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LIBRARY 3 1293 00629 9345
Michigan State
University

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the
dissertation entitled
Characterization of the Vibrational Properties
of Metallochlorins, Chlorophylls and Chlorophyll -

Binding Proteins by Resonance Raman Spectroscopy

presented by

Harold Fonda

has been accepted towards fulfillment
of the requirements for

PHD degree in Chemistry

 

 

 

W47. Aflm/

Major professor

Date M

MS U is an Affirmative Action/Equal Opportunity Institution 0-1277 1

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

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MSU Is An Affirmative Action/Equal Opportunity Institution

 

CHARACTERIZATION OF THE VIBRATIONAL PROPERTIES OF
METALLOCHLORINS, CHLOROPHYLLS AND CHLOROPHYLL-BINDING

PROTEINS BY RESONANCE RAMAN SPECTROSCOPY

BY

Harold Norman Fonda

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1989

@05954

ABSTRACT

CHARACTERIZATION OF THE VIBRATIONAL PROPERTIES OF
METALLOCHLORINS, CHLOROPHYLLS AND CHLOROPHYLL-BINDING
PROTEINS BY RESONANCE RAMAN SPECTROSCOPY
BY

Harold Norman Fonda

Three systems of increasing complexity from metallo-
chlorin model compounds to Chlorophylls to chlorophyll-binding
proteins have been studied. Resonance Raman and IR spectra of
metallo-octaethylchlorins (MOEC) show significant.differences
in the vibrational mode properties of metallochlorins and
metalloporphyrins. The core-size correlation parameters for
Zn, Cu and NiOEC reveal mixing of C.CIII and (3th stretching
character in the high frequency modes. The isotopic frequency
shifts observed for the V3 and um modes of fully and partially
deuterated copper octaethylporphyrin establish that, for a
delocalized vibrational mode, methine d2 deuteration produces
half of the d‘ shift. CuOEC exhibits different patterns of
frequency shifts upon o,fi and 1,6 methine deuteration that
demonstrate the phenomenon of mode localization in metallo-

chlorins.

The vibrational mode characteristics determined for MOEC
provide the basis for the interpretation of the resonance

Raman spectra of metallochlorin t cation radicals and

metal-substituted chlorophyll a. Resonance Raman spectra of
the at cation radicals of CuOEC and selectively methine-
deuterated CuOEC complexes show that unlike the situation in
metalloporphyrins, changes in mode composition occur upon
macrocycle oxidation in metallochlorins. Metal-substituted
chlorophyll a complexes display altered mode compositions
compared to MOEC as a result of addition of the isocyclic
ring. Identification of the core-size sensitive modes in these
complexes provides a reliable method to determine the
coordination state of chlorophyll a in solution and in 2119.
The results obtained for chlorophyll a in solution have been
extended to chlorophyll-binding proteins. Resonance Raman
spectra of the light-harvesting chlorophyll a/b protein
complex (LHC) show four populations of chlorophyll a and two
of chlorophyll b that are distinguished by the stretching
frequencies of their conjugated carbonyl groups. Each of the
Chlorophylls has a S—coordinate Mg atom. The chlorophyll b and
two of the chlorophyll a populations result from H-bonds of
different strength to the carbonyl groups. H-bonding to the
conjugated carbonyls is proposed as a mechanism to account for
the broadened and red-shifted electronic absorption bands of

the Chlorophylls in LHC.

 

To my parents and in memory of my

grandfather, Neil W. Stuart.

iv

ACKNOWLEDGMENTS

I would like to thank Dr. Gerald T. Babcock for his
support and guidance; Dr. George E. Leroi for serving as
Second Reader; Tony Oertling and Asaad Salehi for their
collaboration on the chlorin and at cation radical work; Dwight
Lillie and Matt Espe for maintaining the computer system; and

Bob Kean and Chris Bender for comic relief.

