Two of these
enzymes that contain tyrosine radicals, namely ribonucleotidediphosphate reductase
(RDPR) and Photosystem 11 (PS II), are of particular interest and have been the subject
of extensive research efforts over the last several years (5-7). In prostaglandin H synthase
(PGHS), it has been shown that a tyrosine radical turns over during the catalytic cycle,
indicating its actual involvement in the enzymatic catalysis (4b). In another enzyme,
galactose oxidase (GO), it has been shown that an aromatic free radical, derived from a
tyrosine residue, occurs in the active site (83), and it was suggested that the substrate is

likely to bind to the radical site prior to the enzymatic reaction (8b).
1.1.1 Tyrosine Radicals In The Photosynthetic Oxygen Evolution Process

In cyanobacteria, algae and higher plants, two membrane-bound, multi-subunit
proteins, designated Photosystem I (PS I) and Photosystem 11 (PS H), act in sequence to
oxidize water and reduce carbon dioxide. Photosystem II is the protein in which the
primary photochemistry and the water oxidation takes place (6,7,9,10). Oxygen is

evolved as a by-product of water oxidation. In Figure 1.1, a schematic illustration of the
electron transfer pathway in PS 11 is presented. P680, a chlorophyll molecule, is the

 

 

 

 

QA ’ QB
Fe
300—600ps
Pheo
A
3ps
50-250115 30#s-1ms Mn
P 4 Y2 < ( )4

680

hv

Figure 1.1: Electron transfer pathway in Photosystem II, with electron transfer times
according to references 6 and 11. Variations in electron transfer rates depend

on the S-state of the manganese cluster.

 

 

3
reaction center of Photosystem 11. Upon absorption of a photon, the excited state of P680

is generated, and the oxidized reaction center, P680+ , is formed as a result of electron
transfer from P680‘. The electron is transferred through an intermediate pheophytin
molecule (Pheo) to a protein-bound plastoquinone molecule (QA), then to a second
plastoquinone molecule (Q3). The series of electron transfer steps from the primary

donor (P680) to the quinone site can be represented as follows:

hv
\ + '
P680 Pheo Q A QB , P680 Pheo Q A QB

 

 

 

\ + - ‘ + '-
. P680 Pheo Q A QB . P680 Pheo QAQB

Before another photochemical event can take place, P 680+ must be reduced to P680.
This is achieved as a tyrosine molecule, YZ’ donates an electron to P 680+ , thereby
forming P680 and the tyrosine radical, YZ'. In turn, Yz' is reduced by a tetranuclear
manganese cluster, where the water oxidation takes place (6). Upon absorption of the
next photon by P 680, a second electron is transferred to QB', and the doubly-reduced
quinone (QBZ') then becomes protonated and released from the membrane as a

hydroquinone (HZQ). The cycle is repeated as another quinone molecule binds at the QB

site (10).

As the photon absorption and the electron transfer process go on, four oxidizing
equivalents are accumulated on the manganese cluster, which advances to a higher Sn
state (where n = 0 - 4 represents the number of oxidizing equivalents accumulated on the
manganese cluster) for each photochemical event, and finally water oxidation and

oxygen evolution occurs, as described by the so-called S-state model (12):

 

 

4e-

 

02 + 411* 2H20

Thus, the tyrosine radical YZ.’ acting as an electron transfer mediator between the

water oxidation site and the PS II reaction center, serves two important functions. First, it

couples the one-electron photochemical process taking place at the reaction center to the
four-electron oxidation of water. Secondly, the rate of electron transfer from YZ to
P 680+ is three orders of magnitude faster than that of P 680+Q A’ charge recombination.

Thus, YZ provides an efficient means of maintaining the primary charge separation and
achieving a near—unity quantum yield (6).

A second tyrosine radical, designated YD°, occurs in Photosystem II. The two
tyrosine radicals Y2. and YD. have identical EPR spectra, though they differ in their
kinetic and functional pr0perties. YZ' is directly involved in the electron transfers
between the water oxidation site and the reaction center and its EPR signal is observed
only transrently. The EPR spectrum of Y2 has been desrgnated Signal Hvery fast , Since it
decays on the microsecond time scale in the dark in oxygen-evolving PS 11 particles (13).
YD', on the other hand, can not be directly involved in the electron transfer sequence in
PS 11, as indicated by the slow kinetics of its formation and disappearance. The EPR
signal of YD°, referred to as signal IIslow (Figure 1.2) is stable for hours in the dark at

room temperature (6). It has been proposed that YD° is formed by electron donation from

the reduced tyrosine residue YD to the manganese center in the 82 and 83 states (10). The

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slow decay of YD° is thought to be the result of electron transfer from the manganese S0

state, leading to the formation of the dark-stable S1 state of the manganese. This
oxidation of S0 to S1 by YD. was suggested to stabilize the manganese cluster (14).The

fact that the EPR signals of Y2. and Y]; are identical clearly indicates the identical

chemical nature of the two species (15). It also has other implications related to the

protein environment of the two radicals, which will be discussed below.
1.1.2 Tyrosine Radicals In Ribonucleotidediphosphate Reductase

Ribonucleotidediphosphate reductase (RDPR) is an enzyme that is essential to all
living cells. It catalyzes the reduction of ribonucleotides to their corresponding
deoxyribonucleotides, thereby providing the precursors for DNA synthesis (16). The
enzyme is found in bacterial and mammalian cells, and is also coded for by some
bacteriophages and viruses (17). In 1972, an EPR signal (Figure 1.3) was observed from
the bacterial enzyme (2), which could not be explained on the basis of the known
existence of iron in the sample. Further EPR studies, involving deuterium substitution,

traced the origin of the EPR signal to a tyrosine free radical (18,19). The enzyme is
composed of two non-identical protein subunits, B1 and 32' Each subunit is

enzymatically inactive by itself; the active enzyme is a 1:1 complex of B1 and B2 (17).
The active site of the enzyme, which is believed to occur at the interface of the two
subunits, contains redox active dithiols localized in protein B1, and a tyrosine radical
adjacent to a binuclear iron center in protein B2 (20). Recently, the X-ray crystal

structure of B2 revealed the existence of two binuclear iron centers per B2 subunit (20b).

The two iron centers are 25 A apart, and within each iron center two ferric ions, 3.3 A
apart, are connected by a carboxyl bridge and a u-oxo bridge. The tyrosine radical is 5.3
A away from the nearest iron. The crystal structure also showed that the tyrosine radical

lies along the axis connecting the two antiferromagnetically coupled ferric ions, and that

 

 

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8
leads to significant magnetic interaction between the radical and the iron center (20b,21).
Thus, the spin lattice relaxation rate of the tyrosine radical is enhanced and the EPR line
width increases (5,21). For that reason, ENDOR experiments on the RDPR tyrosine
radical had to be performed at cryogenic temperatures, and no ENDOR was observed
above 110 K (5).

Both magnetic resonance and X-ray crystallography results agree on several major
features of the tyrosine radical of RDPR. First, the phenolic oxygen of the radical is
deprotonated, and is not hydrogen-bonded to any of the surrounding amino acid residues.
The X-ray structure showed that the nearest atom to the tyrosine phenolic oxygen is I
3.3 A away, and that the radical is embedded in the protein (20b). ENDOR experiments
showed no difference in the spectra upon H2O/D20 exchange, indicating that the tyrosine
radical is inaccessible to the solvent (5). Secondly, the conformation of the tyrosine side
chain as determined from the ENDOR data, which places one of the methylene protons

nearly in the plane of the ring, is in excellent agreement with the X-ray structure.

The earlier mechanism that has been proposed for the enzymatic action of RDPR is
one in which the first step is the abstraction, by the tyrosine radical, of a hydrogen atom
from the ribonucleotide (the substrate). This is followed by elimination of OH', yielding
a substrate cation radical, which subsequently undergoes reduction by the thiol groups on
protein B1 to give the product, the deoxyribonucleotide. The tyrosine radical is
regenerated as the parent tyrosine molecule donates one electron during this last step
(22). This mechanism, however, was challenged by the X-ray structure. The authors of
(20b) argue that the tyrosine radical can not participate in direct hydrogen abstraction
from the substrate, since it is embedded 10 A deep into the protein. Instead, they favor a

long-range electron transfer process, probably involving the iron center.

9
1.1.3. Other Biological Systems Containing Tyrosine radicals

Prostaglandin H synthase (PGHS) catalyzes the first step in the biosynthesis of
prostaglandins, prostacyclins, and thromboxanes (23,24). The catalytic action of the
enzyme proceeds via two distinct reactions, denoted as the cyclooxygenase reaction and

the peroxidase reaction. The cyclooxygenase reaction oxidizes arachidonic acid to
prostaglandin 62; the peroxidase reaction reduces hydroperoxides to their corresponding

alcohols, thus forming prostaglandin H2.

The cyclooxygenase reaction is believed to follow a branched chain mechanism,
initiated by hydroperoxide (24). An EPR-detectable free radical species is generated
during the cyclooxygenase cycle, and has been assigned to a tyrosine radical (25).
Subsequent investigations have shown that the tyrosine radical does, in fact, participate
in the cyclooxygenase reaction, and a mechanism that provided a good explanation of the
observed data on PGHS was proposed (4b,c). In this mechanism, the peroxidase
generates an iron (IV) porphyrin cation radical (compound I), which oxidizes a tyrosine
residue to form iron (IV) porphyrin and a tyrosine radical. The tyrosine radical then
abstracts a hydrogen atom from arachidonic acid. In light of this mechanism, PGHS is
analogous to other peroxidase compound I species, namely cytochrome c peroxidase and

horseradish peroxidase.

Galactose oxidase (GO) is a mononuclear copper enzyme excreted by fungi, which
catalyzes the two-electron oxidation of primary alcohols to the corresponding aldehydes,

coupled to the reduction of oxygen to hydrogen peroxide (26,27):

RCHZOH + 02 —> RCHO + H202

Three distinct oxidation states of the enzyme have been identified (26a). The fully

reduced and the fully oxidized forms are EPR-silent. The intermediate state, however,

10
shows a typical Cu (II) EPR signal. The fact that the active, fully oxidimd form of
galactose oxidase contains Cu (II), as demonstrated by X-ray absorption spectroscopy
(26b) indicates that the copper must be spin-coupled to another S = 1/2 species, giving
rise to an EPR-silent state (26a). Using Raman spectroscopy, it was shown that a tyrosine
residue is involved in the active site of galactose oxidase (27). Furthermore, oxidation of
the copper-depleted enzyme gave rise to a g a- 2.005 EPR signal, characteristic of an ‘
aromatic free radical, and, based on isotopic labeling experiments, Whittaker and
Whittaker concluded that the radical is derived from a tyrosine residue (8a). Recently, the
X-ray crystal structure of galactose oxidase has been published (8b), and revealed that
two tyrosine residues, tyr272 and tyr495, are ligated to the copper in the enzyme active
site. Of these, tyrosine 272 is covalently bound to the side chain sulfur of a cysteine
residue, cys228, at the position ortho to the tyrosine phenolic oxygen, thus forming a

thioether linkage between tyr272 and cySm. The assignment of the radical site to either

of the tyrosine residues, however, was yet to be made. This is the subject of Chapter 4 of

the present work, along with an ENDOR investigation of the apoenzyme.

The presence of a stable tyrosine radical in the copper-free galactose oxidase does
not necessarily imply the involvement of the radical in the catalytic activity of the native
enzyme. Although it has been proposed that the substrate interacts directly with the
radical, thus resulting in two-electron oxidation at a mononuclear copper center (8b,26a),
the possibility still exists that the removal of the metal ion may significantly perturb the
structure of the enzyme active site, resulting in radical delocalization in the copper-free

protein (8a).

l l
1.2 TYROSINE— ELECTRONIC STRUCTURE AND SPIN DENSITY

DISTRIBUTION.
1.2.1 Hiickel Molecular Orbital Treatment of Benzene

In order to understand the role that tyrosine radicals play in protein catalysis, it is
essential to understand the electronic structure at the phenolic head group. Regarding
tyrosine as a substituted phenol (or a disubstituted benzene), it is helpful to start by
reviewing the electronic structure of benzene. The three sp2 hybrid orbitals on each of the

benzene carbon atoms are used in a—bonding to a hydrogen atom and two adjacent carbon
atoms. This leaves each carbon atom with one valence electron in its 2pz orbital, and the

w-molecular orbitals are formed as a linear combination of these atomic orbitals :
‘I’i = X cir ¢r
l'

where cir is the linear combination coefficient of atomic orbital ¢r into the ith molecular

orbital. Within the Htickel MO approximation, the orbital coefficients for the benzene

MO's are obtained by solving the secular equation :

E [(Hrs'Eiar-s)cir] = 0 r=l,2,...,6
1'
where firs is the Kronecker delta; (SIS = 1 for r = 3, hrs = 0 for r a! 3. Values of Ei’ the
orbital energies, are the roots of the secular determinant :
| H - E a | = 0
IS IS

where Hrr E a is the Coulomb integral of the rth atom and Hrs a B is the resonance

integral of the Cr—CS bond; Hrs = 0 if Cr and CS are not bonded.

12
The resulting MO's for the benzene molecule are represented in Figure 1.4, with
the orbital energies, orbital coefficients, and symmetry shown for each MO. The six

w-electrons of benzene are assigned to the three bonding orbitals according to the Pauli
principle. In a given MO (\Ifi), the electron density at a certain atom r is the square of the

orbital coefficient (cit)?

The ground state configuration of benzene has two pairs of doubly-degenerate
MO's (see Figure 1.4). When the benzene anion is formed, the unpaired electron is
equally likely to occupy either of the two low-lying antibonding orbitals, resulting in an
average unpaired electron density of 1/6 on each carbon. Experimental data support this
prediction, since the EPR spectrum of the benzene anion indeed shows the expected
equally-spaced seven lines. Any substitution on the benzene ring, however, is expected to
lift the degeneracy of the antibonding orbitals, and the added unpaired electron would
occupy the MO that becomes lower in energy. Which of the two orbitals becomes lower
in energy is determined by the nature of the substituent, whether electron-repelling or
electron-withdrawing, and by the magnitude of the wave function at the position of the

substitution (29).

The electronic structure of benzene discussed so far represents an example of even-
altemant hydrocarbons, the electronic structure of which is remarkably well predicted by
the Hiickel approximation (30,31). Alternant refers to the fact that all the carbon atoms in
the compound can be divided into "starred" and "unstarred" atoms, such that no two
starred (or two unstarred) atoms are bonded together. In even-altemant hydrocarbons,
equivalent structures are obtained by interchanging the starred and the unstarred atoms,
while in odd-alternant systems the two types of positions are not equivalent (32). Thus,
benzene, naphthalene and anthracene are even-altemant hydrocarbons. For hydrocarbon

systems with no heteroatoms, only reactive intermediates (radicals, cations or anions) can

 

13

Figure 1.4: Htlckel molecular orbitals of benzene, where the numbers shown
indicate the orbital coefficients and the dashed lines represent nodal
planes. S and A designate symmetric and antisymmetric orbitals with

respect to the plane of symmetry, respectively (Reference 30).

11712

11712
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15
exist as odd-altemant species (32), e. g., allyl and benzyl radicals. Compounds containing

odd-membered rings can not be classified as altemant systems.

One of the characteristic properties of altemant systems is that, with the exception

of non-bonding molecular orbitals (see below), molecular orbitals always occur in pairs
(31). For every bonding orbital \Iri with energy a + n6 (n > 0), there is an antibonding

orbital ‘1’; with energy a - nB. For each pair of bonding and antibonding MO's the orbital

coefficients are equal at one set of atoms and opposite at the other set of atoms. The
pairing theorem predicts that the unpaired electron density (and hence the EPR hyperfine
splittings) should be equal in both the cation and the anion of a given even-altemant
hydrocarbon molecule — the unpaired electron resides in the highest bonding MO in the
cation and in its "twin" lowest antibonding MO in the anion. Remarkably, observed
hyperfine splittings from several altemant hydrocarbon ions are in close agreement with
those predictions. For example, the observed proton hyperfine splittings for the
anthracene cation are a1 = 3.11, a2 = 1.40, and a9 = 6.65 G. For the anthracene anion, the
hyperfine splittings are a1 = 2.74, a2 = 1.57, and a9 = 5.56 G (30). Although the

hyperfine splittings tend to be slightly higher in the cation, the observed ratios of a92alza2

are very close to 4:2:1, which are those predicted by the simple Hfickel MO calculations.

Odd-altemant hydrocarbons, with an odd number of carbon atoms, have an
additional non-bonding molecular orbital of energy a. In odd-altemant hydrocarbon
radicals, e.g. the allyl and the benzyl radicals, the unpaired electron resides in that non-
bonding molecular orbital. Odd-altemant radicals show some peculiarity when one tries
to interpret their EPR spectra based on simple MO calculations. For the non-bonding MO
in benzyl, simple Htickel MO treatment predicts nodes at the meta-positions, whereas
experimentally, a significant hyperfine splitting (1.75 G) is observed from the meta-

protons (33,34). In the allyl radical, the proton on the central carbon gives rise to a

l6
hyperfine splitting of 4.06 G (35), while half of the unpaired electron density is expected
at each of the terminal carbons; the non-bonding MO is predicted to have a node at the
central carbon. These and other observations on odd-altemant radicals were explained by
taking electron correlation effects into account. That is, the Htickel MO treatment ignores

the interaction of the unpaired electron with the paired electrons in filled MO's.

By assigning different molecular orbitals for electrons with different spins (36,37),
the Hiickel MO theory has been extended to take into account the interaction between the
non-bonding unpaired electron and the bonding electrons in the w-system. In the allyl

radical, for example, the Htickel MO's are (30):

‘11: 1/2(¢1+\/2¢2+¢3) E=a+\/26
r2=1/\/2(¢1-¢3) E=a
\1'3=l/Z(¢1-x/2¢2+¢3) E20: -\/26

which predicts zero unpaired electron density on C2, as indicated by the coefficients of
‘12. If we assign spin a to the unpaired electron in \Irz, and since electrons in different
spatial orbitals will have lower energy if their spins are parallel (38), it follows that the
Coulomb and exchange forces will tend to attract the bonding \Itl electron of spin or

towards the terminal carbons, while the \II1 electron with spin [3 will be forced toward the

center. The result is an excess B-spin (i.e. negative spin density) at the central carbon,
and positive spin densities in excess of 0.5 at each of the terminal carbons. This result

can be obtained in a quantitative manner as follows: by admixing different amounts of
\It3 into \1'1, one can construct two new spatial orbitals for the electrons in \Irl :

‘Ifla = 11+e‘II3

e>0

wlb

‘I/l-e\1'3

17

Substituting the Htickel orbitals into ‘1!“ and \I' we get :

lb’

Wla

l/2(1+e)¢1+ll\/2(l-e)¢2+1/2(1+e)¢3

‘I’lb

l/2(1-e)¢1+1M2(l+e)¢2+1/2(1-8)¢3

\Irla will have a lower energy than ‘1'“), since the electron is more delocalized in

the former. Therefore, \Irla will contain the electron with a spin, while In) will contain

the electron with B-spin. The spin density at each carbon can be estimated by adding the
one-electron density of all orbitals occupied by a-spin electrons (namely \II2 and In)

and subtracting the density of the orbital that has a B—spin elecu'on ( \Irlb) :

p1 p3 = 1/4(1+2£+82)+1/2-1/4(1-2£+82)= l/2+e

p2 1/2(1-28+82)-l/2(l+28+£2)= -2e

Thus, one can distinguish the two terms spin density distribution and unpaired
electron distribution (39). Spin densities, unlike unpaired electron densities, haVe both
magnitude and sign (30). Unpaired electron density, found by applying simple Httckel
MO calculations, predicts zero hyperfine splitting from protons at the unstarred positions
of odd-altemant radicals (e. g. the —CH— proton of the allyl radical and the meta-protons
of the benzyl radical). By taking into account the exchange interaction between the odd
electron and other electrons in the r-shell, one can show that the closed shell w—electrons
are forced to occupy slightly different regions in space (39). The resulting spin density,
now correctly defined, is positive (and slightly higher than HMO predictions) at the
starred atoms, while negative spin density appears at the unstarred atoms, where nodes

are predicted by HMO.