TABLE OF CONTENTS

LIST OF TABLES ix

LIST OF FIGURES Xi

CHAPTER 1 INTRODUCTION
1. The Role of Chlorophyll in Photosynthesis 1
2. The Structure of Metalloporphyrins, Metallochlorins

and Chlorophyll 5

3. The Electronic Absorption Spectra of Metalloporphyrins

and Metallochlorins 9
4. Infrared and Raman Spectroscopy 15
5. Vibrational Mode Assignments for Metalloporphyrins 19

6. The Application of Resonance Raman Spectroscopy to
the Study of Chlorophyll 25

7. Aim and Scope of this Work 26

CHAPTER 2 MATERIALS AND METHODS

1. Materials 27
i. Preparation of Chlorins 27
ii. Preparation of Chlorophyll a and b 28
iii. Preparation of Metal-Substituted Chlorophyll a 29
iv. Isolation of Chlorophyll-Binding Proteins 30

vi

2. Spectroscopic Techniques
i. Electronic Absorption Spectroscopy
ii. Infrared Spectroscopy

iii. Resonance Raman Spectroscopy

CHAPTER 3 VIBRATIONAL PROPERTIES OF METALLOCHLORINS
1. Introduction

2. Results

1. Electronic Absorption Spectra
ii. Resonance Raman Spectra of MOEC
iii. IR Spectra of MOEC

iv. Effects of Methine Deuteration
v. Resonance Raman Spectra of CuECI
vi. Solid State Spectra

3. Discussion

i. C.CIll and Cbe Modes

ii. C,N Modes

iii. CmI-I Modes

iv. Ethyl Group Vibrations

4. Conclusions

CHAPTER 4 VIBRATIONAL PROPERTIES OF METALLOCHLORIN
f CATION RADICALS

1. Introduction

2. Results

3. Discussion

4. Conclusions

31

31

31

31

35

39

39

42

47

51

57

59

62

62

74

76

79

80

82

86

9O

97

CHAPTER 5 VIBRATIONAL PROPERTIES OF METALLOCHLOROPHYLLS

1. Introduction 99
2. Results 103
i. Electronic Absorption Spectra 103
ii. Resonance Raman Spectra 107
3. Discussion 110
4. Conclusions 116

CHAPTER 6 RESONANCE RAMAN SPECTROSCOPY OF

CHLOROPHYLL-BINDING PROTEINS

1. Introduction 118
2. Results 127
i. Electronic Absorption Spectra 127
ii. Resonance Raman Spectra 129
3. Discussion 140

i. Selective Enhancement of Chlorophyll a and

Chlorophyll b 140
ii. Mg Coordination State 144
iii. Carbonyl Stretching Region 147
iv. 28 kDa and Reaction Center Complex Proteins 151
4. Conclusions 153
CHAPTER 7 SUGGESTIONS FOR FURTHER WORK 155
LIST OF REFERENCES 162

viii

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

LIST OF TABLES

Observed and calculated frequencies (cm')
of NiOEP for modes in the region from 900
to 1700 cm (from ref. 55)

Electronic absorption maxima (nm) for MOEC
complexes in benzene solution

Electronic absorption maxima (nm) for MOEP
complexes in benzene solution

Vibrational frequencies (cm“) and Raman
depolarization ratios of MOEC

Vibrational frequencies (cm ), isotope
shifts (cm ) and core-size correlation
parameters for metallochlorin C5; and

Cbe normal modes

Vibrational frequencies (cm ), isotope
shifts (cm ) and core-size correlation
parameters for metalloporphyrin C4; and
Cbe normal modes

Vibrational frequencies (cm ) and isotope
shifts (cm ) for metallochlorin C N modes

Vibrational frequencies (cm') for CuOEC
normal modes in the frequency region below
1350 cm

Predicted and observed frequency shifts for
porphyrin normal modes upon oxidation
(from ref. 99)

Mode characters predicted for CuOEC/CuOECP

ix

21

41

42

44

67

68

75

77

92

94

Table

Table

Table

Table

Table

Table

Table

5.2

5.4

Vibrational frequencies (and) and isotope
shifts (cm”) for the high frequency resonance
Raman modes of CuOEC and CuOEC'

Chlorophylls and chlorophyll-derivatives for
which resonance Raman spectra have been
reported

Electronic absorption maxima (nm) for metal-
substituted chlorophyll a in diethyl ether
solution

Electronic absorption maxima (nm) for
chlorophyll a in various solvents

Resonance Raman frequencies (cm”) and
core-size correlation parameters for metal-
substituted chlorophyll a

Electronic absorption maxima (nm) of
chlorophyll a and chlorophyll b

Chlorophyll a and chlorophyll b carbonyl
stretching frequencies (cm”) in LHC

95

101

105

107

109

127

147

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

LIST OF FIGURES

Structure and labelling scheme for
chlorophyll a

Structure and labelling scheme for
a) metallo-octaethylporphyrin (MOEP) and
b) trans-metallo-octaethylchlorin (MOEC)