18

1.2.2 Tyrosine And Simpler Phenolic analogues

It has been widely accepted that the —-OH group in phenol is co-planar with the
aromatic ring (40). In this planar structure, the compound is stabilized by conjugation of
the lone pair in the oxygen p,r orbital to the aromatic w-lattice. As a result of the
increased r-electron delocalization, the UV spectrum of phenol is 1739 cm‘1 red-shifted
and 14 times more intense relative to that of benzene (41). That is, phenol is another
example of an odd-altemant system, where the substitution of —OH onto the benzene
ring introduces an additional (non-bonding) molecular orbital with two additional
electrons that interact with the ring w-system. When phenol is oxidized, an electron is
lost from the HOMO, and thus one would expect the unpaired electron in phenoxyl
radical to reside in a non-bonding molecular orbital. Clearly, the conjugation of the
phenol oxygen lone pair to the ring introduces a partial double bond character to the
C—0 bond. The amount of the C—0 double bond character depends on the orientation of
the oxygen p,r orbital relative to the pz orbital on the ring carbon. When the two orbitals
are parallel, the conjugation of the oxygen lone pair is maximized and the compound is

most stable, i.e., the energy of the HOMO is lowest (see below).

Fueno, Ree, and Eyring (42), in a LCAO-MO study of numerous phenolic
compounds, treated phenol as a perturbation of its parent hydrocarbon anion — namely
the benzyl anion, which has the same number of r-electrons and the same arrangement of
atomic p7r orbitals — by replacement of the side chain of the benzyl anion by an —OH
group. By a first order perturbation treatment, they expressed the energy of the HOMO
of phenol, which is a non-bonding molecular orbital analogous to that of the benzyl

anion, as:

_ 2
EHOMO ‘ °‘ + (Clix) kx B

 

19
where a and B are the Coulomb integral of a carbon atom and the resonance integral of a

C—C bond, respectively, Cnx is the coefficient of the oxygen p1r orbital in the non-bonding
molecular orbital, and RK is a Coulombic parameter to account for the electronegativity
difference between oxygen and carbon. Thus, since 6 is a negative quantity and kx is a

positive one, the HOMO becomes more stable as the value of (Cm)2 increases. The most

important conclusion that was drawn out of this study is that there is a linear correlation

between oxidation potentials of phenols and the H OM O energies— the more stable the
HOMO (i.e., the higher the value of (Cm)2 ), the higher the oxidation potential of the

compound. Thus, since Cnx represents the degree of conjugation of the phenol oxygen

lone pair to the ring w-system, it follows that the redox potential of the compound is
strongly determined by the orientation of the O—H bond relative to the ring, as the latter
determines the degree of conjugation of the oxygen lone pair. The authors also
investigated the relationship between the efficiency of phenolic compounds as
autoxidation inhibitors and the energy of the HOMO. The inhibition by phenols of
peroxide-initiated autoxidation of organic compounds involves hydrogen atom abstraction

from the phenol to form a phenoxyl radical (43):

kinh

1200' + ArOH > ROOH + Aro'

 

ROO' + ArO' > non-radical products

 

Although the authors of (42) proposed a mechanism for the phenol inhibition of
autoxidation that is contrary to the current belief (see below), they made the valid point
that the efficiency of the phenolic compounds as inhibitors for autoxidation correlates
with the HOMO energy — that is, the higher the HOMO energy of the phenol, the lower

the oxidation potential and the more efficient the compound as autoxidation inhibitor.

20

In a series of articles (43-48), Ingold and co-workers investigated the antioxidation
activity of a-tocopherol (Vitamin E) and related phenols, which inhibit the peroxidation
of lipids in vivo. Stoichiometry of the autoxidation reaction and the kinetic isotope effect
observed upon deuterium substitution of the phenolic hydrogen indicated that the
inhibition proceeds via the above stated mechanism, with the phenoxyl radical formation
being the rate determining step. In addition, their investigation revealed some very
interesting facts: First, the presence of ortho-alkyl substituents sterically protects the
phenoxyl oxygen, resulting in retardation of chain transfer via ArO' ( where ArO'
initiates another chain reaction : ArO' + ROOH ———9 ArOH + ROO' , thus
reducing the antioxidation effectiveness of the phenol ). Secondly, on the basis of
stereo-electronic factors, they explained the wide variation of the phenol antioxidation
activity, in terms of kinh’ and how the substituents on the phenol ring control the
antioxidation activity by varying the degree of delocalization of the unpaired electron in

the phenoxyl radical.

In Figure 1.5, the structures of some of the phenolic compounds studied by Ingold
et al., along with the corresponding values of kinh, are shown. Inspection of the values in
Figure 1.5 shows comparable antioxidation activities of compounds (I) and (H). This

indicates that the length of the side chain ( R in I and II ), has little effect on the
inhibition activity. Compound (III), however, has a value of kinh that is an order of

magnitude lower. The large drop in kinh on going from the heterocyclic arrangement in

(I) and (II) to the related para-methoxy phenol structure (HI) has been attributed to
stereo-electronic effects: the fused ring structure holds the p-type lone pair on the ring
oxygen parallel ( or at a small angle ) to the aromatic ring p1r orbitals, providing for
conjugation of the oxygen orbital with the w-system, thus stabilizing the phenoxyl radicals.
In (III), the on‘ho-methyl substituents force the methoxy group to be twisted out of the

plane of the aromatic ring; the oxygen p-orbital will lie in the plane of the ring,

21

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22

t

and the conjugation of the lone pair to the r—system will be prohibited. X-ray diffraction
data supported this interpretation: the Ar—O—C dihedral angle is about 16° in (H) and
about 90° in (III), i.e. the methoxy group is perpendicular to the ring. In the absence of
alkyl substituents ortho to the methoxy group (IV), the Ar—O—C dihedral angle is 8°,
i.e. the methoxy group is nearly in the plane of the ring. Even better stabilization of the
phenoxyl radical is achieved in the dihydrobenzofuran derivative (V), since 5-membered
rings are more planar than 6-membered ring. In this case, the furan oxygen p-orbital is

perpendicular to the aromatic ring ( dihedral angle Ar—O—C = 0° ), and kinh is enhanced

relative to those of compounds (I) and (H).

The pertinence of the foregoing discussion of phenol electronic structure and redox
potential to the present work becomes clear as we move in our discussion closer to tyrosine
and its simpler para-substituted phenol analogue, para-cresol. Recent cyclovoltametric
studies (49) reported redox potentials of 0.86 V (phenol) and 0.77 V (para-cresol) in
aqueous solution at 25 °C. Judging by the above mentioned relationship between electronic
structure and redox potential, the electronic structure of para-cresol is not expected to be
severely perturbed relative to that of phenol. This assumption is substantiated by the
closeness of the O—H bond dissociation energy of phenol (88.3 k cal/mol) and of para-
cresol (86.5 k cal/mol) (50). Tyrosine, in turn, is a derivative of para-cresol. Horwitz,
Strikland and Billups (51) reported that tyrosine has the same near UV absorption spectrum
as does para-cresol, and they used para-cresol as a model for the phenolic ring of tyrosine
to assign the observed bands to vibronic transitions, assuming a sz local effective symmetry
in both cases. The difference of the redox potential of the tyr/tyr' couple (0.94 V in aqueous
solution at 25° C) from those of phenol and para-cresol has been attributed to different
Hammett coefficients of the para-substituents (52). However, based on spectroscopic
observations (51), the MO structure for tyrosine is expected to be similar to para-cresol and

phenol, i.e. tyrosine can be treated as an odd-altemant system.

23

In summary, tyrosine is an odd-altemant system, the electronic structure of which
resembles that of benzyl anion and phenol. The HOMO in tyrosine is a non-bonding
molecular orbital containing two electrons. When tyrosine radical (R—C6H4—O' ) is
formed, the resulting MO configuration resembles that of the benzyl radical, with the
unpaired electron residing in a non-bonding MO (see Figure 1.6). The extent of
conjugation of the oxygen lone pair in tyrosine (or the unpaired electron in tyrosine
radical) to the ring arr-skeleton will be determined by the orientation of the oxygen p1r
orbital relative to the ring; maximum conjugation occurs when the oxygen orbital is
perpendicular to the ring. Clearly, variation in the orientation of the oxygen p1r orbital
relative to the ring will be reflected as variations in the spin density distribution in the
aromatic ring of the tyrosine radical. Thus, one would expect that any factors that may
change the orientation of the oxygen p-orbital, e.g. hydrogen bonding, will affect the spin
density distribution in the ring. Also, as the degree of hyperconjugation of the tyrosine
methylene protons is determined by the conformation of the side chain relative to the ring,
this will have an effect on the overall delocalization of the unpaired electron, and the spin
density distribution is expected to vary accordingly. The protein environment in the
vicinity of the tyrosine residue determines the orientation of the ring relative to the side
chain, through steric and electrostatic effects, for example. This suggests, then, that the
structure of the tyrosine radical may be controlled in nature to suit its particular function

by building specific constraints into the local environment of the side chain.
1.3 PHOTOCHEMICAL FORMATION OF TYROSINE RADICALS IN VITRO

Radicals of tyrosine and simpler phenolic analogues have been prepared by several
methods, including chemical oxidation, flash photolysis, and photolysis of rigid solutions

(53). When phenol analogues are photolyzed in frozen solutions, phenoxyl radicals
(Ph—O' ) and solvated electrons are formed (53-57). The ground state pKa of the

24

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25
phenolic —OH group in tyrosine is 10.1. However, when phenolic compounds are
photooxidized in neutral solution, the phenol radical cation formed (Pb—OH“) has a
pKal value of approximately -2.0 (58). Land et al. (53) generated phenoxyl radicals by
flash photolysis in aqueous solutions, and did not'observe any shift of the 400 nm
absorption band, characteristic of phenoxyl radicals at acid concentrations up to 12 M
sulfuric acid. That indicated that the phenoxyl radicals are not protonated even at such
high acid concentrations. At pH values higher than 10, the phenoxyl radical (PhO') is
formed directly by electron ejection from the phenolate anion (PhO') (54). Land et al.
(53) proposed that below pH 10, phenoxyl radicals are generated by homolysis of the
O—H bond, with a hydrogen atom being the by-product. They rejected a mechanism in
which a cation radical is formed and rapidly deprotonates, since neutral phenoxyl radicals
are observed in frozen paraffins, where proton release would be unlikely. Feitelson and
Hayon (56) performed a flash photolysis study of tyrosine and related phenolic
compounds in aqueous solutions at different pH values. They concluded that the same
radical (PhO' ) is formed upon flash photolysis, both in neutral and alkaline solutions of
phenols.From measurement of the absorption spectra and decay kinetics, they reported
that for tyrosine at pH 7, O—H bond dissociation accounts for only 23 % of the radicals
formed, whereas the predominant mechanism is electron ejection followed by rapid

deprotonation of the cation radical, in contrast with the earlier proposal by Land et a1.

Summarizing, neutral radicals (R—C6H4—O') are formed upon photolysis of

tyrosine and phenols; the predominant pathway for radical generation being electron
ejection at high pH, while H' atom dissociation and photoionization-deprotonation occur

at neutral pH.

26
1.4 RATIONALE OF THE PRESENT WORK

The foregoing introduction has demonstrated the importance of tyrosine radicals as
intermediates in protein redox chemistry. Some of the tyrosine radical-containing
enzymes are of great interest, and a good understanding of their function could have
potentially important applications, e.g., solar energy conversion utilizing artificial

systems that mimic plant photosynthesis.

The function of tyrosine radicals in protein electron transfer reactions depends on
several factors, the most important of which is, no doubt, the electronic structure of the
radical. Other important factors in this regard include the geometrical structure of the

radical itself and the effect of the local protein environment on this structure.

We have chosen the model compound approach to characterize, under well-
controlled conditions, the structure of tyrosine radicals, and to explore the interactions
that are likely to occur in the more complex in vivo situation. In their protein
environment, tyrosine radicals are immobilized on the magnetic resonance time scale. By
generating the tyrosine radicals in frozen solutions, we have an appropriate model for the
naturally-occurring immobilized radicals. This model compound study complements and
supports an intensive research effort involving several members of our group, as well as

other laboratories, who are studying tyrosine radicals in vivo.

EPR spectroscopy provides a useful means by which to study organic free radicals
and to extract information about the hyperfine interactions, and, in turn, the spin density
distribution in the radicals can be assessed. Furthermore, hyperfine interactions involving
the tyrosine side chain protons can lead to the elucidation of the side chain conformation
with respect to the aromatic ring, a factor that might be relevant to the function of the

radicals in the physiological process. On the other hand, ENDOR spectroscopy, with its

27
inherent higher resolution, has proven very powerful in studying immobilized free
radical species, where EPR line broadening may obscure some useful information. By
using ENDOR spectroscopy, the anisotropic proton hyperfine tensors can be determined.
Assignment of EPR spectral features is aided by the availability of specifically deuterated
tyrosine from commercial sources. The spectra of these isotopically labeled compounds
confirm the assignment of the fully protonated tyrosine spectra and make it possible to
isolate and evaluate the contribution of each set of the tyrosine protons to the hyperfine
coupling. This is further complemented by the computer simulation of the EPR spectra
with the hyperfine tensors obtained from the ENDOR spectra. Since the hyperfine
coupling is directly related to the spin density distribution in the radical, an estimate of
the spin density at various positions of the tyrosine radical can be derived from the
experimentally-measured hyperfine couplings. For the side chain protons, hyperconjuga-
tion is the dominant mechanism contributing to the hyperfine coupling to these protons.
Thus, from the magnitude of the hyperfine coupling to these protons, and since the
degree of hyperconjugation depends on the conformation of the side chain with respect to
the aromatic ring, an insight as to the side chain geometry can be gained. Taken together,
the combination of techniques used in the present study should lead to a better
understanding of the electronic and the geometrical structure of tyrosine radicals in
proteins, contributing to the ultimate goal of establishing a structural-functional

relationship for some of the physiologically important enzymes.

 

28
REFERENCES

l. Finar, I.L. Organic Chemistry, vol. 2; Longman: London; p. 638 (1975).
2. Ehrenberg, A. and Reichard, P. (1972); J. Biol. Chem. 247, 3485.
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8. a) Whittaker, M. M. and Whittaker, J. W. (1990); J. Biol. Chem 265, 9610; b) Ito, N.;
Phillips, S. E. V.; Stevens, C.; Ogel, Z. B.; McPherson, M. J.; Keen, J. N.; Yadev, K.
D. S.; and Knowles, P. F. (1991) Nature 350, 87.

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21. Sahlin, M.; Petersson, L.; Grdslund, A.; Ehrenberg, A.; SjOberg, B.- M.; and
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22. Sjbberg, B.- M.; Graslund, A.; and Eckstein, F. A. (1983); J. Biol. Chem. 258, 8060.
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27. Whittaker, M. M.; DeVito, V. L.; Asher, S. A.; and Whittaker, J. W. (1989); J. Biol.
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28. Babcock, G.T.; El-Deeb, M. K.; Sandusky, P. 0.; Whittaker, M. M., and Whittaker,
J. W.; J. Am. Chem. Soc., submitted.

29. Carrington, A (1963); Quart. Rev. 17, 67.

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32. Smith, W. B. Molecular Orbital Methods in Organic Chemistry; Marcel] Dekker:
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Wertz, J. E. and Bolton, J. R. Electron Spin Resonance: Elementary Theory and
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Radom, L.; Hehre, W. J.; Pople, J. A.; Clarson, G. L.; and Fateley, W. G. (1972); J.
Chem. Soc. Chem. Commun., 308.

Robertson, W. W.; Seriff, A. J.; and Matsen, F. A. (1950); J. Am. Chem. Soc. 72,

1539, and references cited therein.
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Doba, T.; Burton, G. W.; Ingold, K. U.; and Matsuo, M. (1983); J. Chem. Soc. Chem.
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32
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33
CHAPTER 2

THEORY OF EPR AND ENDOR SPECTROSCOPY

2.1 THE ELECTRON PARAMAGNETIC RESONANCE (EPR) EXPERINIENT

The application of electron paramagnetic resonance (EPR) spectroscopy is limited
to paramagnetic systems; i.e. systems with non-zero electron spin angular momentum.
Systems that fall into this category include free radicals, crystals with point defects (e.g.
F-centers), biradicals, triplet state systems, and transition metal ions. One important
application of this technique is the study of biological systems. Metalloenzymes and free
radical intermediates occurring in electron transfer reactions in proteins have been the

subject of a massive body of EPR literature in recent years.
The magnetic moment of the electron is related to the electron spin angular
momentum by:
ac: - 'yeSzh/va- (geeh/4mc)Sz=-geBe Sz
In the above expression, Fe is the 2 component of the electron spin magnetic

moment, gc is a dimensionless quantity referred to as the spectroscopic splitting factor

(gc = 2.0023 for a free electron); 3,. = eh/41rmc is the electronic Bohr magneton; and 76

is the magnetogyric ratio of the electron. The two spin states of the electron, | a ) and

I B ), are eigenfunctions of the operator for the 2 component of the electron spin; that is:

Szla)=+1/2|a)

Sle)='1/2|B)

34
i.e., M5 = 1- 1/2 are the eigenvalues of Sz (in units of h/21r). Thus, we can write:

I‘e='geBeMS

The two spin levels of the electron ( Ms 2’ + 1/2 and Ms = - 1/2 ) are degenerate

in the absence of an external magnetic field. If a magnetic field H is applied along the z-

axis, the energy associated with each spin level is:

w=-uC°H=gCBCMsH

Thus, we have the two Zeeman energies:
Wa=+1l2cheH

W6=-1/2geBcH

which are separated by gOBCI-I . In an EPR experiment, transition between the two

Zeeman levels is induced by the absorption of a photon of energy:

hv=AW=geBeH

which occurs in the microwave region for laboratory fields in the range 3 - 20 kG, which
are typically used. In practice, the paramagnetic sample is placed in a resonant cavity
situated between the poles of an electromagnet, and microwave radiation of fixed

frequency is supplied to the cavity by a klystron. The magnetic field is then swept
through the resonant field H,, and the EPR transition occurs when the resonance

condition, h v = g6 66 H,, is met. The resonance condition implies the selection rule that

MS changes by one unit; i.e., A MS = 1- 1, corresponding to the upward and the

downward transitions between the Zeeman levels.

35
In quantum mechanical terms, the Hamiltonian for the electronic Zeeman

interaction is:

95E:-11¢.H=-yc(h/2w)S.H;geBeS.H=chcHSz
and therefore the two spin states of the electron are also eigenfunctions of if}; :
%E | a)=chcHSz | a)=+1/2chcH

italB>=geaeHszIB>=-1/2g.B.H

i.e., the energy difference between the two electronic Spin states is ge Be H, as concluded

before.

The interaction of the magnetic nuclei in the system with the external magnetic
field leads to the splitting of the nuclear spin levels; the so-called nuclear Zeeman
splitting, which is the basis for NMR. Analogous to the splitting of the electron spin

levels, the Hamiltonian for the nuclear Zeeman interaction is:

%N=-uN.H=-mth/zolfl=-gN6NI.H=-gNBNHIz

The result of the nuclear Zeeman interaction is that each of the electronic spin levels
(defined by MS) is split into sublevels defined by M1, where M1 = I, (I-l), ..... , (-I+1), -I

is the nuclear spin angular momentum. For a nucleus with spin I = 1/2, M1 = t 1/2 and

the splitting of the two nuclear Zeeman levels is gNBNH, where gN and 3N are the

nuclear g factor and the nuclear magneton, respectively. The nuclear Zeeman interaction

contributes to the total spin Hamiltonian, which will be discussed below.