Molecular structure of a) FenOEP and
b) FeHOEC as determined by X-ray
crystallography (from ref. 35)

Electronic absorption spectrum of copper
octaethylporphyrin (CuOEP) in CHZCl2 solution

Porphyrin M.O.'s comprising the Gouterman
four-orbital model (from ref. 42)

Electronic absorption spectrum of copper
octaethylchlorin (CuOEC) in CHzClz solution

Molecular processes associated with
a) Infrared and b) Raman spectroscopy
(from ref. 45)

Atomic displacements in the v2 vibrational
mode of NiOEP (from ref. 55)

Diagram of the apparatus used for the
collection of Raman spectra of KBr disc
samples

Structure and labelling scheme for metallo-
chlorin model compounds. MOEC = t;an_-
metallo-octaethylchlorin and MECI = trans-
metallo-etiochlorin I

xi

10

12

14

16

23

34

38

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

3.10

Electronic absorption spectra of ZnOEC, CuOEC
and NiOEC in benzene solution

Resonance Raman spectra of ZnOEC, CuOEC and
NiOEC in CHzClz solution obtained with Soret
excitation at 406.7 nm. Laser powers: 5, 7
and 20 mW, respectively

Resonance Raman spectra of CuOEC in CIiIzClz
solution obtained with Q, excitation at 488.0
nm and Qy excitation at 615.0 nm. Laser
powers: 100 and 35 mW, respectively

Resonance Raman spectra of NiOEC in CHZCI2
solution obtained with Q, excitation at 488.0
nm and Q, excitation at 615.0 nm. Laser
powers: 70 and 40 mW, respectively

IR spectra of ZnOEC, CuOEC and NiOEC in CCl.
solution

IR spectra of CuOEC-1,6-dz, CuOEC-ofi-d2 and
CuOEC-d, in CCl. solution

Resonance Raman spectra of CuOEC-1,6-dz,
CuOEC-o,fi-dz and CuOEC-d, in CHZClz solution
obtained with Qy excitation at 615.0 nm.
Laser power: 40 mW

Resonance Raman spectra of CuOEP, CuOEP-d2
and CuOEP-d. in CHZCIz solution obtained with
Soret excitation at 406.7 nm. Laser power:
10 mW

Resonance Raman spectra of CuOEP, CuOEP-dz
and CuOEP-d. in CHzClz solution obtained
with visible excitation at 514.5 nm. Laser
power: 50 mW

Resonance Raman spectra of CuECI in C11,,Cl2
solution obtained with Soret excitation at
406.7 nm, Q, excitation at 488.0 nm and Q,
excitation at 615.0 nm. Laser powers: 6,
70 and 40 mW, respectively

xii

40

43

48

49

50

52

53

55

56

58

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Resonance Raman spectra of NiOEC in KBr
obtained with Soret excitation at 406.7 nm,
Q, excitation at 488.0 nm and Q, excitation
at 615.0 nm. Laser powers: 20, 70 and 50 mW,
respectively

Resonance Raman spectra of CuOEC in KBr
obtained with Soret excitation at 406.7 nm,
Q, excitation at 488.0 nm and Q, excitation
at 615.0 nm. Laser powers: 20, 75 and 50 mw,
respectively

Core-size correlation for the high
frequency modes of MOEC

Core-size correlation for the resonance
Raman active (———) and IR active (- -)
high frequency modes of MOEP

Structure of metallo-methyloctaethylchlorin
(MMeOEC)

Electronic absorption spectrum of CuOEC”
in CHzCl2 solution

Resonance Raman spectra of CuOEC, CuOEC-
1,6-d2, CuOEC-a,fi-d2, CuOEC-d. and CuECI
in CHzCl2 solution obtained with Soret
excitation at 363.8 nm

Resonance Raman spectra of CuOEC“, CuOEC-
1,6-d; , CuOEC-a,p-d; , CuOEC-d3“ and

CuECI ' in CHZCIz solution obtained with Soret
excitation at 363.8 nm

Electron density contour maps for the am
and a“ orbitals of a) free base porphine and
b) free base chlorin (from ref. 123)

Electronic absorption spectra of chlorophyll
a and Zn, Cu and Ni-substituted chlorophyll
a in diethyl ether solution