36
2.2 ELECTRON-NUCLEAR HYPERFINE INTERACTION

In addition to the interaction of the unpaired electron and the nuclei with the
external magnetic field, the electron interacts with the magnetic nuclei ( nuclei with

I 2 1/2 ) in its vicinity. The effect of the so-termed hyperfine interaction is that the
resonant field, i.e., the field experienced by the electron, will be the vectorial sum of the
external field and the local field generated by the surrounding nuclei. Electron-nuclear
hyperfine interactions fall into one of two categories: i) isotropic, or Fermi contact
interaction, and ii) anisotropic, dipolar interaction between the electron and the

surrounding nuclei.
2.2.1 Dipolar Hyperfine interaction

The classical expression for the electron dipole-nuclear dipole interaction energy is:
wdip = Hlocal ”e

zueuN (1-3c0320)/r3

In this expression, it is assumed that both dipoles are oriented along the external
magnetic field (see Figure 2.1). Hlocal is the local magnetic field at the electron,

generated by the nuclear dipole; r is the distance between the two dipoles; and 0 is the
angle between the external magnetic field and the vector connecting the electron and the
nucleus. Clearly, the dipolar interaction will depend strongly on 0, i.e., it is anisotropic.
Since the electron is not localized in space, the average energy is calculated by averaging
cos2 0 over all possible positions of the electron. For a hydrogen atom, for example, the
electron is in a spherically symmetric 1s orbital. Averaging cos2 0 over a sphere yields a
value of 1/3, i.e., the diplolar energy vanishes. This is consistent with the expectation that

in a hydrogen atom, where the 1s orbital is spherically symmetric, all orientations of the

37

 

 

 

 

 

Figure 2.1: Illustration of the electron-nuclear dipolar interaction (after Bolton, J .R.
in Biological Applications of Electron Spin Resonance; Schwartz, H. M.
et al., eds. ; 1972).

 

38
vector r (see Figure 2.1) with respect to the external field are equally probable, whereas
the dipolar interaction energy must be anisotropic, according to the above expression.
Thus, dipolar interactions average to zero for free radicals in solutions of low viscosity,

where molecular tumbling is sufficiently rapid, rendering the system purely isotropic.
A more general expression for the dipolar interaction energy is given by (1):
Wdip=(|.tc.ttN)/r3 - 3(uc.r)(ttN.r)/r5
If we substitute into this expression the dipole moment operators:
116 = - ge 3e 8
PN = 8N 3N I
then we can express the Hamiltonian for the dipolar interaction as follows:

llD=-gecegNaN[(s.I)/rB-3(s.r)(r.r)/r5]

Expanding the vectors into their components, we get:

((r2 ~3x2)/r5) -(3xy/r5) -(3xzjr5) Ix
%D=1s.,sy,s.1 - (-ge6egr~16N)° -<3xy/r5> «.2- 3y2>lr5> -<3y2/r5> °Iy

-(3xflr5) -(3yflr5) ((r2- 322)/r5) 12
or %b=hS.II

where the angular brackets indicate an average over the unpaired electron distribution.
Thus, it is clear that the dipolar coupling tensor T will depend on the properties of the
orbital in which the unpaired electron resides. It is also important to note that the dipolar

tensor is traceless — i.e., the sum of the diagonal elements is zero. Therefore, for

isotropic systems, the dipolar interaction vanishes, as noted above.

 

39
2.2.2 Isotropic Hyperfine Interaction

For an unpaired electron in a spherically symmetric s-orbital, the isotropic
hyperfine interaction is what gives rise to the observed hyperfine splittings. The energy

of isotropic hyperfine interaction was given by Fermi (2) as:

Wiso=-(81r/3) a, ”N | \Ir (0) |2

where ‘1! (0) is the electronic wave function evaluated at the nucleus; i.e., I ‘1' (0) I 2 is

the probability of finding the electron at the nucleus. Accordingly, isotropic hyperfine
interaction exists only when there is a finite electron density at the nucleus, hence the
term "contact interaction". This is a property unique to s orbitals, since p, d, f, . . .
orbitals all have nodes at the nucleus and thus will exibit only anisotropic hyperfine

interaction.

Substituting the dipole moment operators into the expression for isotropic

interaction energy yields the Hamiltonian (3):
iiiSO=(81r/3)gcccgN5N | r (0) |28.1

=hAOS.I

where A0 = 81r/3h ge Be gN 6N I ‘II (0) I 2 is the isotropic hyperfine coupling constant

( in Hz ).
2.3 MECHANISM OF HYPERFINE INTERACTION IN n-RADICALS

In conjugated systems, e. g. benzenoid and semiquinone radicals, the unpaired

electron resides in molecular orbitals that are usually modelled by linear combination of
atomic pz orbitals. Such molecular orbitals typically have nodes in the molecular plane,

therefore one would expect no spin density at the carbon nuclei, and hence no isotr0pic

 

40
hyperfine interaction with aromatic protons. Nevertheless, isotropic hyperfine
interactions with aromatic protons do occur. Two distinct mechanisms provide for
isotropic interaction in such systems, depending on the type of proton considered. In EPR
terminology, an a-proton is a proton bound directly to an atom carrying unpaired spin
density. A B-proton is two bonds away from the atom with unpaired spin density.
Examples of a- and B-protons are the ring protons and the side chain protons in the
benzyl radical, respectively. The mechanism of isotropic hyperfine interaction to each

class of protons is considered below.

2.3.1 Isotropic Hyperfine Coupling to or-Protons— Spin Polarization

In Chapter 1, it has been shown that electron correlation effects render some of the
"paired" electrons in a conjugated system slightly unpaired, as a result of the interaction
with the unpaired electron. We have also defined the spin density at a certain region of
the molecule as the difference between the probability densities of electrons with a- and
B-spins. Now, consider a C—H fragment of an aromatic radical. In this case, the
hydrogen atom is bound to one of the three sp2 orbitals on the carbon. The two
possibilities for assigning electron spins in the C—H o—bond, shown in Figure 2.2, would
be equally probable (i.e., the spin density at the proton would be zero) if there was no
interaction with the electron in the carbon pZ orbital. This is not the case, however, since
the configuration where the two electrons on the carbon atom have parallel spins (Figure
2.2a) is more stable (Hund's rule), and hence more probable. Thus, if we assign spin a to
the carbon pz electron, and since configuration a is more probable, we will have more
centers with B-spin at the hydrogen and a-spin at the carbon sp2 orbital. That is to say,
there will be a net negative spin density at the proton, and a positive spin density at the
carbon nucleus. For this reason, the isotropic hyperfine coupling to a-protons in

conjugated systems is negative. This mechanism of isotropic hyperfine interaction to

 

41

2P:
0
0
l a l l 19 a

 

(a) (bl

Figure 2.2: Spin polarization of a C—H bond,leading to negative spin density at the

a-proton (Reference 1).

 

 

42
a-protons is known as spin polarization; that is, the C—H o-bond is polarized by the
unpaired electron in the carbon 2p, orbital. Clearly, the magnitude of the a-proton
hyperfine splitting will be proportional to the spin density on the a-carbon. This

proportionality is expressed by the well-known McConnell relationship (4):
A0“ = Q p"

where A0“ is the a-proton hyperfine splitting, p1r is the spin density in the p, orbital of

the a-carbon, and Q is a proportionality "constant". Estimates for the value of Q for
aromatic free radicals range from -22.5 to -27 .4 G (3). Although Q is not a universal
constant, it is generally assumed that radicals with similar structure have similar Q
values, and thus the McConnell relationship provides a useful means of interpreting
isotropic a-proton hyperfine couplings in rr-radicals. For the tyrosine radical in RDPR,
an estimate of Q = -24.9 G has been obtained by Bender et al., from a rigorous
calculation of the spin density distribution and the isotropic a-proton hyperfine couplings
measured by ENDOR (5). This value should be similar for tyrosine radicals in other
systems, and will be used in our treatment of the model tyrosine radicals considered in

the present work.

In addition to the isotropic hyperfine interaction, a-protons in rr-radicals exhibit an
anisotropic dipolar coupling component, the magnitude of which is typically 50 % of the
isotropic coupling. As indicated above (Section 2.2.1), the dipolar coupling tensor is
traceless; typical principal values in the x-, y-, and z-directions are -0.5 A550, 0.5 Aim,
and 0, respectively (1). In Figure 2.3, the effect of the field orientation on the value of
(l - 3 cos2 0) is shown (6). When the magnetic field is parallel to the C—H bond (Figure
2.3a), the spin density is mostly in the region where (1 - 3 cos2 0) is negative, leading to
a positive dipolar hyperfine interaction. With the field perpendicular to the C—H bond in
the plane of the radical (Figure 2.3b), (1 - 3 cos2 0) is positive and the dipolar coupling is

 

43

\ /’

\~\\® C : ’//

\ e I.

(O) \\lh’\’£I-3coszO-O
[I \\ h

\\\
\\ l/’
b \H/
\
t 1 ,, \
I, ‘
I
I

 

 

Figure 2.3: Anisotropic hyperfine interaction with a-protons; the dotted lines represent

the region where 3 cos2 0 = 1 (Reference 6).

 

44

negative. In Figure 2.3c, the magnetic field is perpendicular to the plane of the radical,
(1 - 3 0032 0) is approxirnately zero, and so is the third principal value of the dipolar

tensor. Thus, taking into account both the isotropic and the dipolar couplings, typical
ratios of the principal values of the total a-proton hyperfine tensor in w-radicals are

1.5 Aim : 0.5 Aim : A350 in the x-, y-, and z-directions, respectively, where the y-axis is

along the C—H bond and the z—axis is perpendicular to the molecular plane.
2.3.2 Isotropic Hyperfine Coupling to B-Protons— Hyperconjugation

In aromatic rr-radicals, B-methyl protons are coupled to the spin density in the
a-carbon 2p, orbital via a hyperconjugation mechanism. Since rr-orbitals are
antisymmetric with respect to the molecular plane, the atomic orbitals of the three

B—hydrogen atoms must form a molecular orbital with the same symmetry as the

rr-orbital. This is achieved by a linear combination of the hydrogen ls orbitals ( ‘l’i ):
‘1’=Cr¢1'02(¢2+¢3)

to form the pseudo w-orbital \II, depicted in Figure 2.4, which becomes part of the
rr—system. Thus, the spin density at the methyl protons is positive, and therefore the

B-proton hyperfine coupling is positive, unlike that of a-protons (6).

For a freely rotating methyl group, a hyperfine splitting of 70 MHz is observed, but
a wide variation of B-proton hyperfine couplings, ranging from 14 MHz to > 130 MHz,
is not uncommon (6). This variation has been explained on the basis of the dependence of
the B-proton hyperfine coupling on the conformation of the B-protons relative to the

T-Ol‘bltal on the oz-carbon, described by the Heller-McConnell expression (7a):

A0l5‘=(B0+Bzc0320)p7r

45

 

Figure 2.4: Hyperfine interaction of B—methyl protons arising from hyperconjugation

with the aromatic ring (Reference 1).

 

 

 

46
where A0? is the B-proton isotropic hyperfine coupling constant, B0 and 32 are constants,

p1r is the spin density at the a-carbon, and 0 is the dihedral angle between the plane
containing the a-carbon pz orbital and the Ca—CB bond, and the Ca_CB"HB plane. B0 is

much smaller than B2, and is usually neglected (7b). That leaves us with the simpler

expression:

A03 = B2 p cos2 0

where B2 is assumed constant for radicals with similar electronic structure. For tyrosine

radicals, a value of B2 = 162 MHz has recently been reported (5).

The dependence of the isotropic coupling on cos2 0 implies a dependence on the

degree of conjugation between the B—hydrogen ls orbital(s) and the w-system, which
provides a partial double bond character to the Ca—CB bond (8). Maximum conjugation,

and hence maximum hyperfine coupling, is achieved when 0 = 0°, i.e. when the Ca—HB

bond is parallel to the pz orbital on Ca. When 0 = 90°, the B-proton is in the nodal plane

of the w-system, and no hyperfine coupling occurs.

Thus, unlike a freely rotating methyl group, where all the methyl protons are
equivalent, restricted rotation about the Cor—CB bond renders the B-protons inequivalent,
and different hyperfine couplings are observed for different protons. Several instances of
this situation have been encountered in free radicals in single crystals, in disordered
solids, and in proteins (9). Anisotropic dipolar interactions of B-protons are typically
510 % of the isotropic interaction— i.e., hyperfine coupling to B-protons is almost
isotropic. Thus, from a measurement of the isotropic hyperfine coupling constant, an
insight as to the conformation of the B-protons can be gained, given the magnitude of the

Spin density at the oz-carbon.

 

47
2.4 THE SPIN HAMILTONIAN

For the simplest case of one electron interacting with one proton, the spin

Hamiltonian can be written as:

%=%E+%N+%iso
= 13654-11 - gNBNI.H+hAOS.I
= BCS.g.H - gNBNI.H+hA0(SxIx+Sny+SzIz)

where the first two terms represent the electron- and the nuclear Zeeman interactions,
respectively; and the last term represents the Fermi contact interaction. Dipolar

interactions will be dealt with later in this section.

Generally, g is a tensorial quantity; significant deviation from the free electron
g-value (gc = 2.0023), as in the case of transition metal ions, results from spin-orbit
coupling, which depends on the directional properties of the orbital containing the
unpaired electron with respect to the applied magnetic field (10). For isotropic systems,
this coupling averages out to zero, and the g factor can be treated as a scalar quantity.
This is generally a good approximation for organic radicals, which exhibit very small

g-anisotropy (6). In such cases, the electronic Zeeman term can be simply written as
g6 66 S . H.

For the moment, we will ignore the nuclear Zeeman term, since it does not affect
the energies of the EPR transitions (it does, however, shift the absolute energies of the
spin states). Furthermore, when the external field is applied along the z axis, then if we
assume that the hyperfine interaction is much smaller than the Zeeman splitting, we can
ignore lex + Sy Iy . The last approximation simply implies that the quantization of the
electron spin angular momentum along 2 is not perturbed by the nuclear hyperfine

interaction, which is a very good approximation unless the hyperfine interaction is

 

48
exceptionally large (see below). Thus, applying the simplified spin Hamiltonian :

%=geBeHSz+hAOSzIz

to the four spin functions ¢1 = I ac aN), 492 = I ac 3N), 4>3 = I Be (IN), and

¢4 = I Be 5N), we obtain the following energies of the four spin levels:

81=(acaNIchcHSz+hAOSzIZIacaN)=1/2chcH+l/4hA0
£2=(aeBNIcheHSz-I-hAOSzIZIacaN):1/2geBeH-1/4hA0

e3=(6caNIchCHSz+hAOSzIZIacaN)=-ll2chcH-1/4hA0
e4=(BcBNIchcHSz-+hAOSzIzIacozN)=-l/2geBcH+1/4hA0

Figure 2.5 shows the energy level diagram for a system of one unpaired electron
interacting with one proton, and the two allowed EPR transitions which satisfy the
selection rules A M5 = i- 1 and A M1 = 0. The frequencies of the two transitions are given
by:

v1=1/h(81-£3)=1/hchcH+AO/2
”2:1/h(82'€4)=1/h8chH‘A0’2

i.e., the two transitions will be centered about the resonance frequency, ge Be H/h, and

split by A0, the isotropic hyperfine coupling constant expressed in Hz. Practically, the

microwave frequency is kept constant and the magnetic field is varied. The two EPR
lines are then separated by a = hAolgeBe, where a is the hyperfine coupling constant in

Gauss.

Next, we proceed to the more general case where the dipolar hyperfine interaction

and the nuclear Zeeman interaction are incorporated in the spin Hamiltonian, while

retaining the assumption that ge is a scalar. The dipolar hyperfine interaction term

(section 2.2. 1) and the contact interaction term (section 2.2.2) can be combined such that:

49

 

 

 

 

 

 

 

/ / fi ‘
a , /
c ,
/ / / \ h
a 9 B / /
C 6 /
‘ \ \ v1 v2
\ \ P
\ / ’
\ \ fie / /
‘ Y
H = 0 Electron Isotropic
Zeeman hyperfine

Figure 2.5: Energy level diagram for a system of one electron interacting with one proton

(S = 1/2, I = 1/2) in a static magnetic field H. v1 and v2 are the frequencies of

the two allowed EPR transitions.

50
%HF = xiso + %D
where yin].— represents the total hyperfine interaction energy. Thus, Wm: can be expressed

as:

llHF=hs.A.I

where A is the total hyperfine coupling tensor, which combines both the isotropic and the
dipolar components. By proper choice of the axis system, the dipolar tensor T can be

diagonalized such that:

S.T.I=TxxSxIx+Tnyny+TzzSzIz

Therefore, we can write:

S.A.I=AxxSxIx+Anyny+AzzSzIz

where Axx = Txx + A0, Ayy = Tyy + A0, and A22 = Tzz + A0 are the principal values of the

hyperfine coupling tensor. Recalling that the dipolar tensor T is traceless, it follows that
(Axx + Ayy + Aug/3 = A0, the isotropic hyperfine coupling constant

Now, with the assumption that the electron Zeeman term is the dominant term in
the Hamiltonian (i.e., that S is quantized along 2, the magnetic field direction), we write

the full Hamiltonian:

%=geBcHMs+hS.A.I—gNBNH.I

The hyperfine interaction term may be thought of as the energy of the nuclear dipole in
the local hyperfine field (HHF) generated by the electron. Thus, the effective field at the

nucleus (Hcff) is the vectorial sum of the applied Field (H) and the hyperfine field

(HHF), and we can write:

 

 

51
%=ge5cHMS‘gN3NHHF-I-gNBNILI

or ll=cheHMS-gNBNHCff.I

.—

where Hcfi = H + HHF

Thus, the nuclear spin will be quantized along the direction of H63, i.e., along the

resultant of H and HHF- This does not necessarily coincide with the direction of H

(i.e., the z axis), where the electron spin is quantized. Depending on the relative

magnitudes of H and HHF, one of three situations will arise ( see Figure 2.6 ):

A) I H I > > I HHF I : In this case, Hcff will be approximately parallel to H

(see Figure 2.6a). Therefore, both the nuclear— and the electron spin will be quantized

along H, and the spin Hamiltonian can be written as:

%=chCHMS+hS.A.I-gNBNH.I
=chcHMS+thM1A-gN6NHMI

=cheHMS+hMSMII°A’l-gNBNHMI

where 1 is a unit vector in the direction of the external magnetic field. Recalling that A
combines both the isotropic and the dipolar components of the hyperfine interaction, we

can express the hyperfine term as follows:

thM11.A.1 =thMI[A0+l..Tl]

=hMSMI[A0-geBcgNBN( (1'2'322)/1'5)]

If the unpaired electron is in a p-orbital centered on the interacting nucleus, we can

substitute 2 = r cos 0, and we get:

52

 

 

Figure 2.6: The variation of the direction of the nuclear spin quantization (Heff) with

respect to the applied field (H), depending on the magnitude of the hyperfine
field at the nucleus (HHF) (Reference 1).

53
hMSM11.A.1=hMSMI[A0+B(3c0320-1)]

where B is the anisotropic hyperfine coupling constant and 0 is the angle between the
magnetic field direction and the p orbital where the unpaired electron resides. Thus, the

full spin Hamiltonian representing this case is:

li=gCBCHMS+hAOMSMI+hB(3c0820-1)MSMI

The energies of the spin levels in the one electron - one proton system are:

81:1/2chcH+1/4hA0+l/4hB(3cosZO-l)
82:1/2chcH-1/4hA0-1/4hB(3cos20-1)
e3=-1/2chcH-1/4hA0-1/4hB(3c0320-1)
£4:-1/2chcH-t»l/4hA0+1/4hB(3c0320-1)

Figure 2.7 shows energy level diagrams corresponding to the two limiting cases
where 0 = 0° (Figure 2.7a) and 0 = 90° (Figure 2.7b). It is clear that larger hyperfine
splitting arises when the field is applied parallel to the p-orbital ( 0 = 0°; Figure 2.7a )

and a smaller splitting is observed for 0 = 90‘> (Figure 2.7b ).

B) | H | s | HHF I : In This case, the direction of Heff is different for MS = + 1/2

and M5 = - 1/2, i.e., the nuclear spin will be quantized in a direction determined by the

electron spin state (Figure 2.6b). The result is the occurrence of "forbidden transitions",

where both the electron- and the nuclear spin change sign, and these appear as satellite
lines in the EPR spectrum. If the two nuclear spin states corresponding to Ms = +l/2

(i.e., those quantized along Heffm— see Figure 2.6b) are denoted by | a'N) and | al.,),
and those corresponding to Ms = -l/2 are denoted by I a"N) and I 3"le then by applying
the spin Hamiltonian on page 51 to the four spin functions d>1 = I ate a'N),

¢2 = I “e B'N), ¢3 = I Be OWN), and ¢>4 = I13‘3 B"N), we get the corresponding energies:

 

 

54

Figure 2.7: Energy level diagram for a one electron - one proton system where the
hyperfine field is much smaller than the external field, when the orbital

containing the unpaired electron is A) parallel to the external field; and

B) perpendicular to the external field.

55

A25 on—

0

Aze 3
AZ?

20
A.;;

cannon».—
omaeuofli

 

 

 

 

 

 

 

 

 

 

 

56

A28 on—
Azo .3

AZ}
A....._

 

eaten?
939583

 

 

 

 

 

 

 

 

 

 

 

 

57
31 = “2 8e Be H ’ “2 8N 5N Heff(+)
82 = “2 8e Be H + “2 3N 3N Heff(+)
83 = - 1/2 ge Be H - 1/2 gN 5N Heff(-)
84 = ' “2 8e Be H + “2 8N 3N I'Iert(-)

The energies of the four possible transitions between these spin levels, shown in Figure

2.8, are:

Wa = 82 - 83 = gc Be H +1/2 8N 5N ( Heff(+) + Hcff(-))
Wb = 62 - a4 = ge fie H +1/2 3N 3N ( Heff(+) ' Hrcff(-))
Wc = 81 - s3 = g,3 3., H -1/2 8N 3N ( Heff(+) ' Heff(-))

Wd = 81 - s4 = gc Be H -1/2 8N 3N ( Heff(+) + H1==1’f(-) )

where Wa and Wd correspond to the normal "allowed transitions", and Wb Wc are the

"forbidden transitions", which usually have much smaller intensity. In effect, this

situation resembles the mixing of the spin states I ac BN) and I Be aN), whereas

a a ) and B B ) are unaffected (see Figure 2.5); i.e., M is no longer a good
e N e N I

quantum number.

C) I H I << I HHF I : Here, Hcff is almost parallel to HHF, while the external

field H is in a different direction (Figure 2.6c). Thus, S and I are quantized in two
different directions. Taking S to be quantized along the external field (i.e., along the
z-axis), and assuming axial symmetry, we can substitute 2 = r cos 0 and x = y = r sin 0 in

the dipolar Hamiltonian (page 38), and the full spin Hamiltonian becomes:

ll=chcHMS+hMS[A0+B(3cos20-1)Iz+3Bcos0sin01x]

=geBeHMS'gNBN[HII Iz+H_L Ix]

 

 

58

 

 

 

 

 

 

“c x” ‘+ IacaN> 3
‘x_ _1
5‘;— IacflN> 31
“a 5. : E
i a Ib Ic
v E_
B //’P I Ipe Rl> 84
e r '
\\\*v * 8
Inc “14> 3
H = 0 Electron Isotropic
Zeeman hyperfine

Figure 2.8: Energy level diagram for a one electron - one proton system, where the

hyperfine field is comparable to the external field (Reference 1).

59
where HII =-th/gNBN[A0+B(3c0820-1)]

and H_L=-hMS/gNBN[3Bsin0cos0]

If I “N ) and I BN ) were the nuclear spin functions for I quantized along z, then the

Hamiltonian matrix can be written as:

-

”(0N WI 0N) (“N WI 5N)

 

 

_<3N WI 0N) (BN WI [3ng

chcMs-I/ngBNHII -1/2gNBNHl

'l/ZgNfiNHi cheHMS-I'l/ngBNHH

Diagonalizing this matrix (since I is not actually quantized along 2) yields the energies
for a given value of MS:

8=geBeHlVIS11/2gNBN V(HII2+H.LZ)

=geBcHMS¢1/2hMS V[(A0+B(3c0820-1))2+9stin7-0 c0520]

=cheHMS:1/2hMS x/[(AO-B)2+3B(2A0+B)cos20]

The dependence of second order effects on the relative magnitudes of the external
field and the hyperfine field is best demonstrated in the classical work of Miyagawa and
Gordy (l 1), where they studied radiation-damaged single crystals of alanine at several
microwave frequencies. The radical they characterized, CH3—C°H—R, exhibited an
isotropic hyperfine coupling of 26 G, due to three equivalent methyl protons. The methyl
proton coupling, being isotropic, was found independent of the microwave frequency.

The a-proton coupling, on the other hand, has both an isotropic component and a dipolar

 

60
component. The dipolar coupling of the a-proton led to the observation of satellite lines
in the EPR spectra at certain orientations of the crystal in the magnetic field. The
intensity of these satellite lines, which arise from second order, or "forbidden"
transitions, was low at X- and Q-band frequencies (9 GHz and 35 GHz, respectively). At
intermediate frequencies (K—band; 24 GHz), the hyperfine field and the external field
became of comparable magnitude (case B, above), and the "forbidden" transitions were
as intense as the normal "allowed" transitions. Miyagawa and Gordy also showed that the
latter effect occurs as the relative intensities of the two doublets a,d and b,c (Figure 2.8)

are reversed on going from the low frequency limit to the high frequency limit (11).
2.5 THE ELECTRON-NUCLEAR DOUBLE RESONANCE (ENDOR) EXPERIMENT

ENDOR (electron-nuclear double resonance) is a technique where two radiation
fields, microwave and radiofrequency, are applied to the sample, and the change in the
intensity of a partially saturated EPR transition is detected as the RF is swept through the
resonance frequency. That is to say, ENDOR can be described as "EPR-detected NMR"
(12). ENDOR has the advantage of much greater sensitivity over NMR— the energy of
the microwave quantum is three orders of magnitude larger than that of the RF quantum.
On the other hand, ENDOR provides much higher resolution than EPR: a hyperfine
splitting of l G, which usually can not be detected in a powder EPR experiment,
corresponds to 2.8 MHz (for g = 2.00). Furthermore, the number of ENDOR lines
increases additively with the number of nuclei, whereas in an EPR experiment, the
number of lines increases multiplicatively. Thus, ENDOR is a powerful tool for the study
of immobilized radicals, where most of the hyperfine information is obscured by the EPR

line broadening (8, l3).

61

For the simple, isotropic system of one electron - one proton (S = 1/2, I =l/2 ), and
with the assumption that both MS and MI are quantized along the external magnetic field

(taken as the z-direction), the energy levels are given by the spin Hamiltonian:

%=geBcH.S+h AS.I-gNBNH.I

where A is the isotropic hyperfine coupling constant (in Hz). The energy levels are given
in Figures 2.9 a and b for ”N > N2 and VN < A/2, respectively (where the nuclear

frequency, vN = gN 5N H/h). The ENDOR transitions shown in Figure 2.9 are governed
by the selection rule A M1 = i 1. For vN > A/2 (Figure 2.9a), the two ENDOR
transitions occur at ”N 1- N2; i.e., the two lines are separated by A and centered at ”N:
For ”N < A/2 (Figure 2%), the ENDOR transitions are observed at A/2 : ”N3 i.e., the

two lines are centered at N2 and split by 2uN. In general, the ENDOR frequencies are
given by:
where the relative magnitudes of vN and N2 determine the splitting and the center of the

transitions.

In the solid state, dipolar interactions are not averaged out, and we have to take

hyperfine anisotropy into account. In this case, the ENDOR frequencies are given by:

V+= | VNiR/ZI

where R is an effective hyperfine coupling for a particular orientation of the radical with
respect to the magnetic field. That is, in the strong field approximation (where both MS

and M1 are quantized along the external field direction, i.e., the z—axis), the observed

hyperfine splitting is:

 

62

Figure 2.9: Energy levels (Hz) for S = 1/2 and I = 1/2 system, for a) ”N > N2; and
b) ”N > A/2 (Reference 13).

m.=-_%

ml”

 

 

w“...

fill-0%

63

.. __ fits". thvnofi .A'

 

 

 

 

2 MIL-J—
y" ”a"? ENDORo
'1'- 1 £hV.-%hvn-*lm
ESR
.-1_-'__\ —- We'lhvn-llAl
V" vat? ENDOR' 4
..1 1.x _-,...,-I..,.I...
2

 

_f__fiF—_‘T—

'g'wn ENDOR.

—%y.'-4LIAI'_;_ y"

 

ESR
I \. '%V0’11Al‘%yn
I
I "3"; y" ENDOR-
—-t

 

64
R = Aiso + Bxx 1,,2 + 13yy 1y2 + 1322122

where Bxx, B”, and B22 are the principal components of the dipolar coupling tensor, and

1x, 1’, and 12 are the direction cosines of the external magnetic field with respect to the

xyz principal axis system of the hyperfine tensor. Thus, for axial symmetry, and if z is
the symmetry axis, then Bzz = -2Bxx = -2Byy. If the external magnetic field (H) is applied

in the yz plane, and if 0 is the angle between H and the z-direction, we have 1x = 0,

1y = sin 0, and 12 = cos 0. Then:
R = Aiso +1/2 B22 (3 cos2 0 - 1)

If the field is in the z-direction, then 0 = 0, and we get:

R=Aiso+Bzz=A||

Similarly, if we consider the case of H applied in the xy plane, we obtain:
R=Aiso+Bxx (forHIIx)
R=AiSO+Byy (forH II y)

where Aiso + Bxx = Aiso + Byy = A J.

This treatment is used in the calculation of the expected 170 ENDOR frequencies, in

Chapter 3.

 

65

References

1. Wertz, J. E. and Bolton, J. R. Electron Spin Resonance: Elementary Theory and
Practical Applications; McGraw-Hill: New York (1972).

2. Fermi, E. (1930); Z. Physik 60, 320.

3. Carrington, A. and McLachlan, A. D. Introduction to Magnetic Resonance; Harper

and Row: New York (1967).

4. McConnell, H. M. (1956); J. Chem. Phys. 24, 764; McConnell , H. M. and Chestnut,
D. B. (1957); J. Chem. Phys. 27, 984; McConnell, H. M. and Chestnut, D. B. (1958);
J. Chem. Phys.28, 107.

5. Bender, C. J.; Sahlin, M; Babcock, G.T.; Barry, B. A.; Chandrachekar, T. K.; Salowe,
8.; Stubbe, J.; Lindstrom, B.; Petersson, L.; Ehrenberg, A.; and Sjbberg, B.- M.
(1989); J. Am. Chem. Soc. 111, 8076.

6. Morton, J. R. (1964); Chem. Rev. 64, 453.

7. a) Heller, C. and McConnell, H. M. (1960); J. ChemPhys. 32, 1535; b) Stone, E. W.
and Maki, A. H. (1962); J. ChemPhys. 37, 1326.

8. Kurrek, H.; Kirste, B.; and Lubitz, W. Electron-Nuclear Double Resonance of
Radicals in Solution; VCH Publishers: New York (1988).

9. Barry, B. A.; El-Deeb, M. K.; Sandusky, P. O.; and Babcock, G. T. (1990); J. Biol.
Chem. 265, 20139.

10. Abragarn, A. and Bleany, B. Electron Paramagnetic Resonance of Transition Ions;

Clarendon Press: Oxford (1970).

 

66
11. Miyagawa, I. and Gordy, w. (1960); J. Chem Phys 32, 255.

12. Babcock, G. T., personal communication.

13. Kevan, L. and Kispert, L. Electron-Nuclear Double Resonance Spectroscopy; Wiley-

Interscience: New York (1976).

 

67
CHAPTER 3

EPR CHARACTERIZATION OF IMMOBILIZED TYROSINE
RADICALS: A MODEL FOR THE TYROSINE RADICALS
IN PHOTOSYSTEM II

3.1 INTRODUCTION

In oxygen evolving photosynthesis, chemical energy, produced in the light-driven
water oxidation process, is utilized to reduce carbon dioxide. The electron transfer
reactions are canied out by two membrane-bound proteins, Photosystem I (PS I) and
Photosystem H (PS II) (1). Photosystem II is the membrane complex that oxidizes water
and reduces bound quinone. Details of the electron transfer pathway from the water

oxidation site to the bound quinone site in PS II were given in Chapter 1.

Recently, Barry and Babcock (2) have shown that Photosystem II contains two
redox active tyrosine residues, YD and Y2. The two tyrosine radicals, YD. and YZ', have

identical EPR spectra, even though their functions and their catalytic behavior are
different. Y2. mediates the electron transfer from the manganese cluster, where the

water oxidation takes place, to the oxidized reaction center chlorophyll, P680+ (3,4). The

function of YD° is uncertain, but it has been shown that it is formed by electron donation

from YD to the manganese S2 and S3 states (see Chapter 1), and decays by electron
donation from the manganese SO state (5,6). Thus, the interconversion of the YD/YD'

couple is thought to play a role in stabilizing the manganese cluster by forming the dark-
stable 81 state (6).

 

68

The fact that the EPR spectra of YD' and Y2. are identical indicates that the two
species have identical chemical structure. Other naturally occurring tyrosine radicals,
however, have strikingly different EPR line shapes and line widths (5,7). While the EPR
spectra of the PS H radicals show a single line with partially resolved hyperfine structure,
the radicals in ribonucleotidediphosphate reductase (RDPR) and prostaglandin H
synthase (PGHS) are dominated by a large doublet hyperfine splitting (7-9).
Furthermore, the spectral width of the Y]; tyrosine radical in PS II is less than 20 G,

whereas that of the RDPR radical is close to 40 G (8,10). The tyrosine radicals in PGHS

 

and galactose oxidase (GO) exhibit EPR linewidths of approximately 35 G (9,11). These
variations in the spectral properties of tyrosine radicals can be explained if one takes into
account the effect of the protein steric factors and their influence on the structure of
tyrosine radicals (12-14). Clearly, the EPR spectra of the tyrosine radical species are
sensitive to variations in the protein environment, and therefore can provide information
about the spin density distribution and the geometry of the radical. By UV photolysis of
frozen solutions, we have generated a series of tyrosine radicals in vitro, specifically
deuterated at the B—methylene group and at different ring positions. These frozen radicals
provide an appropriate model for the naturally occurring tyrosine radicals, since the latter
are immobilized in their protein environments. The EPR data on the specifically
deuterated model tyrosine radicals make it possible to assess the magnitude of the
hyperfine coupling to the individual tyrosine protons, and, in turn, to predict the spin
density distribution around the phenol ring and the conformation of the methylene group.
The properties of the unperturbed frozen tyrosine radicals can be compared to those of

the corresponding protein-immobilized YD° radicals in Photosystem H (7) and of the

RDPR tyrosine radical (10). Thus, the spin density distribution and the geometry of the
in vitro tyrosine radical, Y', can be compared to those of the Photosystem II YD'IYZ'

tyrosine radicals as well as to other biologically relevant tyrosine radicals.

 

69
3.2 MATERIALS AND METHODS

Materials. L- and DL-tyrosine were purchased from Sigma. The specifically deuterated
tyrosines: DL-[B,B-2H] tyrosine; L-[3,5-2H] tyrosine; and L-[2,6,y-2II] tyrosine, and the
fully deuterated tyrosine were purchased from MSD Isotopes. The isotopic purity was
98% in all cases. L-tyrosine (35 - 40 % phenol-170) and water (30 % 170) were from
Cambridge Isotope Laboratories. Fast Atom Bombardment-Mass Spectroscopy
(FAB-MS) was performed on the 17O-enriched tyrosine to verify the 170 content. The
FAB-MS data is shown in Figure 3.1. From the relative heights of the peaks at m/z = 181
and m/z = 182 (Figure 3.1), and taking into account the natural abundance of 13C, we
calculate an isot0pic enrichment of 46 % in our 17O-labeled tyrosine, which is higher

than the value reported in the manufacturer's specifications.

Tyrosine Radical Generation. A solution of the respective tyrosine (ca. 5 mM) was
made in 12.5 mM borate buffer, pH 10.0. The solution was degassed by bubbling with
nitrogen or argon, and ca. 0.3 ml were loaded into a 4 mm (O.D.) EPR tube and frozen in
liquid nitrogen. Tyrosine radicals were then generated by irradiation of the EPR sample
at 77 K, with the full spectrum of a 1000 W Hg-Xe lamp for 1 min (15). No qualitative
change in the EPR spectra were observed upon varying the tyrosine concentration

(0.5 - 10 mM) or illumination time (0.5 - 20 min). Because the specifically deuterated
tyrosines were supplied as either the L- or the DL-isomers, the EPR spectra of both
isomers of the fully protonated species were compared and found to be identical.
Samples of the fully protonated tyrosine were also prepared in unbuffered aqueous acidic
(12 M H2S04) and alkaline (saturated KOH or NaOI-I) media, and the tyrosine radical
EPR signals were identical in all cases. We conclude that the technique we use, namely

UV photolysis of frozen aqueous solutions, generates the same radical species (viz. the
neutral tyrosine radical, R—CGHr—O', see below) over the entire pH range, and

 

70

 

658.5 vocab—v-02 «c 88827. m2-m<n_ ”fin charm

 

 

 

 

{a ..
QGN Ema . 8mg awn awn can Gm Gm
I I P a r I > h > t 4 l P l > I n . .u I 4. P - lvl. 0 P L I .I bl o< rqi
A _F :Ht . am.“ _ . _: __ mW. mm =—
_ n .— m. a m 3”. Am
A
4 am ram
1 t. m
1 mt
A n.
n ,6? «Au
A w
A m
60
. m
. 9
1 _
4 48¢
kflul mad.

 

 

 

71
regardless of whether the matrix is glassy or polycrystalline. Glassy samples prepared by
incorporating 40 % (v/v) ethylene glycol in the borate buffer exhibited EPR spectra that
were severely broadened by an unidentified radical species that was not observed in the
aqueous samples, either polycrystalline or glass. The photochemical generation of this
spurious radical species was probably sensitized by the tyrosine itself, since no EPR
signal was observed from the ethylene glycol-borate buffer alone after UV illumination
at 77 K. Thus, the use of organic solvents as glassing agents was ruled out, and we
continued our investigation with the powder samples prepared in borate buffer, where
only the desired tyrosine radicals are generated (see below). Typical EPR spectra of the

fully protonated tyrosine radicals in different solvents are depicted in Figure 3.2.

17O-Enriched tyrosine Radicals. The EPR spectrum of the 17O-enriched tyrosine
radicals was recorded from a sample prepared in 17O-enriched water (30 % 17O) and
containing potassium chloride (4 M) and potassium hydroxide (0.1 M). Potassium
hydroxide was added to the sample from a concentrated solution so that the smallest
possible volume could be used in order to retain the highest isotopic enrichment in the
final solution. Surprisingly, the use of isotopically enriched water was the only way we
could observe any broadening of the 17O-enriched tyrosine radical spectrum; samples
prepared in unlabeled water showed spectra that were identical to those of the unlabeled
tyrosine radical. This observation shows that the tyrosine phenolic oxygen is
exchangeable with the solvent, a phenomenon that has only a few precedents in the

literature (16).

EPR Measurements. The EPR spectra reported here were recorded at 120 K on a Bruker
ER 200D spectrometer, equipped with 100 kHz modulation and a TE102 cavity. Identical

spectra were recorded at temperatures as low as 77 K. The temperature at the sample was

regulated by using a home-built nitrogen flow system, and the temperature directly below

 

72

Figure 3.2: X-band EPR spectra of photochemically-generated tyrosine radicals in a)
borate buffer, pH 10.0; b) saturated KOH; c,d)l2 M H2SO4. Temperature:
120 K; modulation amplitude: 0.5 G; microwave power: 0.5 mW; scan
width: 100 G (a-c), 600 G ((1). Spectrum 6 is an expansion of the feature at

the low field wing of spectrum d (see text).