Electronic absorption spectra of chlorophyll
a in acetone, methanol and pyridine solution

xiii

60

61

65

66

84

87

88

91

104

106

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

5.3

5.4

6.1

6.2

6.3

6.4

6.5

Resonance Raman spectra of chlorophyll a
in acetone solution and Zn, Cu and Ni-
substituted chlorophyll a in diethyl ether
solution obtained with Soret excitation at

406.7 nm. Laser powers: 35, 20, 20 and 20 mW,

respectively

Resonance Raman spectra of chlorophyll a
in acetone, pyridine, methanol and diethyl

ether solution obtained with Soret excitation

at 441.6 nm. Laser powers: 20, 15, 15 and
15 mW, respectively

Proposed arrangement of the polypeptides
and electron transfer components in the
PS II/OEC complex (from ref. 154)

Arrangement of pigments in the L and M
subunits of the reaction center from

Rhodopseudomogas yirigig (from ref. 158)

Electronic absorption spectrum of LHC
isolated from pea leaves

Electronic absorption spectra of LHC,
the 28 kDa chlorophyll a-binding protein
and the LHC-depleted Photosystem II
reaction center complex isolated from
spinach

Resonance Raman spectra of LHC at -125W:
obtained with 413.1 and 441.6 nm excitation.
Laser powers: 28 and 25 mW, respectively

Resonance Raman spectra of LHC at -125%:
obtained with 472.7 and 514.5 nm excitation.
Laser power: 45 mW

Resonance Raman spectra of chlorophyll a
in acetone at -125°C obtained with 413.1

and 441.6 nm excitation. Laser powers: 19
and 22 mW, respectively

xiv

108

111

121

123

128

130

131

132

133

Figure

Figure

Figure

Figure

Figure

6.8

6.10

Resonance Raman spectra of chlorophyll b

in acetone at -125°C obtained with 441.6

and 472.7 nm excitation. Laser powers: 20
and 40 mW, respectively

Resonance Raman spectra of LHC, the 28 kDa
protein and the reaction center complex
obtained with 406.7 nm excitation. Laser
power: 20 mW

Resonance Raman spectra of LHC, the 28 kDa
protein and the reaction center complex
obtained with 441.6 nm excitation. Laser
power: 22 mW

Resonance Raman spectra of LHC, the 28 kDa
protein and the reaction center complex
obtained with 476.5 nm excitation. Laser
power: 25 mW

Calculated excitation profiles for a 1600
cmq'mode and a 300 cm' mode of chloro-
phyll a (-—-) and chlorophyll b (- -) in LHC

135

136

138

139

143

CHAPTER 1
INTRODUCTION
1. The Role of Chlorophyll in Photosynthesis

In the process of photosynthesis in higher plants, light
energy absorbed by chlorophyll is utilized to convert water
and carbon dioxide into oxygen and stored chemical energy in
the form of carbohydrate (1,2). Ingenhousz in 1779 showed that
it is the green parts of plants that are involved. The
structure of the green pigment chlorophyll (Figure 1.1) was
determined by Willstatter and Stoll in the early 1900's: at
the same time organomagnesium reagents were being developed
by Grignard. This coincidence led to the (incorrect) idea.that
photosynthesis occurred by the photodecomposition of carbon
dioxide bound to chlorophyll. More recent work has shown that

the photosynthetic process is considerably more complex.

In our present understanding of photosynthesis in higher
plants, there are two photosystems (called Photosystem I and
Photosystem II) that operate in series to drive a sequence of
electron transfer reactions (3). These reactions culminate in

the splitting of oxygen from water and the formation of NADPH

 

Figure 1.1 Structure and labelling scheme for chlorophyll a

3
(= reduced nicotinamide-adenine dinucleotide phosphate) and

ATP (= adenosine triphosphate) to be used for the subsequent
reduction of carbon. dioxide. The components of the two
Photosystems in the electron transfer chain are arranged
within protein complexes embedded in the photosynthetic
membrane. At the heart of these Photosystems are chlorophyll
a molecules that form the reaction centers, P-700 (P-700
refers to a pigment with an electronic absorption maximum at
700 nm) of Photosystem I (4) and P—680 of Photosystem II (5).
The primary energy conversion processes in photosynthesis

occur at these sites.