73

 

l l l l I

I l l I '
3390 3410 3430 3450

Maonetic Field (G)

r l
3350 3370

 

 

 

74

 

 

 

 

 

 

 

 

”I [J I“

 

 

 

 

 

 

I ' I ' I I I I ' i I I I I
3050 3150 3250 3350 3450 3550 3650 3750

 

 

I

I I I
3120 3140 3160

Maonetic Field (G)

I
3100

75
the sample tube was measured by using a copper-constantan thermocouple connected to
an Omega 199 temperature read out. The temperature thus measured was found to be
within 05° from the sample temperature. Magnetic field strength and microwave
frequency were measured directly by using a Bruker ER-035M NMR Gaussmeter and a
Hewlett-Packard 5255A frequency converter/5245L counter, respectively. This enables
us to calculate g values accurate to the fourth decimal place. The spectrometer was
interfaced to a microcomputer equipped with an IBM data acquisition board, which we

used for data collection and storage.
3.3 RESULTS

Specific Deuteration Effects on the EPR Spectra of Tyrosine Radicals. Figure 3.3
shows the tyrosine numbering convention we use in this work. In Figure 3.4, the EPR
spectra of tyrosine radicals generated by UV photolysis of frozen borate-buffered
solutions are presented. The EPR spectrum of the fully protonated radical (Figure 3.4a)
shows partially resolved hyperfine structure, with a g value of 2.0047 and a peak-to-
trough line width of 24 G. By virtue of the generation technique, the tyrosine phenolic
—OH group is deprotonated at pH 10, and upon UV irradiation the neutral radical is
formed by electron abstraction (17-20). As pointed out in the introduction (Section 1.3),
neutral radicals are formed by UV illumination, even at neutral or acidic pH. This is

confirmed by the similarity of the EPR spectra obtained from radicals prepared in 12 M
H2SO4 and in alkaline media (Figure 3.2). Furthermore, a 600 G-wide spectrum of the

tyrosine radical generated in 12 M H2804 (Figure 3.2d) shows two multiplets at the

wings, separated by 507 G, in addition to the tyrosine radical signal at the center. Clearly,
the two features with the 507 G splitting are due to H' hyperfine splitting, indicating that

in this case, O—-H bond homolysis contributes to the formation of neutral radicals

(cfi Section 1.3). The additional splitting of these features, clearly observed when the

 

76

 

Figure 3.3: Tyrosine numbering convention used throughout this work.

 

 

 

77

Figure 3.4: X-band EPR spectra of photochemically- generated model tyrosine radicals,

specifically deuterated as indicated, in borate buffer (pH 10.0). Temperature

120 K; modulation amplitude: 0.5 G; microwave power: 0.5 mW; scan
width: 100 G.

78

g=2.0047

  
  
   
    
 

A, Protonoted tyrosme

B. 2.6- Deuteroted tyrosine

C 3.5- Deuteroted tyrosine

D Methylene- deuterated
tyrosme

E Per-deuterated tyrosme

 

F ' I ' I r I ' I ' I
3350 3370 3390 3410 3430 3450

Maonetic Field (G)

79

spectrum wing on either side is expanded (Figure 3.2e) follows a 1 : 4 : 6 : 4 : 1 intensity
pattern, typical of splitting from four equivalent protons. This probably indicates
association of the H' atom with a tyrosine ring in its vicinity, although we did not confirm
this possibility. The appearance of the low-intensity features, which arise from an
unidentified species, next to the main tyrosine peak in Figure 3.2e does not alter the fact
that the tyrosine radical signal is similar in shape to those observed at high pH, confirming

that the same radical is formed in both cases.

For comparison, Figure 3.5 represents analogous EPR spectra of specifically deutera-
ted YD° radicals in Photosystem II. These spectra, taken from reference 7, were recorded
from whole cells of the cyanobacterium Synechocystis 6803, by Dr. Bridgette Barry
(University of Minnesota) during her postdoctoral fellowship at MSU. The EPR spectrum
of the fully protonated YD° species (Figure 3.5a) has a line width of 18 G and a g-value of

2.0041. The small deviation of the latter value from the 2.0046 value observed in purified

PS II preparations is attributed to a slight contamination from P700+, the oxidized reaction

center of Photosystem I, and/ or sample orientation effects in the magnetic field (7).

Deuterium substitution at the 2,6 positions results in very little change in the

spectrum of the model tyrosine radical (Figure 3.4b) relative to that of the fully protonated
compound. Similarly, little effect was detected for the 2,6-deuterated YD. radical (Figure

3.5b). This indicates that in both the model compound and the YD. radical, the coupling of

the unpaired spin density to the 2,6 protons is small. Deuteration at the 3,5 positions,
however, changes the EPR spectra of the model tyrosine and of the YD. radical
dramatically. Both spectra show a doublet structure, which is more resolved in the model
tyrosine spectrum (Figure 3.4c). The latter shows a total line width of 21 G and a splitting
of 14 G, resulting from hyperfine coupling to one of the B—methylene protons (see below).
In the case of the YD. radical (Figure 3.5c), the total line width is decreased to 16 G and

the doublet splitting is only 10.5 G (7).

 

80

Figure 3.5: X-band EPR spectra of specifically deuterated YD' radicals in Photosystem II
(7).

81

g = 2.0041

A. Protonoted tyrosine

   
  
 
  

B. 2.6- Deuteroted tyrosine

C. 3,5- Deuteroted tyrosine

D. Methylene-deuterated tyrosine

E. Per-deuterated tyrosine

 

I l

‘ l

3390 3210 343
Madnetic Field (G)

I
3370

 

82
The doublet splitting that results from hyperfine coupling to the B-methylene

protons is removed upon specific deuteration at the —CH2— position. Figure 3.4d shows

the EPR spectrum of the methylene-deuterated model tyrosine radical. The correspond-
ing spectrum of the B-deuterated YD° radical is presented in Figure 3.5d. Comparison of
the methylene-deuterated radical spectra in Figures 3.4d and 3.5d reveals a striking
similarity. Both spectra have an overall line width of about 16 G. Moreover, the partially
resolved hyperfine structure in the two spectra is quite similar. This result demonstrates

that the magnitude of the hyperfine coupling to the 3,5 protons is similar in both species.

The spectrum of the fully deuterated model tyrosine radical, shown in Figure 3.4e,
shows a single line with a peak-to-trough line width of 8 G, similar to that of the fully
deuterated YD' radical (Figure 3.5e). The similar line width of the spectra of both fully
deuterated species demonstrates that the radical we have generated in vitro is, in fact, a
tyrosine radical, and that the spectra in Figure 3.4 are free from contamination from other
paramagnetic species that might be expected to be produced by the UV illumination
technique. The narrowing of the tyrosine radical spectra to 8 G upon deuteration also

shows that the major splittings in these spectra result from hyperfine coupling to protons.

The EPR Spectrum of 17O-Enriched Tyrosine Radicals: Spin Density at the

Phenolic Oxygen. The spin density at the tyrosine phenolic oxygen is, no doubt, an
important factor in determining the redox potential of the radical (cfi Section 1.2.2),
which is key to its function in protein redox chemistry. As we noted above, the phenolic
group in our tyrosine models is deprotonated, so we do not observe proton hyperfine
splitting from an —OH proton, from which the spin density at the oxygen could be
estimated. In this case, direct evaluation of the oxygen spin density requires measurement

of hyperfine coupling to 17O, the only oxygen isotope with non-zero nuclear magnetic

83
moment. As the natural abundance of 170 is only 0.037 %, the use of 17O-enriched

compounds is necessary for 17O hyperfine splitting to be observed in an EPR experiment.

The tyrosine 170 spin density is related to its hyperfine coupling by an equation of

the form:
a0 = roO Po + Qcoo PC

where p0 and pC are the spin densities in the p1r orbitals of the oxygen atom and its
neighboring carbon, respectively, a0 is the isotropic hyperfine coupling to the oxygen,
and roo and QCOO are o-ir interaction parameters, that describe the interaction of the
C—0 a-bond with the rr-spin densities at the oxygen and the carbon, respectively (21).
This equation was originally developed by Karplus and Fraenkel (21) to describe 13C
hyperfine interactions, and was later employed for the treatment of hyperfine splittings
from other second-row nuclei, namely 14N, 17O, and 19F (22). It has been shown that the

dominant contribution to the oxygen hyperfine coupling comes from the spin density in
the p1r orbital on the oxygen atom itself; the contribution to a0 from the p1r spin density
on the neighboring carbon being much smaller (22-28). For semiquinone radicals, for

example, Silver and Luz (24) reported that the o-rr interaction parameters QOCO and

QCOO are -40.5 G and 6.4 G, respectively (the opposite signs were attributed to the fact

that the gyromagnetic ratio of oxygen is negative, while that of carbon is positive). Thus,
the form of the Karplus-Fraenkel equation has been simplified, and Dirnroth et al. (22)

have successfully interpreted the 170 hyperfine coupling observed in 17O-enriched 2,4,6-
triphenyl phenol radicals by assuming a direct proportionality between the 170 hyperfine

coupling and the spin density at the oxygen:

a0=QOPO

84
In this McConnell-type equation, the value of Q0 (which corresponds to QOCO in

the original Karplus-Fraenkel expression), was taken as -40 :i: 4 G (22). Melrnaud and
Silver (26), utilizing literature data on several 17O-e'nriched organic and inorganic
w-radicals, provided a test for the validity of this Simple expression. By correlation of

observed hyperfine splittings with spin densities estimated from dipolar tensors as

measured in single crystals, these authors have shown that the 170 hyperfine coupling is,
in practice, proportional to the 1r-spin density at the oxygen, with the proportionality
constant equal to -41 1 3 G; the contribution of the spin density at the neighboring

carbon was taken to be negligibly small (26).

In an effort to measure the spin density at the oxygen in our tyrosine model
compounds, we recorded the EPR spectrum of 17O-enriched model tyrosine radicals, and
that of the unlabeled species, under the same conditions. The two spectra are compared in
Figure 3.6. The spectrum of the 17O-enriched species is noticeably broadened relative to
the unlabeled compound, indicating significant spin density at the oxygen. Similar
instances, where 170 enrichment resulted in EPR line broadening, have been reported in
the literature (29). Unfortunately, the broadening shown in Figure 3.6 does not provide a
reliable measure of the oxygen spin density (see below), and we have to rely on the
literature data for a quantitative estimate of the latter. We are confident, however, that
the broadening we observe (Figure 3.6) is a demonstration of a significant spin density at
the oxygen, and is not a spectral artifact, as we could not detect any broadening of the

17O-enriched spectrum when the same experiment was carried out in unlabeled water, as

we noted above.

Bender et al. (10), in a self-consistent calculation, determined a value of 0.16 for
the spin density at the phenolic oxygen in the RDPR tyrosine radical. A similar value

(0.174) was reported for the spin density at the oxygen in the analogous radical of

85

Figure 3.6: The broadening of the EPR spectrum of 17O-enriched tyrosine
radical (—) relative to the unlabeled compound ( - - - - ).

Temperature: 120 K; modulation amplitude: 5.0 G; microwave

power: 0.5 mW; scan width: 100 G.

86

A0. 0.0.“. 2.9.032
om¢m

omvm omwm
. _

ovwm
_

omwm
_

_ _

 

 

6:52.: 3.32-0
t.

0250...: 3.2.5.2: .............

 

_

owmm
.

comm
L

 

 

87
2,4,6-tri-t.butyl phenol (30). We expect a comparable spin density at the oxygen for our
model tyrosine radical, and thus we will use the value of Bender et al. in the discussion

that follows.

The isotropic and anisotropic hyperfine coupling constants for 170 were included

in the tables published by Morton and Preston (31). The anisotropic coupling constant for
170, B0, is -l68.4 MHz (for unit spin density in the oxygen p, orbital). Therefore, for a

spin density of 0.16, we expect an anisotropic coupling to the 17O nucleus, B = -26.9
MHz = -9.6 G. The isotropic coupling to the 170 can be evaluated by using the above
mentioned McConnell-type equation. Using the proportionality constant of Melrnaud and
Silver (-41 G) and the oxygen spin density found in the RDPR tyrosine radical (0.16), the

isotropic hyperfine coupling to the 170 nucleus in the model compound radical is

expected to be:
aim = -41 p0 = -41 (0.16) = -6.6 G.
Thus, since the dipolar coupling constant is traceless (cf. Chapter 2), the hyperfine
coupling tensor components of the 170 nucleus are:
AII=aiso+2B='25°8G

A_L=aiso-B=3.0G

where axial symmetry is assumed, since the unpaired electron is in a p-orbital. Such high

anisotropy of 170 hyperfine tensors is not unusual, and usually results in the observation
of the larger component (All) as satellite lines; the other component is usually less than

the line width of the unlabeled radical signal (32-34).

In the case of the model tyrosine radical (Figure 3.6), the total broadening of the

17O-enriched spectrum, measured at half-amplitude of either side of the spectrum,

88
is ~ 2.5 G. Although slightly less than the calculated value of A ..L (3.0 G), we interpret

this broadening as being the result of the perpendicular component of the 17O hyperfine

coupling tensor; the difference being probably due to the fact that we have only ~ 40 %

17O enrichment in the sample. The parallel compOnent of the 170 hyperfine coupling
tensor, I Al I0 I = 25.8 G, is larger than any of the proton couplings we observe in the

model tyrosine radical. Thus, Al I0 is expected to give rise to six lines, split by ~ 25.8 G
(170, I = 5/2), and these lines will be further split by the tyrosine protons, i.e., each of the
Al I0 satellites will have the shape of the unlabeled tyrosine signal. However, we were
unable to observe the 17O satellites, probably because of the large 170 anisotropy and the
much smaller intensity of these satellites relative to the unlabeled tyrosine signal. In cases
where the 170 satellites can not be resolved, as it became evident for the 17O-enriched
tyrosine model, ENDOR spectroscopy, with its inherent higher resolution, may seem to
be a feasible way if one is to determine the magnitude of the hyperfine coupling to the
oxygen, and hence its spin density (35-38). Drawing on the value of the oxygen spin
density in the RDPR tyrosine, we expect the model compound 170 ENDOR resonances
to be centered about 36 MHz (All) and 4 MHz (A _L) (39). Our attempts to detect 170
ENDOR transitions in the model compound tyrosine radical were unsuccessful, although
we tried different temperature, microwave power, and RF power conditions. This is not
unusual, considering the unfavorable combination of the large 170 hyperfine anisotropy
and the existence of quadrupolar coupling, which renders ENDOR detection difficult
(40,41). Thus, although we have demonstrated the occurrence of a significant spin
density on the tyrosine phenolic oxygen, our data do not provide a direct measure of the
magnitude of this spin density. In the discussion below, we will use the value provided in
the literature (0.16) for the oxygen spin density as found in the RDPR tyrosine radical
(10).

89
3.4 DISCUSSION

Tyrosine radicals have now been identified in several redox active enzymes,
including Photosystem II (2,14), prostaglandin Hsynthase (9), ribonucleotidediphosphate
reductase (8,10), and galactose oxidase (1 1). Of all these tyrosine radical species, only
the two tyrosine radicals occurring in PS H, YD° and Yz°, have identical EPR spectra
(1,2,7 ,14). All the remaining biologically active tyrosine radicals, and the immobilized
model tyrosine radicals in single crystals (42) and in powders, which we investigate in
the present work, have different EPR line shapes and line widths. Model tyrosine radicals
have been studied in solution (13,43,44) and in single crystals (42,45). Sahlin et al. (15)
reported generation of tyrosine radicals by UV illumination of frozen solutions, but no
EPR spectrum was shown, and no analysis of the hyperfine couplings was presented. The
model compound - specific deuteration approach we have taken in the present study
enables us to isolate the hyperfine coupling to each of the tyrosine protons, and hence to
assess the spin density distribution in the aromatic ring. We have also undertaken a
comparative study of the EPR characteristics of the model compound radical, the
tyrosine radical in RDPR, and the tyrosine radicals in PS II. The radicals in RDPR and
PS II have been chosen since EPR data on specifically deuterated tyrosine radicals is
available for these two systems (7,10), and Bender et al. (10) have also performed an
ENDOR study on the RDPR tyrosine species. Since the RDPR tyrosine radical has been
fully analyzed (10), a comparative EPR study involving the latter species, the
Photosystem II YD' tyrosine radical, and the unperturbed, frozen tyrosine model radical,
makes it possible to compare the hyperfine coupling pattern and the spin density
distribution in each species. That, in turn, should lead to an assessment of the structure of
the in vitro radical, as well as that of the PS 11 tyrosine radicals, and an understanding of

how the protein environment affects the structure of the latter species.

90
Figure 3.7 compares the EPR spectra of the fully protonated YD° tyrosine radical

in PS II (7,14), the model tyrosine radical, and the tyrosine radical in RDPR, published
by Bender et al. (10). The variation in the line shape and line width of the tyrosine
radicals in these different systems is evident from these data. We propose that the
drastically different EPR spectral characteristics exhibited by radicals having the same
chemical identity can be explained on the basis of geometrical factors— namely the
conformation of the B-methylene group relative to the aromatic ring; the spin density
distribution being essentially similar in all species. This interpretation is in line with

earlier predictions by Ehrenberg and co-workers (46) and by Barry and Babcock (14).

For aromatic free radicals, the electron spin is delocalized over the ring, and the
spin distribution at the various ring carbons determines the magnitude of the hyperfine
coupling to each proton. As discussed in Chapter 1, tyrosine radicals are expected to
follow an odd-altemant spin distribution pattern, with large spin density at carbon 1, and
smaller —but significant — spin densities at the oxygen and at carbons 3 and 5 (see

Figure 3.3). Much smaller, negative spin densities are expected at carbons 2, 4, and 6.

The occurrence of considerable spin density on the oxygen atom is demonstrated
by the broadening of the EPR spectrum of the 17O-enriched model tyrosine radical,
relative to the unlabeled compound (Figure 3.6). As we noted above, these data do not
provide a quantitative estimate of the magnitude of the oxygen spin density. For the
tyrosine radical in RDPR, which is a neutral, non-hydrogen-bonded species, similar to
our model tyrosine radical, Bender et al. have calculated a spin density of 0.16 for the
oxygen atom (10), and we assume that we can use that as a good estimate for the oxygen

spin density in the model compound case.

The spin density on the oxygen will depend, of course, on the orientation of the

oxygen p1r orbital with respect to the aromatic ring; i.e., on the extent of conjugation of

 

 

 

 

 

91

Figure 3.7: X-band EPR spectra of a) the fully protonated YD' tyrosine

radical in Photosystem II (7), b) the model tyrosine radical,
and c) the tyrosine radical in RDPR (10).

93
the oxygen orbital to the ring ir-skeleton (47,48). Maximum conjugation, and highest
oxygen spin density, are achieved when the oxygen p1r orbital is parallel to the pz orbital
on the ring carbon (49). The extent of conjugation, which will determine the degree of
double bond character of the C—-O bond, is directly related to the barrier to the rotation
about the C—0 bond (50). The latter has been estimated at 3.36 k cal/mol for phenol
(51). r-Donating para-substituents, however, are expected to decrease this barrier, and
Radom et al. (49) calculated a decrease of 0.32 k cal/mol of the barrier height upon para-
metlryl substitution on the phenol ring. We expect a similar effect from the tyrosine side
chain-— i.e., a lowering of the C—0 bond rotation barrier by ca. 0.3 k cal/mo] relative to
phenol, and hence we predict that the barrier would be on the order of ca. 3 k cal/mol in
the tyrosine radical. A lower C—O rotational barrier implies that the oxygen p-orbital
can be twisted out of the ring—orthogonal orientation more easily, which would lead to
lower conjugation, i.e., less C—O double bond character. Hydrogen bonding to the
phenolic oxygen is an important factor in this regard. Our prediction of the height of the
C—0 rotation barrier in the tyrosine radical (ca. 3 k cal/mol) is comparable in magnitude
to the energy of a typical hydrogen bond. Therefore, even if a hydrogen bond formation
requires that the oxygen p-orbital be twisted away from the maximum conjugation
orientation, the hydrogen bond would sufficiently compensate for the energy involved in
that twist. Thus, hydrogen bonding is expected to change the extent of double bond
character of the C—-O bond, and that, in turn, will have an effect on the spin density
distribution (16). Intuitively, one would expect the spin density on the oxygen itself to be
affected by hydrogen bond formation. One report, however, contradicts this prediction
(28), and current investigations in our laboratory, both experimental and by
semiempirical MO calculations, are aimed at testing this effect. The Photosystem H

tyrosine radical YD° is hydrogen bonded at the phenolic oxygen (14), and thus the

oxygen spin density is likely to be slightly different from that in the RDPR tyrosine

94
radical and the model species. This will not, however, have a major effect on the proton

hyperfine splittings discussed below.