The exact nature of the P-680 and P-700 reaction center
Chlorophylls.haslbeen.difficult.to ascertain. P-680 is thought
to be a ligated monomeric chlorophyll a (6) whereas P-700 may
have a dimeric structure (7-14). other structures for P-700
that have been suggested are the enol form of chlorophyll a
(15), 6-chloro-substituted lo-hydroxy-chlorophyll a (16) or
chlorophyll a' (the C10 epimer of chlorophyll a) (17). The
reaction center Chlorophylls have electronic absorption maxima
in the visible region that are red-shifted compared to the 662
nm absorption maximum of monomeric chlorophyll a in acetone
solution. Further' modification of the jproperties of’ the
reaction center Chlorophylls is seen by comparison of their
mid-point potentials. The mid-point potential, E; of chloro-
phyll a in an organic solvent is in the region of +0.8 V (6)

but for P-7oo/P-7oo“, a, = +0.49 v (13) and for P-680/P-680“,

4
E, = +1.1 V (19). The high redox potential of P--680+ is

necessary for the oxidation of water: 2H20/02 + 41?, at pH 7.0,
E:m = +0.82 V or under estimated physiological conditions at pH

5.0, E, = +0.93 v.

The reaction center chlorophylls constitute less than 1%
of the total number of chlorophyll molecules in the plant. The
vast majority of chlorophylls are involved in light absorption
and transfer of the excitation energy to the reaction centers.
The major chlorophyll-binding protein is the light-harvesting
chlorophyll a/b protein complex (LHC) , which serves both
Photosystem I and Photosystem II (20,21) . LHC contains
chlorophyll a and chlorophyll b molecules (chlorophyll b has
a formyl group in the 3—position instead of a methyl group as
in chlorophyll a) that absorb maximally at 677 and 652 nm,
respectively. Additionally, there are separate sub-antenna
complexes of chlorophyll a serving each Photosystem. The
chlorophylls absorb maximally in the range 670 to 683 nm for
the Photosystem II subantenna and 680 to 695 nm for the
Photosystem I subantenna. This arrangement of light-harvesting
and reaction center chlorophylls ensures that the light energy
is distributed to both Photosystems and that energy transfer
is "downhill" energetically to the reaction center. Directing
the light energy absorbed by a large number of antenna
chlorophylls to a single reaction center chlorophyll raises
the photochemical turnover rate thereby increasing the

efficiency of photosynthesis (22).

5
2. The Structure of Metalloporphyrins, Metallochlorins and

Chlorophyll

A metalloporphyrin is a tetrapyrrole macrocycle with an
extensively delocalized w electron system. Metalloporphyrins
with a range of structures play important roles in numerous
and diverse biological processes (23). The structure of a
typical synthetic metalloporphyrin, that of metallo-octa-
ethylporphyrin (MOEP) is shown in Figure 1.2(a). Without the
central metal ion, hydrogens are bonded to two of the pyrrole
nitrogens and the structure is known as the free base. In the
labelling scheme, C; and Cb1refer to the carbon atoms of the
pyrrole rings and C5 to the methine bridge carbons. The four
methine bridge carbons are labelled a, B, 1 and 6. The
core-size of a metalloporphyrin is defined as the distance
from the center of the macrocycle to the pyrrole nitrogen
atoms (cg-N distance). The core-size depends on the size and
coordination state of the central metal ion and is determined
from the X-ray crystal structures of representative metallo-

porphyrin complexes.

A metallochlorin is a derivative of a metalloporphyrin
in which a Cbe bond of one of the pyrrole rings has been
reduced (24) . Figure 1.2(b) shows the structure of trans-
metallo-octaethylchlorin (MOEC). Both gig- and trans-
configurations of the reduced Cbe bond are possible. In

nature, metallochlorins occur as the magnesium-containing

\ \ \
\ N ’N\ (O)
8 \ \IM” 3 MOEP
,, \ /
N N
\ l
\ Cg/Ca\cb/
X )
(b)
MOEC

 

Figure 1.2 Structure and labelling scheme for a) metallo-
octaethylporphyrin (MOEP) and b) trans-metallo-octaethyl-
chlorin (MOEC)

7
chlorophyll pigments (25) and in iron-containing leukocyte

myeloperoxidase (26,27) and bacterial heme d enzymes (28-30).
Several studies have been directed towards elucidating the
structural, redox and ligand-binding properties of metallo-
chlorins (31-34) to determine the factors that make a chlorin
the preferred macrocycle in these instances. A comparative
study of FenOEP and FenOEC by X-ray crystallography (35)
showed that the average iron-nitrogen.distance is*very similar
in the two complexes (1.996 A for FenOEP and 1.986 A for
FenOEC) . However, the porphyrin macrocycle in FenOEP is
planar but the chlorin macrocycle of FeHOEC is S, ruffled
(Figure 1.3). The core-size of the chlorin macrocycle in the
planar conformation is intrinsically larger than the corre-
sponding porphyrin macrocycle. Ruffling of the chlorin
macrocycle reduces the core-size (and hence the metal-nitrogen
distance) compared to the planar conformation. This distortion
is possible because the chlorin, being less aromatic, has
increased flexibility. Similar conformational flexibility was
observed for nickel tetramethylchlorin compared to nickel

tetramethylporphyrin (36).

The two main chlorophyll pigments in higher plants are
chlorophyll a and chlorophyll b. A key structural feature of
chlorophyll is the presence of a chlorin macrocycle with a
trans-configuration of the hydrogens about the reduced our
bond of ring IV. Fused onto the chlorin macrocycle is a fifth

ring containing a keto carbonyl group. The X-ray crystal

(a)
Fen OEP

( b)
F enOEC

 

Figure 1.3 Molecular structure of a) FeHOEP and b) FenOEC as
determined by X-ray crystallography (from ref. 35)

9
structure of ethyl chlorophyllide a dihydrate (37) (where R

= Cfig in Figure 1.1) shows that the effect of the fifth ring
is to reduce the amount of buckling of the macrocycle. This
in turn increases the extent of conjugation of the keto
carbonyl with the main 1 electron system. In the naturally-
occurring' chlorophyll pigments the central metal ion is
magnesium. The free base form of chlorophyll is called a
pheophytin. Photosynthetic bacteria utilize bacteriochloro-
phyll pigments in which both rings II and IV are reduced
(25,38). Bacteriochlorophyll a has the same pattern of
peripheral substituents as chlorophyll a except for
replacement of the vinyl group by an acetyl group in the 2
position. Chlorophyll clland chlorophyll C, are another type
of naturally-occurring chlorophyll pigment found in diatoms
and brown algae (25,39,40). Chlorophylls c have the same

oxidation state as that of a porphyrin.

3. The Electronic Absorption Spectra of Metalloporphyrins

and Metallochlorins

The electronic absorption spectrum of a typical metallo-
porphyrin of D“, symmetry, copper octaethylporphyrin, in CH2C12
solution is shown in Figure 1.4. The.observed.bands correspond
to in-plane 1r - 1* electronic transitions which for a D“1
metalloporphyrin are of E; symmetry. The intense transition

around 400 nm is called the Soret or B band. The two weaker

10

 

 

 

 

 

 

_ B
LU
0
Z
<
m
m
C
U)
m
4
4 I k A J
300 400 500 700

WAVELENGTH, nm

Figure 1.4 Electronic absorption spectrum of copper octa-
ethylporphyrin (CuOEP) in CHzCl2 solution

11
transitions in the visible region are labelled the a and 8

bands or Q00 and Q01. The 8 band is a vibronic overtone of the

a band.

The theoretical interpretation of the electronic absorp-
tion spectra of metalloporphyrins is based on the four-
orbital model of Gouterman (41,42). M.O. calculations (43)
predict two HOMO's and two LUMO's as shown in Figure 1.5.
Under D,h symmetry the two HOMO's, labelled b1 and b2, are of
an and am symmetry, respectively. The a2u orbital is
calculated to be higher in energy than the an orbital. The
two LUMO's, labelled c1 and c2 are a degenerate pair of e,
symmetry. This M.O. treatment identifies the visible bands
with the transition a2,ll -> e, and the Soret band with a,“ -o e,.
Because the transition dipoles are almost exactly equal the
visible and Soret bands are predicted to be of equal
intensity. In Gouterman's development of the four-orbital
model, the aZu and am orbitals are assumed to be degenerate.
The singly excited configurations (a2u1e81) and (alulef) have
the same symmetry and undergo configuration interaction to
produce the excited states that correspond to the B and Q
transitions. For the 8 state, the transition dipoles are
additive hence the strong Soret absorption and in the Q state
the transition dipoles subtract, leading to the weakly allowed

visible bands.