The hyperfine coupling to a-protons, i.e. those bound directly to the ring, is

described by the McConnell relationship:
A0“ = Q to1r

where A0“ is the a-proton hyperfine splitting, p“r is the spin density in the p1r orbital of
the a-carbon (52). The proportionality constant, Q, was determined for tyrosine radicals,

and found to be equal to 24.9 G. For B-protons, i.e., those two bonds away from the ring

carbon, the hyperfine coupling depends on both the spin density at the ring carbon, p1r ,
and dihedral angle, 0, defined by the projection of the Cg—HB bond and the ring carbon

pz orbital axis, on a plane perpendicular to the Cg—HB bond (53):
A05=(B0+B2 c0320) p1r

As noted above (Chapter 2), B0 is much smaller than B2 and thus can be neglected. Thus,

the simple expression:

A05 = B2 p1r cos2 0

accounts satisfactorily for B-proton hyperfine couplings in aromatic w-radicals. For
tyrosine radicals, the value of B2 has been estimated at 58 G (10).

Our results on the specifically deuterated tyrosine radicals show that in these
species, the unpaired electron is delocalized over the aromatic ring, with the majority of
the spin density at carbons 1, 3, and 5 (see Figure 3.3), in accordance with earlier reports

on naturally-occurring tyrosine radicals (14,46). The lack of significant spectral change
upon deuterium substitution at the 2,6 positions of either the model tyrosine or the YD°

95

radical indicates that the spin density at these positions is negligibly small in both species.
Having established the similarity of the spin density at the oxygen and at the 2,6 carbons

in both the model tyrosine and the YD' radical, we can then compare the spectra of the B-

deuterated radicals to assess the spin density at the 3,5 positions. For clarity, the spectra

of the methylene-deuterated model tyrosine and YD° radicals are reproduced in Figure

3.8, along with the corresponding spectrum of the RDPR tyrosine radical (10), also
deuterated at the methylene position. The similarity of the spectra of the YD° radical
(Figure 3.8a) and the model tyrosine radical (Figure 3.8b) is quite clear; the spectrum of
the corresponding RDPR radical differs only by showing higher resolution of the spectral
features, but the overall appearance is retained in all cases. Thus, we conclude that the
spin density at the 3,5 positions is comparable in all species. For the RDPR radical, an
EPR/ENDOR analysis has established a hyperfine coupling of 6.5 G (10), corresponding
to a spin density of 0.26 at the 3,5 carbons. From the similarity of the spectra in Figure
3.8, we conclude that this value is a valid estimate for the spin density at these positions in
the model tyrosine radical and in YD'. This conclusion, which implies that the difference
in the side chain conformation does not drastically affect the spin density distribution in
the ring, is supported by the measurements of similar 3,5 proton hyperfine couplings in
tyrosine radicals in other systems. For the YD° radical in spinach Photosystem II
preparations, 3,5 protons have a hyperfine coupling of ~ 6 G, as measured by ENDOR
(54). Moreover, in both single crystal and solution studies of tyrosine free radicals, the 3,5
proton hyperfine couplings have been shown to be ~ 6.2 G; coupling to the 2,6 positions

was negligibly small in both systems (13,42).

Thus, in agreement with the odd-altemant electron spin distribution model, the spin
density distribution in all the tyrosine radical species that have been studied is very
similar. A value of 0.49 for the spin density at C1 in the RDPR tyrosine radical has been
deduced by analyzing the dipolar tensors of the ring protons (10). Drawing on the

similarity of the spin density at the 2, 3, 5, and 6 positions, and with the assumption that

 

96

Figure 3.8: X-band EPR spectra of the methylene-deuterated tyrosine radicals:
a) the Photosystem 11 YD' radical (7); b) the model tyrosine radical;

and c) the tyrosine radical in RDPR (10).

98

the ring spin density remains fairly constant in species with different side chain

conformations, we expect a value close to 0.49 for the spin density at carbon 1 in our

tyrosine free radical and in the YD° species as well. Because the magnitude of the
methylene proton hyperfine coupling depends. not only on the spin density at C1 but also
on the conformation about the C1—C3 bond, changes in the conformation of the

methylene protons can have a large effect on the EPR spectral line shape, even if the spin
density at C1 remains fairly constant. This seems to be the major factor behind the wide

variation of the spectral line shapes of tyrosine radicals in different systems.

Of all the studies on tyrosine radicals reported to date, only the in vitro tyrosine
radical, studied in solution at elevated temperatures, showed the triplet splitting expected
from two equivalent methylene protons, indicating free rotation about the C1—C3 bond
(13). In all other tyrosine radical species, the EPR spectrum is dominated by a large
doublet splitting, indicating strong hyperfine coupling of the spin density at C1 to only one
of the methylene protons. This implies, quite clearly, that the conformation of the
—CH2— group with respect to the ring is such that 0 for one proton is large, which gives
rise to a small hyperfine coupling, whereas 0 is substantially smaller for the second,
strongly coupled proton; 0 being the dihedral angle referred to in the Heller-McConnell

expression for the B-proton hyperfine coupling, above.

The spectra of the 3,5-deuterated tyrosine model and the YD. radical, and, for

comparison, the spectrum of the 3,5-deuterated RDPR tyrosine radical, are reproduced
in Figure 3.9. Although all spectra show doublet splittings-— less resolved in the YD. case,

it is clear that the doublet splitting and the overall spectral extent increase progressively
in the order YD. ( model tyrosine ( RDPR tyrosine. From these spectra, we infer that the
inequivalence of the two methylene protons is evidently the case in all these systems, and

the differences in B-proton hyperfine coupling between these various tyrosine species can

be explained on the basis of its cos2 0 dependence; that is, one of the two —CH2—

 

99

Figure 3.9: X-band EPR spectra of the Photosystem II YD° tyrosine radical (a), the

model tyrosine radical (b), and the tyrosine radical in RDPR (c), all

deuterated at the ring 3,5 positions.

101
protons has a small dihedral angle and is strongly coupled to the C1 spin density, and the
dihedral angle of the strongly coupled proton is different for tyrosine radicals in different
systems. For the RDPR radical, the doublet splitting in Figure 3.9c is about 20 G (10),

indicating that in this radical, one of the methylene protons makes a dihedral angle of
32° with respect to the pz orbital on C1, while the second proton is situated nearly in the

plane of the ring, i.e., 0 5 90° (10,12). The doublet splitting in the YD° radical species

(Figure 3.9a) is 10.5 G, reflecting a dihedral angle of 52° for the strongly-coupled
methylene proton; the second methylene proton is calculated to have a hyperfine
coupling of about 4 G. The strongly coupled B-proton in the model tyrosine radical has a
dihedral angle of 45°, as calculated from the observed 14 G doublet splitting of the 3,5-
deuterated species (Figure 3%). Figure 3.10 provides a model for the conformation of
the —CH2— group in the model tyrosine radical and in the YD. species. These
conformations, as well as those for other biologically relevant and model tyrosine
radicals, are summarized in Table 3.1. Values of 01, the dihedral angle for the strongly-
coupled methylene proton, were computed from hyperfine coupling values, A1, utilizing
the simplified Heller-McConnell equation, in which the term in B0 is neglected. The
values of 02 were then deduced from 01 on the basis of the tetrahedral geometry of the
sp3-hybridized B—carbon (i.e., 01 + 92 = 120°), and from these the values of A2 were
computed. In all cases, we employed what we believe are the most reliable values of

B2 = 58 G and p1r = 0.49, which were determined in a rigorous manner for the tyrosine

radical in RDPR (10). We were thus able to predict accurate values of 01 and 02 for the

tyrosine radical in solution, where the two methylene protons are equivalent, as reported
by Sealy and co-workers (13). The conformation predicted for the RDPR tyrosine radical
from the magnetic resonance data (10) has recently been confirmed by the results of an
X-ray crystallographic study of the protein (55). The case of tyrosine radicals in single

crystals provides an estimate of the uncertainty in the results of our procedure for

102

Table 3.1: Isotropic hyperfine couplings and corresponding dihedral angles for the

methylene protons in different tyrosine radical species.

 

 

Species Conditions A1 (G) 91 A2 (G) 92 Reference
Tyr° model frozen aqueous 14 45° 2 75° This Work
solution; 120 K
Tyr° model single crystal; R.T. 14 45° 2 75° 42
Tyr° model aqueous solution; 7.5 60° 7.5 60° 13
333 K
YD'/ YZ' whole cells of cyano- 10.5 52° 4 68° 7
bacteria; R. T.
RDPR isolated enzyme; 10 K 20 32° — 88° 10
PGHS isolated enzyme; R. T. 19 35° — 85° 59
PGHS isolated enzyme; R. T. 14 45° 2 75° 59

+ indomethacin

103

(a)

(b)

 

Figure 3.10: A model for the methylene group conformation in the model tyrosine radical

and the Photosystem II YD° radical: a) side view; b) end view, looking along
the C3—C1 bond.

104
calculating 01 and 02. In this case, our values deviate by ~ 8° from the crystallographic
results (56,57). Although this deviation could merely reflect a slight perturbation of the
structure of the radical relative to the unionized molecule, for which the crystal structure
was determined, other factors probably contribute to this inaccuracy, e.g., small variations

of the spin density at C1 or our neglecting of the B0 term and the dipolar contribution to

the hyperfine coupling.

The observation of the well resolved doublet of the 3,5-deuterated model tyrosine
radical is of particular interest and merits further discussion. First, barriers to rotation
about Csp3—Csp2 bonds are generally on the order of 1 - 3 k cal/mol (13), and Shimoda
et al. (58) have estimated a barrier of 1.1 k cal/mol for the rotation of the ethyl group in
2,6-di-t-butyl-4-ethyl phenol radical. We expect a barrier to the rotation of the tyrosine
radical side chain of a similar magnitude. Thus, under our experimental conditions, the
tyrosine side chain is frozen (kT E 0.24 k cal/mol at 120 K). One might expect that the
B-CH2 group would freeze at random orientations, in which case a broad, unresolved
EPR spectrum would be observed. Our results contradict this expectation, as the well-
resolved, 14 G doublet in Figure 3.9b indicates that there is considerable homogeneity in
the methylene conformation with respect to the ring. We calculate that in this
conformation the strongly coupled proton makes a 45° angle with respect to the ring

(Figure 3.10 and Table 3.1), which suggests that a minimum in the potential surface that

describes rotation about the Ca—CB bond occurs near this angle. Second, this conclusion

is reinforced by the analysis of tyrosine radicals in single crystals, where only one of the
two B-methylene protons is strongly coupled and gives rise to a coupling (14 G) that is
essentially the same as that observed for the powder species. Thus, the powder
conformation is similar to that which occurs in the single crystal, again indicating that
this geometry is a low energy structure. Third, the tyrosine radicals that occur as

enzymatically active moieties in proteins have conformations about the methylene group

105
that deviate from the low energy structure observed for the tyrosine models (Table 3.1).
The YD° radical in PS H has a conformation that is relatively close to the low energy
structure, whereas the radicals in prostaglandin H synthase and in ribonucleotide-
diphosphate reductase deviate more substantially, suggesting a higher energy

conformation.

An indication that the conformation about the methylene group may be important
for enzymatic catalysis comes from a recent PGHS work by Palmer et al. (59). The EPR
spectrum of the native enzyme, which retains high cyclooxygenase activity (Chapter 1),
shows a 35 G-wide spectrum with a doublet splitting of 19 G, from which the geometry
in Table 3.1 was calculated. Reaction of the enzyme with indomethacin or with
tetranitromethane inhibits cyclooxygenase catalytic activity, although a tyrosine radical
may still be generated. The EPR spectrum, however, is significantly altered relative to
the native enzyme. The linewidth has decreased to 26 G and the overall spectrum is
quite similar to our model tyrosine radical spectrum (Figure 3.4a).This observation
indicates that addition of the inhibitor allows the tyrosine radical to relax to a lower

energy conformation and may be the basis for the inhibition observed.

Our assignment of the hyperfine couplings in the model tyrosine radical are further
supported by the simulation of the EPR spectra of the 3,5-deuterated and the methylene-
deuterated models (Figure 3.11). In the simulation of the 3,5—deuterated radical (Figure
3.11a), a 14 G isotropic coupling to one methylene proton (corresponding to a dihedral
angle of 45°), was assumed. The simulation of the spectrum of the methylene-deuterated
compound (Figure 3.11b), involves two a-protons, with isotropic coupling of 6.7 G, and
50 % anisotropy (39, 41). The g-values were determined for the tyrosine radical in PS H

(14). The simulation parameters are listed in Table 3.2.

106

Figure 3.11: Computer simulations of the EPR spectra of a) 3,5-deuterated, and
b) methylene-deuterated model tyrosine radicals, with the parameters

listed in Table 3.2.

107

 

 

 

 

 

l l l l l

I I I
3410 3430 3450

Magnetic Field (G)

I I I
3350 3370 3390

108

Table 3.2: Parameters used to simulate the EPR spectra of the 3,5-deuterated and the

methylene-deuterated model tyrosine radicals.

 

 

Radical gxx, gyy, gzz l.w. (G) Axx, A”. Azz (G) Proton(s)
3,5-deuterated Y° 2.0074, 2.0044, 4.0 14.0, 14.0, 14.0 Ho
2.0023
B,B—deuterated Y° 2.0074, 2.0044, 4.0 11.4, 3.8, 7.6 H33

2.0023

 

 

109
In conclusion, we have shown that the local protein environment plays a key role in

maintaining the conformation of the tyrosine side chain. This is well demonstrated by the
situation in Photosystem H. Two distinct tyrosine radicals occur. The first, YD°, gives

rise to a stable EPR signal; the second, Yz°, is only observed transiently and is involved
in the electron transfer events that link reaction center photochemistry to water oxidation
(1, 4, 5). Despite the difference in function, the EPR spectra of the YD° and Yz° radicals
are identical. Thus, the conformation of the side chain must be identical, which implies a
similar local protein environment. The crystal structure of RDPR (55) shows that in this
protein, the amino acids in contact with the tyrosine radical are contributed from several
neighboring helices. Presumably, these steric interactions are responsible for fixing the
conformation of the radical. Using this as our model, we conclude that the three-
dirnensional protein structure in the vicinity of the YD° and YZ° must be very similar.
This structure most probably involves interactions with amino acids that are contiguous
in sequence with the redox active tyrosines and with amino acids in neighboring helices.
YZ° and YD° occur on the third membrane spanning helices of the PS 11 reaction center
polypeptides, D1 and D2, respectively, and the amino acid sequence contiguous to the
two tyrosine radicals is conserved between the two polypeptides (1).This sequence
similarity, in addition to conserved three-dimensional structure, provides a rationale for

the identical conformations for the two radicals.

110

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6. Styling, S. and Rutherford, A. W. (1987); Biochemistry 26, 2401.

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37. Kennedy, M. C.; Werst, M.; Telser, J.; Emptage, M. H.; Beinert, H.; and Hoffman,
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115
CHAPTER 4

EPR AND ENDOR CHARACTERIZATION OF FROZEN PHENOL
ANALOGUES: A MODEL FOR THE DERIVATIZED TYROSINE
RADICAL IN GALACTOSE OXIDASE

4.1 INTRODUCTION

In Chapter 1, it has been noted that galactose oxidase (G0), a mononuclear copper
enzyme, can exist in three distinct redox forms (1). X-ray absorption spectroscopy was
used to show that the copper exists as Cu (H) in the active, fully oxidized form of the
enzyme (2). The fact that no Cu (II) EPR signal is detectable in this form suggested that
formation of the latter involves oxidation of an amino acid residue in the protein, and that
the copper is spin-coupled to the enzyme radical, giving rise to the EPR-silent state (1,2).
A tyrosine residue has been identified in the radical site of the enzyme by resonance
Raman spectroscopy (3), and an EPR study on the copper-free form of the enzyme
resulted in the proposal that the radical is derived from a tyrosine residue (4). The X-ray

structure of galactose oxidase showed that among the ligands to the copper are two
tyrosines, tyr272 and tyr495 (5). Of these, tyr272 was found to be covalently-bound at the

ortho-position to the sulfur atom of a cysteine residue, CYS228. The sulfur atom involved
in this unusual tyrosine-cysteine thioether linkage lies nearly in the plane of the tyr272
aromatic ring, suggesting partial double bond character for the Y272— 8 bond.
Furthermore, located directly above the sulfur atom, at a distance of 3.84 A, is the phenyl
ring of a tryptophan residue, trp290, while the tryptophan indole ring is stacked on the

tyr272 phenol ring. These structural observations suggested to the authors of (5) that the

 

116

galactose oxidase tyrosine radical is likely to be associated with tyr272, with the w-system

probably extended, through the sulfur, to the stacking tryptophan.

Our interest in the structure-function relationship of tyrosine radicals in biological
systems prompted us to extend our investigatiOn to the radical in galactose oxidase.
Although Whittaker and Whittaker (4) have demonstrated that the radical derives from a
tyrosine residue, the identification of two tyrosine residues in the copper ligation sphere
leaves the definitive location of the radical an open question. It is also of particular
interest to note that the spectral characteristics of the galactose oxidase radical are
distinct from those of the tyrosine radical in RDPR (6). In order to pinpoint the origin of
the galactose oxidase radical, i.e., to determine whether it is the cysteinyl-substituted
tyr272 or the unsubstituted tyr495, we have carried out a set of EPR and ENDOR
experiments on phenol analogues, with and without ortho-thioalkyl substitution. These
experiments were designed specifically to test the effect that a thioether substitution
would impose on the spin density distribution and EPR characteristics of a tyrosine
radical. The model compound study, in conjunction with an ENDOR study of the
apogalactose oxidase radical (7), lead to the conclusion that the radical is localized
mainly on the ring of the thioether-substituted tyr272. The ortho-cysteinyl thioether
linkage does not severely perturb the spin density distribution pattern in the apoenzyme
tyrosine radical, and we can apply the same treatment that we applied for unsubstituted
odd-altemant tyrosine radicals (Chapter 3) to deduce the side chain geometry of the

apogalactose oxidase tyrosine-derived radical.

4.2 MATERIALS AND METHODS

Materials. Phenol, 4-methyl phenol, 2-methylmercaptophenol, and 2,6-di-t-butyl-4-
methyl phenol were from Aldrich and were used as supplied (in our discussion of the
phenol model compounds throughout this chapter, we use the regular numbering
convention in which the phenolic group is bonded to carbon 1 of the aromatic ring). 2-
Methylmercapto-4-methyl phenol was a generous gift from Dr. J. Whittaker (Carnegie-
Mellon University).