12

 

 

 

 

b1 (a2u)

 

Figure 1.5 Porphyrin M.O.'s comprising the Gouterman four-
orbital model (from ref. 42)

13
The nature of the central metal ion influences the energy

and intensity of the B and Q transitions. The metal 1 orbital
can conjugate with the 1r electron system of the porphyrin
macrocycle. The an orbital, with electron density on the
pyrrole nitrogens, has the appropriate symmetry for such
interaction, but the a1n orbital does not. As the metal becomes
more electropositive the energy of the a2n orbital is raised.
This results in a red-shift of the electronic absorption
spectrum and, for alkyl-substituted porphyrins, a decrease in

the relative intensity of the Q band compared to the Soret.

Reduction of a metalloporphyrin to a metallochlorin
lowers the molecular symmetry from D,h to C2, taking into
account S, ruffling, where the x,y degeneracy has been
removed. Configuration interaction produces four states, two
of which comprising the Soret band are accidentally degenerate
and strongly allowed (44) . The other two states are Q, and Q,.
The Q, transition is at lower energy and is weakly allowed
whereas the Q, band is almost exactly forbidden. The Q, band
of a metallochlorin is stronger and red-shifted compared to
the corresponding Q band in a metalloporphyrin. These effects
are clearly seen in the electronic absorption spectrum of

copper octaethylchlorin in CHZClz solution (Figure 1.6) .

14

 

 

 

    

 

 

 

_ B
LLI
0
Z
<
m
(I:
O
(D
2
Qy00
1 l L i
300 400 500 600 700

WAVELENGTH, nm

Figure 1.6 Electronic absorption spectrum of copper octa-
ethylchlorin (CuOEC) in CHzCl2 solution

15
4. Infrared and Raman Spectroscopy

Molecular"vibrations fall into 'the frequency' region
between 100 and 4000 cm”. Two techniques available for the
characterization of the vibrational modes of a molecule are
infrared (IR) and Raman spectroscopy. The molecular processes
involved in these two methods are.depicted in Figure 1.7 where
|m> and |n> denote different quantum states of a vibrational
mode in the ground electronic state of the molecule. The
energy separation between the states |m> and |n> corresponds
to the vibrational frequency um of that particular vibrational
mode. In IR spectroscopy, direct absorption of a quantum of
frequency um can promote the transition from state |m> to
|n>. The vibrational mode is IR active if there is a change

in the molecular dipole moment during the vibration.

When a beam of light impinges on a sample of molecules,
some is absorbed, some is transmitted and some is scattered.
Most of the scattered light is unchanged in frequency
(Rayleigh scattering). In Raman scattering, incident light of
frequency uL is scattered by a molecule with a concomitant
increase or decrease in frequency by an amount corresponding
to the vibrational quantum.umv The process depicted in Figure
1.7(b) is referred to as Stokes (Raman) scattering because the
transition from |m> to |n> results in a decrease in the
frequency of the scattered light. The state |e> in Figure

1.7(b) is a virtual state representing a sum over all excited

16

a) IR b) Raman

h3>"jT--T--

 

 

|n> |n>
I an
|m>

|m>

 

Figure 1.7 Molecular processes associated with a) Infrared
and b) Raman spectroscopy (from ref. 45)

17
electronic states of the molecule. The Raman scattering event

can be envisioned as two simultaneous transitions: absorption
from |m> to |e> and emission from |e> to |n>. Anti-Stokes
(Raman) scattering is associated with the transition |n> to
|m> where the molecule has lost a vibrational quantum of
energy and the scattered light.hasta higher frequency. At room
temperature, most molecules are in the ground vibrational
state so that Stokes scattering is more intense than anti-
Stokes scattering. A vibrational mode is Raman active if there
is a change in the ‘molecular' polarizability' during the

vibration.

The intensity of scattered radiation, Im, in photons per
molecule per second for the Raman transition |m> to |n> is

given by (45-48):

I... = 19232315 v.‘ I. 2.. I (as)... I2

where u, is the frequency of the scattered radiation (for
Stokes scattering, v, = uL - um), IL is the intensity of the
incident radiation in photons per second and ((190),, is the

path component (p,o = x,y,z) of the polarizability tensor.