 

117
Phenol Radical Generation. Solutions for EPR measurements were prepared by
dissolving the respective compound (10 mM) in an aqueous solution of potassium
hydroxide (0.1 M). The radicals were generated by UV illumination for 2 min at 77 K
(8), and the samples were kept in liquid nitrogen until used. No qualitative change in the
EPR spectra were observed upon varying the illumination time (1 min - 10 min).
Identical EPR spectra were obtained for the phenol samples prepared as described above
and for samples prepared in benzene or chloroform. We also checked the effect of salt
concentration on the EPR spectra of phenol radicals that were generated in aqueous
samples. Figure 4.1 represents EPR spectra of the radical derived from 2-methyl-
mercapto-4-methyl phenol in chloroform (a), 1 mM aqueous potassium hydroxide (b),
0.1 M aqueous potassium hydroxide (c), 0.1 M aqueous potassium hydroxide containing
4 M potassium chloride ((1) or 12 M lithium chloride (e). The similarity of the spectra in
Figure 4.1a-e indicates that the identity of the radical and its spin density distribution are
not altered by the base concentration that we routinely use in our experiments (0.1 M) or
by the presence of high concentrations of potassium chloride or the glass-forming salt,
lithium chloride. The structure of the diamagnetic phenol precursors was also established
by NMR spectroscopy on samples prepared in chloroform and in aqueous solutions with
different base concentrations. Typical NMR spectra are shown in Figure 4.2 for 2-
methylmercapto-4—methyl phenol in CDC13 (a) and in 0.1 M NaOD/DZO (b). The NMR
spectra confirm that no chemical change was caused by base concentrations in the range
used for the EPR and ENDOR samples. Only at high base concentrations (2 10M) and
under aerobic conditions did the EPR spectrum of the 2-methylmercapto-4-methyl
phenol radical change (Figure 4.11), and under these conditions it resembled that of the
4-methyl phenol radical, particularly on the low-field side of the spectrum. This probably
indicates a substitution of the thiomethyl group by a hydrogen. This effect was not

observed for any of the other phenol model compounds we used. The EPR spectrum of

 

 

118

Figure 4.1: X-band EPR spectra of the radical derived from 2-methylmercapto-4—methyl
phenol by UV illumination in a) chloroform; b) 1 mM KOH;
c) 0.1 M KOH; d) 0.1 M KOH + 4 M KCL; e) 0.1 M KOH +12 M LiCl; and
f) 10 M KOH. Temperature: 120 K; modulation amplitude: 0.5 G;

microwave power: 0.5 mW.

119

 

 

l l l

I I ' I I I
3390 3410 3430 3450

Magnetic Field (G)

f I
3350 3370

 

120

 

1 fl 1 l l

I I I I
3390 3410 3430 3450

Magnetic Field (G)

I I
3350 3370

121

Figure 4.2: 300 MHz NMR spectra of 2-methyl mercapto-4-methylphenol
in CDC13 (a) and in 0.1 M NaOD/DZO (b).

122

 

 

 

 

123

 

 

lbll £59.. 7”

r-

 

 

 

I I I1 I

‘

 

124
the 4-methyl phenol radical,for example, remained unchanged even when high base
concentrations, up to saturated potassium hydroxide, were used. With the change we
observe in the 2-methylmercapto-4-methyl phenol case, we ruled out the use of
concentrated base solutions to form the model radicals in glassy matrices. Instead, we
used concentrated lithium chloride as a glassing agent, since the latter did not induce
chemical changes in any of our compounds, as we noted above. Forming glasses was
crucial for the success of our model compound ENDOR experiments to achieve higher
radical concentrations upon UV irradiation. A glassy matrix also provides favorable
relaxation properties for ENDOR detection, as the radicals retain some mobility in a

glass, but not in a more rigid polycrystalline matrix.

EPR and ENDOR Measurements. EPR and ENDOR spectra were recorded on a Bruker
ER 200D spectrometer equipped with a Bruker ER 250 ENDOR accessory, with 100 kHz
and 12.5 kHz field modulation used for the EPR and the ENDOR experiments,
respectively. A TE102 cavity was used for recording the EPR spectra. For the ENDOR
measurements, RF irradiation was generated by a Wavetek 3000-446 frequency
synthesizer, and supplied through an ENI 3100L amplifier to a Bruker ER250 ENB
ENDOR cavity with coils that we constructed as described in Reference 6. A Teflon rod,
having the exact outer diameter as the quartz Dewar insert, and supported by a coaxial
steel rod, was chilled in a freezer, and two brass cylinders were positioned on the rod so
that their spacing matches the dimensions of the ENDOR cavity. Silver wire (0.018"
diameter) was then tightly wrapped on the Teflon rod and soldered to the brass cylinders.
After adjusting the spacing of the coil turns, a section of Teflon heat-shrink tubing was
fitted over the coil and the ends of the brass cylinders, and a heat gun was used to evenly
shrink the tubing while the coil— still fitted on the Teflon rod -—was turning on a lathe.
The silver wire is thus embedded into the Teflon tubing, and the turns of the coil are held

at regular distances. The coil was then pulled off the Teflon rod by cooling in liquid

125
nitrogen. With the coil mounted into the cavity, the RF circuit was run through the coil
via spring-loaded BNC connectors, in contact with the brass collets at the ends of the
coil, and terminated into a 50 0 load. Sample temperature and measurement of
microwave frequencies and magnetic field strengths were performed as described in
Chapter 3. EPR spectral simulations were done by using the program described
elsewhere (9), that was implemented on a microcomputer by Dr. C. W. Hoganson in our

laboratory.
4.3 RESULTS

As we noted above, the crystal structure of galactose oxidase indicated partial
double bond character of the C—S bond that bridges cysteine 228 to the ortho-position

of the tyr272 phenol ring (5). The Ca—S—CHZ bond angle was found to be 105.1 °,
which is decreased from the regular tetrahedral angle (109.47°) by repulsion of the two
lone pairs in the sulfur sp3 orbitals. The lowest energy conformation of Ar—S—R
molecules is the planar one (10a,b), where conjugation of the sulfur lone pair to the
aromatic ring provides for partial Ca—S double bond. The conjugation of the sulfur lone
pair in this class of compounds, however, is less than that of the oxygen lone pair in
phenolic compounds, which accounts for the lower barrier to internal rotation in
benzenethiol relative to phenol (10c). The nearly planar structure of the tyr272—S—R, as
shown by the crystal structure, is close to the low energy conformation, and therefore the
suggestion of a partial C04 double bond by Knowles et al. (5) is reasonable. In the
apoenzyme, however, the partial Ca—S double bond character will exist only if the
radical retains the same geometry around this bond as found in the native enzyme. The
partial double bond character of the Ca—S bond might be expected to perturb the spin
density distribution significantly, and, if a substantial spin density is located at the sulfur,

the g-tensor will be altered accordingly (see below).

126

In order to probe the effect of an ortho-cysteinyl linkage on the electronic structure
and the EPR pr0perties of the tyrosine radical in galactose oxidase, we generated a series
of phenol radicals in vitro, with and without thioether substitution. The thioalkyl-
substituted model compounds are expected to duplicate the planar arrangement of the
—SR side chain with respect to the phenol ring (10a,b) that was observed in the crystal
structure of the enzyme (5). The X-band EPR spectra of these radicals at 120 K are
shown in Figures 4.3 and 4.4. The powder spectrum of the phenol radical (Figure 4.3a)
shows a partially resolved five-line structure. This is consistent with the expected
unpaired spin density distribution for this radical— namely an odd-altemant spin
distribution pattern, with large spin density at the para-position and smaller spin densities
at the ortho-positions. Previous results by other authors also show that proton hyperfine
couplings measured in phenol and substituted phenol radicals are in agreement with the
odd-altemant spin distribution pattern (Table 4.1). Thus, the features produced by the
coupling to the para-proton are further split by the two ortho-protons, to give rise to the
spectrum in Figure 4.3a. The ten-line spectrum of the 4-methyl phenol radical (Figure
4.4a) is produced by a large coupling to the para-methyl protons and a smaller, but
appreciable, coupling to the two ortho-protons. In solution, these couplings are expected
to give rise to a twelve-line spectrum; in the powder spectrum in Figure 4.4a, however,
anisotropy in both the g- and the A-tensors produce line broadening and spectral overlap,
so that only ten lines are clearly discernible. The coupling to the ring meta-protons is
small for both phenol radicals, and these two nuclei contribute only to the line
broadening. Substitution of a methylrnercapto group at the ortho-position (Figure 4.3b
and 4.4c) eliminates the moderately strong coupling to the proton in this position, and
some of the features resolved in Figure 4.3a and 4.4a collapse. Accordingly, the EPR
spectral widths of the sulfur-substituted compounds are smaller than those of the

corresponding underivatized radicals. The g-value measured at the zero-crossing for the

127

Figure 4.3: X-band EPR spectra of the radicals derived from phenol (a) and 2-
methylrnercapto phenol (b). Temperature: 120 K; modulation amplitude: 0.5

G; microwave power: 0.5 mW.

128

EPR
T=120K

 

 

 

l l l l

I I I I I
3380 3400 3420

Magnetic Field to)

I I
3360 3440

 

129

Figure 4.4: Experimental and simulated EPR spectra of 4-methyl phenol (a,b) and 2-
methylmercapto-4-methyl phenol (c,d). Experimental conditions are the same

as in Figure 4.3. The simulation parameters are listed in Table 4.2.

130

' g=2.0060

 

 

 

 

 

 

 

 

 

EPR

T=120K
a II II EXP.

0I
b SIM. CH3
c EXP; O
‘SCI‘U
0 SIM. CH3
r I I I ' I ' I f I I l

3360 3380 3400 3420 3440 3460

Magnetic Field (G)

131

Table 4.1: Proton hyperfine coupling constants (Gauss) in 2,6-R1-4-R11-phenol radicals.

 

 

R1, RII a4“ 32,6 a” Ref.
H, H 10.1 6.9 1.9 11
H, H 10.2 6.6 1.8 12
H, H 10.1 6.65 1.8 13
H, H 10.2 6.65 1.85 14
H, H'1 10.7 5.3 0.8 14
H, H 10.22 6.61 1.85 15
H, H) 10.03 6.69 1.98 16
H, Herb 10.69 5.16 1.2 16
H, H 10.0 6.5 1.8 17
Me, H 10.13 7.22c 1.99 18
Me, H 9.5 6.5c 1.65 13
Et, H 9.35 5.7c 1.65 13
Fri, H 9.5 3.7c 1.8 13
But, H 9.62 — 1.95 18
Bu’, H 9.6 — 2.0 13
H, Me 12.4 — -— This work‘
H, Me 12.3 6.1 1.4 11
H, Me 11.95 6.0 1.45 13
H, Me 12.7 6.1 1.4 14
H, Mea 15.1 4.5 0.05 14

 

Table 4.1 (cont'd.):

132

 

 

RI, RH 34* 32,6 a3,5 Ref.
H, Me 11.86 5.93 1.47 18
Me, Me 11.95 6.0C 1.4 13
Me, Me 12.5 6.1c 1.4 12
Bu’, Me 11.2 — —— This work"
But, Me 11.2 — 1.68 19
But, Me 11.22 — 1.63 20
Bu‘, Mea 14.19 — 0.89 21
Bu’, Me 107 — 1.8 22
But, Me 11.11 — 1.78 23
But, Me 11.0 — 1.65 24

 

 

“ Values of A4 are for the p-proton or the li-proton(s) in the p-alkyl substituent.

° See Figures 4.5 and 4.6.

‘ Values reported are for the cation radical, ¢OH + '.

5 Estimated using SCF calculations.

’3 Values reported are for the B-proton(s) in the ortho-alkyl groups.

 

133

spectra shown in Figures 4.3 and 4.4 are 2.0054 (phenol), 2.0057 (2-methylmercapto
phenol), 2.0060 (4-methyl phenol), and 2.0059 (2-methylmercapto-4-methyl phenol).
Qualitatively, the small shift in g-value in the thioether derivatives relative to the
unsubstituted compounds indicates that the sulfur substituent does not severely perturb

the spin density distribution of the radical.

To test the effect of thioether substitution on the spin density distribution of
phenol- and tyrosine-like radicals quantitatively, we used EN DOR spectroscopy to
measure the methyl proton splittings in the 4-methyl phenol and the 2-methylmercapto-
4-methy1 phenol radicals. Figure 4.5 represents the ENDOR data for these radicals at

115 K. Both radicals exhibit axial line shapes, typical of B-proton coupling (25). The
spectrum of 4-methyl phenol (Figure 4.5a) shows transitions at 31.5 MHz (A J_) and

33.2 MHz (Al I) that we assign to the 4-methyl protons. This is a case where A/2 is

larger than the proton Larrnor frequency (14.7 MHz), and the transitions observed in

Figure 4.5a provide hyperfine coupling values for the methyl protons in 4-methyl phenol
as follows: Al I = (33.2 - 14.7) x 2 = 37.0 MHz, A J. = (31.5 - 14.7) x 2 = 33.6 MHz,

and Aiso = (A1 I 4» 2A J. )/3 = 35.0 lVH-Iz = 12.4 G. The isotropic hyperfine coupling of

the methyl protons determined here by ENDOR for the radical in a frozen matrix is in
good agreement with the result obtained for the same neutral radical in solution (Table
4.1). This indicates that the radical we generated is neutral, as expected from our
radical generation technique; cation radicals show significantly higher hyperfine
coupling to the para-methyl protons (Table 4.1). Furthermore, this agreement shows
that the spin density distribution and hyperfine coupling are not significantly perturbed

by immobilization.

For the 2-methylmercapto-4-methyl phenol radical, an axial ENDOR line is
observed (Figure 4.5b), with transitions corresponding to A .L and Al I at 27.1 MHz and

28.3 MHz, respectively. From these values, we obtain A .L = 25.2, Al I = 27.6, and

 

134

Figure 4.5: ENDOR spectra of the radicals of 4-methyl phenol (a) and 2-methy1—
mercapto-4-methyl phenol (b), showing the 4-methyl proton axial features.
Temperature: 120 K; microwave power: 10 mW; RF power:

40 W; RF modulation: 150 kHz.

135

 

 

29

 

 

24

. j T T . . r
25 26 27 28 29 30 31

Frequency (MHz)

136
Aiso = 26.0 MHz = 9.3 G. We were unable to detect the coupling to the ortho-protons by
ENDOR for either model phenol compound, despite the fact that the spectral simulations
below indicate substantial coupling to these protons. This is not unusual, as a-proton
ENDOR resonances are more difficult to detect, Owing to their large hyperfine

anisotropy, which leads to substantial broadening of their ENDOR lines (25).

The data in Figure 4.5 show that the hyperfine coupling to the 4-methyl protons
decreases by ~ 25 % when one of the ortho-protons is substituted by a thioether linkage.
The interpretation of this observation is contingent upon the status of the para-methyl
group, whether freely rotating or immobilized. If the methyl group were immobilized,
the three methyl protons would be inequivalent. On the basis of the Heller-McConnell
relationship (26), the observed difference of the methyl proton hyperfine coupling would
then be attributed to different dihedral angles of the more strongly coupled methyl proton
in each compound, and/or a difference in the spin density at the para-position, to which
the methyl group is bound. On the other hand, for a freely rotating methyl group, the sole
factor that can account for the change in the methyl proton hyperfine coupling is a
change in the spin density at the para-position. That the para-methyl group in our phenol
models is freely rotating under our experimental conditions is indicated by several
observations. First, this fact is clearly demonstrated by the EPR spectrum of 2,6-di-
t.butyl-4-methyl phenol, recorded under the same conditions (Figure 4.6). The four-line
spectrum in Figure 4.6 results from coupling to the 4-methyl protons; coupling to the
meta-protons and to the ortho-t.butyl protons is too small to resolve, and contributes only
to the line broadening. The observed relative intensities of 1 : 3 : 3 : 1 indicates that the
para-methyl group is freely rotating, rendering the three methyl protons equivalent.
Shimoda et al. (20) studied the temperature dependence of the B-proton hyperfine
coupling in 2,6-di-t.butyl-4-a1kyl phenol radicals, and reported a value of 1.1 k cal/mol
for the rotational barrier of the 4-ethy1 group, and 1.6 k cal/mol for both the 4-isopropyl

137

 

I l I H l

I I I
3410 3430 3450

Magnetic Field (G)

I I I
3350 3370 3390

Figure 4.6: X-band EPR spectrum of the radical derived from 2,6-di-t.butyl-
4-methyl phenol. Temperature: 120 K; modulation amplitude: 0.5 G;

microwave power: 0.5 mW.

 

138
and the 4-cyclohexy1 groups. For the 4-methyl substituted compound, however, no
detectable change of the methyl proton hyperfine coupling was observed, and therefore
the methyl group was assumed to be freely rotating over the entire temperature range
(-45° C to +20° C) investigated (20). Furthermore, the methyl proton hyperfine
splitting that we measure from Figure 4.6 is 11.2 G. This is in excellent agreement with
the value reported by Shimoda et al. (20) for the same radical in solution (11.22 G).
This agreement, along with the implication of the relative line intensities we noted
above, show that under our experimental conditions, the para-methyl group is rotating
in the 4-methyl phenol and the 2-methylmercapto-4-methyl phenol radicals (Figure 4.5),
and that the ortho-thioalkyl substitution did not affect the rotation status of the para-
methyl group in the latter compound. Secondly, we recorded the EPR spectrum of the
4-methyl phenol radical at 10 K, and the same hyperfine structure shown in Figure 4.4a

was observed, indicating free rotation of the methyl group even at this temperature.

From Figure 4.5, we observe a 25 % decrease in the para-methyl proton hyperfine
coupling upon ortho-thioether substitution. If we assume that the sulfur substitution does
not induce a drastic change in the BZ-factor in the Heller-McConnell equation, we
interpret this result to indicate a corresponding reduction of the spin density at the ring
4-position. Using the ENDOR data for the methyl proton coupling, and hyperfine
coupling tensor components for the ortho-protons consistent with their a- proton character
and with their isotropic coupling constants reported in the literature (Table 4.1), we were
able to obtain good simulations of the EPR spectra of 4-methyl phenol and its thioether
derivative. These simulations are shown in Figure 4.4b and 4.4d, respectively, and the

simulation parameters are listed in Table 4.2.

 

139

Table 4.2: Parameters used in the simulation of the EPR spectra of 4—methyl phenol and

2—methylmercapto-4-methyl phenol radicals.

 

 

Radical gxx’ gyyr gm l.w. (G) Axx’ Ayy» An(G) Proton(s)

4-methyl phenol 2.006, 2.005, 2.0023 1.6 132,120,120 P-CHs
9.75, 3.25, 6.5 ths)
2.55, 0.85, 1.7 H36)

2-n1ethylrnercapto 2.0095, 2.0067, 2.8 9.86, 9.0, 9.0 P-CH3

2.0023

-4-methyl phenol
9.75, 3.25, 6.5 H6
255,085, 1.7 H3
3.0, 1.0, 2.0 H5

 

140
4.4 DISCUSSION

The free radical that has been detected in galactose oxidase (4,7) shows unique EPR
spectral characteristics. Although Whittaker and Whittaker's observation of alteration of
the radical's EPR signal upon incorporation of methylene-deuterated tyrosine in their
cultures suggested that the galactose oxidase radical is derived from a tyrosine (7), the
GO radical signal is different from those observed in other naturally-occurring tyrosine
radicals. In RDPR, for example, the tyrosine radical EPR signal is ~ 40 G wide, and is

dominated by a large doublet splitting (6). The signal observed in galactose oxidase,

 

however, has a triplet structure, and the overall line width is ~ 33 G (7). The results of
the X-ray crystal structure study of galactose oxidase suggested the possibility of the
radical spin density being delocalized over the tyrosine 272 ring, the cysteine 228 sulfur
atom, and the tryptophan 290 phenyl ring, which is located directly above the cys223
sulfur (5). If that were the case, then one would expect the spin-orbit coupling of the
sulfur to cause a substantial shift of the GO radical g-value from the free electron g—value
(2.0023). The zero-crossing g-value of the galactose oxidase radical, however, is only
2.0055 (7), which is only slightly different from that of the tyrosine radicals in RDPR (6)
and in PS 11 (8). One can argue, then, that the radical observed in galactose oxidase is the
cysteinyl-substituted tyrosine 272, with the spin density confined mainly to the tyrosine

ring, hence the small effect of the sulfur spin-orbit coupling on the g-tensor. The

occurrence of a second tyrosine residue, Y495, in the copper coordination sphere, and the

suggestion, based on the X-ray crystallographic data, that the Y272—S bond acquires

partial double bond character (5) indicate that further investigation was necessary to
clarify the situation regarding the origin of the galactose oxidase radical. An in vivo
ENDOR investigation of the apoenzyme radical, performed by P. O. Sandusky in our

laboratory and reported elsewhere (7), showed axial features characteristic of B-proton

coupling, from which a value of Aiso = 14.6 G was assigned to a tyrosine methylene

141
proton. The ENDOR data also provided evidence for a hydrogen-bonded proton, with
hyperfine tensor components of AZ = + 3.1 MHz, and Ax = Ay = - 1.4 MHz. In addition,
some moderately-strongly coupled features were observed and attributed to a-proton
coupling, but a full assignment of the a-proton hyperfine coupling tensor was not
achieved since its largest component, being masked by the intense B-proton features, was
not detected (7). The only way to simulate the EPR spectrum of the galactose oxidase
radical, using the ENDOR parameters for the (Ii-proton coupling, was to include one
B-proton and one anisotropically coupled a-proton; all attempts to simulate the EPR
spectrum while assuming an underivatized tyrosine were unsuccessful (7). Thus, based
on the ENDOR and the EPR simulation results on the enzyme radical, tyrosine 495 was
ruled out as the origin of the GO radical. The question remained, however, as to the spin
density distribution in the cysteinyl-substituted tyrosine 272 radical, its geometry, and
how to rationalize the unique EPR properties of the radical on the basis of its geometrical
and electronic structure. The model compound EPR/ENDOR results we present here

provide answers to these issues.

The analysis of the galactose oxidase EPR spectrum and its simulation parameters
indicate that the ortho-cysteinyl tyr272 radical retains an odd-altemant spin distribution

about the tyrosine ring, which causes strong hyperfine coupling to one B-methylene
proton and to the ring proton bound to C5 (see Figure 3.3 for the tyrosine numbering
convention used here). The presence of heavy elements— such as chlorine, bromine, or
sulfur— in organic radicals induces a substantial deviation of the g-tensor components
away from the free electron g-value (27). An example of this effect is the cysteine
radical, which has principal g-tensor components of 2.003, 2.029, and 2.052, and 8180 of
2.028 (28). In odd-altemant radicals, however, the effect of heavy atom substitution on
the g-tensor appears to be much less pronounced. The isotropic g-values of the 2-

chlorophenol and the 4-methylmercapto benzyl radicals, for example, are 2.0054 and

142
2.0049, respectively (12,29). Thus, the zero-crossing g—value of the galactose oxidase
Y272 radical, 2.0055, is typical for this class of compounds. This g—value is substantially
lower than that of the cysteine radical, suggesting that the radical spin density is localized
mainly on the tyrosine ring, with very small spin density on the ortho-cysteinyl sulfur, as
can be expected from the relatively low degree of conjugation of the sulfur lone pair to
the aromatic ring (10c). This argument is supported by the closeness of the g-value of the
GO radical to those observed in other systems containing unsubstituted tyrosine radicals
(6,8), and by our observation that the g-values of the methylmercapto-substituted model

compounds are close to the corresponding parent phenols (Figures 4.3 and 4.4).

It has been shown that ortho-substituted phenol radicals retain the odd-altemant
spin distribution pattern, with large spin densities at positions 4 and 6, and small densities
at positions 3 and 5, even when the substituent is a strong rr-electron donor such as a
methoxy group (30). Furthermore, it has been shown that thioether substitution does not
severely perturb the spin density distribution of the benzyl radical; the latter and its para-
methylrnercapto derivative have essentially the same proton hyperfine coupling at the
ortho- and the meta-positions (5.1 G and 1.7 G, respectively) (29). The retention of the
odd-altemant spin distribution pattern in ortho—thioalkyl-substituted phenol and tyrosine
radicals is supported by our successful simulations of the EPR spectra of the 4-methyl
phenol radical and its ortho-thiomethyl substituted derivative (Figure 4.4b,d) on the basis
of this assumption. In each case, parameters for the strong hyperfine coupling to three
equivalent para-methyl protons were taken from our ENDOR data (Figure 4.5), and
moderately strong, anisotropic hyperfine coupling to two ortho-protons (Figure 4.4b) or
one ortho-proton (Figure 4.4d) were consistent with typical values reported in the
literature for phenol radicals in solution (Table 4.1) and with the anisotropy usually

exhibited by a-proton hyperfine coupling tensors (25). The g-tensors employed in these

143

simulations reproduce the zero-crossing g-values observed in the EPR spectra
(Figure 4.4), and were oriented such that gxx was along the C—0 bond and gzz was

perpendicular to the ring plane. The A-tensor for the a-protons was such that the smallest
component, A”, was along the Ca—Ha bond, and the largest component, Ann was
perpendicular to it in the aromatic plane (cf. Section 2.3.2). The simulation parameters
(Table 4.2) imply that the isotropic hyperfine coupling to the ortho-protons in the 4- ‘
methyl phenol radical and its thioether derivative is 6.5 G. Using a value of 25 G for the
McConnell Q-factor, this corresponds to a spin density of 0.26 at the ortho-carbons in

both radicals.

Our model compound ENDOR data show that the introduction of an ortho-
methylrnercapto substituent to the ring in 4-methyl phenol reduces the hyperfine
coupling to the 4-methyl protons by 25 %. With the establishment of the free rotation of
the methyl group, we conclude that the spin density at C4 in the 2-methylmercapto-4-
methyl phenol radical is 25 % lower than the corresponding spin density in 4-methyl
phenol. In galactose oxidase, since the native enzyme acquires a low-energy, nearly
planar conformation (as shown by the crystal structure, as discussed above), the
extension of the methylrnercapto side chain to include the rest of the cysteine molecule is

not expected to change this effect— i.e., we expect the spin density at C1 in the

galactose oxidase radical (see Figure 3.1) to be approximately 25 % smaller than that in
an unsubstituted tyrosine radical. A spin density of 0.49 occurs at C1 of the RDPR radical

(6); this value is typical of the spin density at this position in phenol-like radicals. Thus,

we predict that the spin density at C1 of the galactose oxidase radical will be ~ 0.37.

In Chapter 3, we used the spin density at C1 and the methylene proton couplings
measured for our model tyrosine radicals, the tyrosine radical in RDPR (6), and the YD°

tyrosine radical in Photosystem II (8), to deduce the geometry of the B-CHZ group

 

144
relative to the tyrosine ring in each species, utilizing the Heller-McConnell relationship.
With the same procedure, and using the scaled spin density at C1 in Y272 in apogalactose
oxidase, we calculate a dihedral angle 0 of 34° for the more strongly coupled B-proton in
this case (0 is the angle between the C1—Cg—H3 plane and the plane formed by the

Cl-pz orbital axis and the C1—CI3 bond). This calculation suggests that rearrangement of

the tyrosine phenol head group occurs upon removal of the copper, as the dihedral angle
reported by Ito et al. (5), from the crystal structure of the metal-containing protein, is
16°. Moreover, simulation of the apoenzyme radical EPR spectrum shows that only one
methylene proton is strongly coupled to the ring spin density (7), indicating that the
second methylene proton is nearly in the plane of the ring, as predicted by our
calculation. In Figure 4.7, we present a model for the conformation of the tyrosine 272
side chain with respect to the plane of the ring in apogalactose oxidase. Comparison of

the crystal structure of the holoenzyme (5) with the model shown in Figure 4.7 suggests
that a rotation of ~ 50° occurs about the C5-C1 bond upon removal of the copper.

Although the magnetic resonance parameters of the galactose oxidase radical are
not strongly perturbed by the thioether substitution on the tyrosine ring, the chemistry of

the radical rrright be more dramatically affected. The redox potential of the apogalactose
oxidase tyr272 is ~ 600 mV (4), which is 250 to 500 mV lower than the redox potentials
of the YD° and YZ° tyrosine radicals in Photosystem II (31). This lower potential is, at

least in part, a consequence of the ortho-cysteinyl linkage. r-Electron donating
substituents have been shown to lower the redox potential of substituted toluenes (32)
and phenols (33). Thus, a thioether substituent, acting as a w-electron donor (29,34), is
expected to lower the redox potential of tyrosine. The w—electron donating ability of
thioether substituents is largest in the planar conformation (10a). Accordingly, since the

sulfur atom of the cysteinyl bridge to tyrosine 272 in G0 is nearly in the plane of the

 

145

 

 

 

Z-axis Z-axis
I
HI! I
\2 I
C : C ‘ D 0
/ :
R I
I

SR

 

Figure 4.7: A model for the o-cysteinyl-substituted tyrosine radical in apogalactose
oxidase; a) side view; b) end view, looking along the C5-C1 bond. The

dihedral angle for the more strongly coupled proton, 01, is 34°.

 

146
tyrosine aromatic ring (the Y272—S bond is only 7° above the ring plane), as indicated

by the X-ray structure (5), it is reasonable to conclude that the tyr272 redox potential is

lowered by the cysteinyl substitution. This will also be the case for the apoenzyme,
provided that the conformation of the thioalkyl group with respect to the tyrosine ring
does not change. The situation is more complicated than that, however, since the

w—stacking interaction that involves tryptophan 290 (5), may play a role in this regard.
It is also likely that the conformation of the B-CH2 group is an important factor in

determining the redox potential of tyr272. The effect of stereo-electronic factors on the

redox potentials of organic compounds has been well demonstrated in the case of a-
tocopherols (35-40), where the redox potential is determined by the conformation of of
the para-OR groups with respect to the phenol ring, as the latter factor dictates the
degree of conjugation of the oxygen lone pair. In Photosystem H, the two redox active
tyrosine radicals, YZ° and YD° , differ in their redox potentials by 250 mV, although the
side chain conformation of both radicals is identical (Chapter 3). Clearly, the factors that
determine the redox potentials of redox active tyrosine species in proteins are not yet well
understood. In galactose oxidase, the tyr272 redox potential observed in the apoenzyme is
expected to be different from that in the native enzyme, as the ligation to the copper, no

doubt, constitutes a significant perturbation.

We conclude that the cysteinyl-substituted tyrosine 272 in galactose oxidase is an
odd-altemant radical species, similar to those found in RDPR, PS H, and prostaglandin

H synthase, at least in the apoenzyme.

147

References
l. Whittaker, M. M. and Whittaker, J. W. (1988); J. Biol. Chem 263, 6074.

2. Clark, K.; Penner-Hahn, J.; Whittaker, M. M.; and Whittaker, J. W. (1989); J. Biol.
Chem 264, 6433.

3. Whittaker, M. M.; DeVito, V. L.; Asher, S. A.; and Whittaker, J. W. (1989); J. Biol.
Chem 264, 7104.

4. Whittaker, M. M. and Whittaker, J. W. (1990); J. Biol. Chem 265, 9610.

5. Ito, N.; Phillips, 8. E. V.; Stevens, C.; Ogel, Z. B.; McPherson, M. J.; Keen, J. N.;
Yadev, K. D. 8.; and Knowles, P. F. (1991); Nature 350, 87.

6. Bender, C. J .; Sahlin, M; Babcock, G.T.; Barry, B. A.; Chandrachekar, T. K.; Salowe,
S.; Stubbe, J.; Lindstrhm, B.; Petersson, L.; Ehrenberg, A.; and Sjbberg, B.- M.
(1989); J. Am Chem Soc. 111, 8076.

7. Babcock, G. T.; El-Deeb, M. K.; Sandusky, P. O.; Whittaker, M. M.; and Whittaker, J.
W. J. Am Chem Soc., submitted.

8. Barry, B. A.; El-Deeb, M. K.; Sandusky, P. O.; and Babcock, G. T. (1990); J. Biol.
Chem 265, 20139.

9. Brok, M.; Babcock, G. T.; DeGroot, A.; and Hoff, A. J. (1986); J. Magn. Reson. 70,
368.

 

 

10.

11.

12.

l3.

14.

15.

16.

17.

18.

19.

20.

21.

22.

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a) Bemardi, F; Mangini, A.; Guerra, M.; and Pedulli, G. F. (1979); J. Phys. Chem
83, 640; and references cited therein; b) Ichikawa, T.; Aoki, K.; and Iitaka, Y.
(1978); Acta Cryst. B34, 2336; c) Parr, W. J. E. and Scaefer, T. (1977); J. Magn.
Res. 25, 171.

Dixon, W. T. and Norman, R. O. C. (1964); J. Chem Soc., 4857.

Dixon, W. T.; Moghimi, M.; and Murphy, D. (1974); J. Chem Soc., Faraday Trans.
II 70, 1713.

Stone, T. J. and Waters, W. A. (1964); J. Chem Soc., 213.
Dixon, W. T. and Murphy, D. (1976); J. Chem Soc., Faraday Trans. II 72, 1221.
Neta, P. and Fessenden, R. W. (1974); J. Phys. Chem 78, 523.

Dixon, W. T.; Kok, P. M.; and Murphy, D. (1977); J. Chem Soc., Faraday Trans. II
73, 709.

Shiga, T. and Imaizumi, K. (1973); Arch. Biochem Biophys. 154, 540.
Weiner, S. A. (1972); J. Am Chem Soc. 94, 581.
Weiner, S. A. and Mahoney, L. R. (1972); J. Am Chem Soc. 94, 5029.

Shimoda, F.; Hanafusa, T.; Nemoto, F.; Mukai, K.; and Ishizu, K. (1979); Bull.
Chem Soc. Japan 52, 3743.

Nemoto, F.; Tsuzuki, N.; Mukai, K.; and Ishizu, K. (1981); J. Phys. Chem 85, 2450.

Becconsall, J. K.; Clough, S.; and Scott, G. (1960); Trans. Faraday Soc. 56, 459;
Proc. Chem Soc. (London) ( 1959), 308.

 

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Bennett, J. E. (1960); Nature 186, 385.

Kevan, L. and Kispert, L. Electron-Nuclear Double Resonance Spectroscopy; Wiley-

Interscience: New York (1976).

a) Heller, C. and McConnell, H. M. (1960); J. ChemPhys. 32, 1535; b) Stone, E. W.
and Maki, A. H. (1962); J. ChemPhys. 37, 1326.

McConnell, H. M. and Robertson, R. E. (1957); J. Phys. Chem 61, 1018.
Box, H. C; Freund, H. G. and Budzinski, E. E. (1966); J. Chem Phys. 45, 809.
Wayner, D. D. M. and Arnold, D. R. (1984); Can. J. Chem 62, 1164.

a) Smith, I. C. P. and Carrington, A (1967); Mol. Phys. 12, 439; b) Stone, E. W. and
Maki, A. H. (1964); J. Am Chem Soc. 87, 454.

Boussac, A. and Etienne, A. L. (1984); Biophys. Biochem Acta 766, 576.

Pryor, W. A. Free Radicals; McGraw-Hill: New York (1966).

Bordwell, F. G. and Cheng, J.-P. (1991); J. Am Chem Soc. 113, 1736.

Tagaki, W., in Organic Chemistry of Sulfur; an, 8., ed.; Plenum Press: NY. (1977).

Burton, G. W.; Le Page, Y.; Gabe, E. J.; and Ingold, K. U. (1980); J. Am Chem Soc.
102, 7791.

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37. Burton, G. W.; Hughes, L.; and Ingold, K. U. (1983); J. Am Chem Soc. 105, 5950.

38. Doba, T.; Burton, G. W.; and Ingold, K. U. (1983); J. Am Chem Soc. 105, 6505.

39. Doba, T.; Burton, G. W.; Ingold, K. U.; and Matsuo, M. (1983); J. Chem. Soc. Chem.

Commun., 461.

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151
CHAPTER 5

CONCLUSIONS AND FUTURE WORK

The present work was aimed at determining the electronic and the geometrical
structure of frozen tyrosine and model phenol radicals, and to provide models for the
naturally occurring, redox active tyrosine radicals in their protein environment. The
technique we employed for generating the radicals, namely UV irradiation of frozen
powders or glasses at 77 K, yields radicals that are stable for several months when the
samples are kept in liquid nitrogen, and thus provides a versatile means for studying this
class of radicals in disordered solids. All previous EPR/ENDOR investigations of
tyrosine radicals were performed on radicals generated in solution by chemical oxidation
of a rapid flow (1), or on radiation—damaged single crystals (2). The model compounds
we investigated provide an appropriate model for the naturally occurring tyrosine

radicals, since the latter are immobilized on the magnetic resonance time scale.

We have demonstrated that the spin density distribution in the model tyrosine
radicals, and in the tyrosine radicals in Photosystem II and in apogalactose oxidase,
follows an odd-altemant pattern, with major spin densities at the phenolic oxygen and at
carbons 1, 3, and 5 of the aromatic ring. This spin density distribution pattern is similar
to that found in the tyrosine radical in RDPR (3). In all cases, the EPR spectral

characteristics are dominated by the large coupling of one of the methylene protons to the

large spin density at C1. Thus, we attribute the spectral differences between the various

tyrosine radical species to different conformations about the C1—C5 bond,which

determines the magnitude of the hyperfine coupling. We also presented models for the
conformation about the C1—C3 bond in the model tyrosine radicals and in the natural

 

152
radicals in Photosystem H and in apogalactose oxidase. This side chain conformation is
likely to be dictated by the protein environment, and, in turn, it controls the degree of
hyperconjugation of the methylene protons to the ring. Combining our results on the
model compound radicals with those on the tyrosine radicals in RDPR and in
Photosystem H, we conclude that the spin density is not altered by the change in the
B-methylene group conformation in the three systems. To further support this conclusion,
a future ab initio calculation of the spin density distribution in tyrosine radicals with

different side chain conformations is needed.

The spin density at the tyrosine phenolic oxygen still needs further investigation, as
our attempts to detect 170 ENDOR transitions were unsuccessful. A modified ENDOR
technique, involving the use of a pumped-helium cryostat to reach temperatures as low as

1.6 K, magnetic field modulation, and rapid scanning of the radiofrequency (several

MHz/s) — which allows averaging several thousand scans in reasonable time —has
been employed for detecting 17O ENDOR transitions in the enzyme aconitase (4).
Although implementing this technique on our ENDOR spectrometer requires major
modifications of the electronics, it may prove useful for detecting the 17O ENDOR
transitions in labeled tyrosine radicals. If successful, this experiment can then be
extended to assess the oxygen spin density in naturally occurring tyrosine radicals. It has
been possible to incorporate deuterium-labeled tyrosine in RDPR (3) and in Photosystem
H of the cyanobacterium Synechocystis (5), and similar techniques can be employed to
incorporate 17O-enriched tyrosine in these two systems. This should also be of interest in
regard to the fact that the Photosystem II tyrosine radical YD° is hydrogen-bonded,
whereas the RDPR radical is not. Thus, the effect of hydrogen bond formation on the
oxygen spin density can be studied. The spin density at the phenolic oxygen is an
important factor in determining the redox potential of the radical, which is key to its

function in the protein redox chemistry.

 

 

153

For the ortho-cysteinyl substituted tyrosine radical in apogalactose oxidase, the
spin density at C1 is 25 % lower than that found in other tyrosine radical systems, as
demonstrated by our results on the thioalkyl—substituted phenol models. In spite of this,
however, we have shown that the radical in the apoenzyme retains the odd-altemant spin
distribution pattern, and that the spin density is mainly confined to the tyrosine ring, with
very little density at the sulfur. The investigation needs to be extended to the native
enzyme, however, as the removal of the copper constitutes a major perturbation to the
radical site. As the radical in the oxidized native enzyme is ligated to the Cu (11) center,
spin coupling of the two species renders the native enzyme diamagnetic. Therefore,
biochemical manipulation, to trap the radical ligated to a diamagnetic metal, is a
prerequisite to any EPR or ENDOR study of the radical site in the native enzyme. One
approach to this problem would be to substitute Zn (dlo, S = 0) for the copper center.
Another way is to oxidize the enzyme in the presence of cyanide, which ligates to Cu (1)
preferentially over Cu (II), thus shifting the equilibrium of the Cu (ID/Cu (I) couple
towards the diamagnetic Cu (I) state, and allowing the detection of the radical signal.
Either of these two approaches should lead to a state of the enzyme that resembles the
native form more closely than the copper-depleted form which has been characterized by

EPR and ENDOR.

154

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5. Barry, B. A.; El-Deeb, M. K.; Sandusky, P. 0.; and Babcock, G. T. (1990); J. Biol.
Chem 265, 20139.

 

 

NICHIGRN STQTE UNIV

"III
31

IIIIIIII IIIIIIII III III III

 

2.'"r..y.
......rt'