18
From second-order' perturbation theory, the Kramers-

Heisenberg dispersion formula shows the polarizability tensor

to have the form:

(02M)Inn = 1 M|e><e|g > + <n|p_¢,|e><e|g,|m>
h ° Von

- UL + 1F. um + UL + ll".

where |m>, |n> and |e> correspond to the states in Figure
1.7(b), uw,is the energy of the transition from |m> to |e>,
in.is the a th component of the dipole moment operator and F.
is the homogeneous linewidth of the excited state |e>. The
summation is carried out over all excited states |e> of the
molecule. In this expression, the first part corresponds to
a resonant term whereas the second part corresponds to a
non-resonant term. As the incident laser frequency, v,
approaches the energy of an electronic transition, u” the
sum-over-states will be dominated by that single electronic
state. The contribution of the first term to the scattering
intensity increases as the energy separation Iv... - uLl is
minimized. This resonance condition results in an enormous
enhancement of the intensity of Raman scattering compared to
non—resonance (or normal) Raman scattering. The key,
therefore, to a resonance Raman experiment is to match the
frequency of the incident laser radiation to an electronic
absorption band of the molecule under study. Vibrations
associated with the chromophore of the molecule will be

enhanced.

19
Resonance Raman spectroscopy is particularly suitable for

the study of molecules of biological interest (49-51). Selec-
tive enhancement of the resonance Raman spectrum of a protein—
bound chromophore is possible by excitation into an electronic
absorption band of the chromophore. Additional advantages are
that samples may be studied at low concentration and in

aqueous solution.

IR and Raman spectroscopy can be used to determine the
vibrational frequencies of a molecule in its ground electronic
state. For a molecule with a center of symmetry, the selection
rules prohibit the vibrational modes from being simultaneously
IR and Raman active. The IR active modes are antisymmetric
with respect to inversion but the Raman active modes are
symmetric. The vibrational intensities are usually different
in the IR and Raman spectra so that both techniques are
required for full characterization of the vibrational modes

of a molecule.

5. Vibrational Mode Assignments for Metalloporphyrins

Metalloporphyrins and hemoproteins have been studied
widely by resonance Raman spectroscopy (45,52-54) . The system
of vibrational mode assignments for porphyrins currently in
use is based on the normal coordinate analysis of NiOEP

(55,56). The choice of NiOEP for the normal coordinate

20
analysis was made for several reasons: 1) the complex is

stable and experimental data are available not only for NiOEP
but also for its methine-deuterated (NiOEP-d0 (57-59) and
15N-substituted (NiOEP-15m) (59) derivatives: 2) the X-ray
crystal structure of NiOEP is known (60); 3) the symmetrical
structure of NiOEP simplifies the symmetry classification and
assignment of the observed vibrational bands: and 4) the
internal vibrations of the ethyl groups, which are not conju-
gated with the « electron system of the porphyrin macrocycle,
do not appear in the resonance Raman spectra so that assign-

ment of the porphyrin skeletal vibrations is made easier.

In the normal coordinate calculation of the in-plane
vibrations, the structural parameters used are based on the
X-ray crystal structure of the triclinic form of NiOEP (60),
which have been slightly modified to maintain Dm symmetry.
The peripheral ethyl group is treated as a point mass of 15
amu. In this model, the in-plane vibrations are factorized
into 35 gerade (9A1, + 9818 + 8A2, + 982,) resonance Raman active
modes and 18 ungerade (13,) IR active modes. The A1, and A2,
modes produce polarized (p) and anomalously polarized (ap)
bands, respectively. The Bu and Ba modes produce depolarized
(dp) bands. Table 1.1 lists the observed and calculated
frequencies of NiOEP for modes in the region from 900 to 1700
cm"1 (55). The observed resonance Raman frequencies are for

NiOEP in CHZClz (59) solution. The frequencies of the E, modes

are obtained from.the IR spectrum of NiOEP in a 051 disk (58).

21

Table 1.1 Observed and calculated frequencies of NiOEP for
modes in the region from 900 to 1700 cm'1 (from ref. 55)

 

 

Symmetry No. Obs. Calc. Potential energy
distribution (%)
A1, 1!; 1602 1591 u(Cbe)60, v(Cb-Et)19
V3 1519 1517 u(C.Cm)41, u(C.Cb)35
in, 1383 1386 u(C.N)53, 6(C.-C,)21
V5 1025 1048 v(Cb-Et)38, u(C,Cb)23
315 um 1655 1656 y(C,C,,)49, u(C.Cb)17
un 1576 1587 u(Cbe)57, u(Cb-Et) 16
v12 1348* 1351 y(C,N)63, u(Cbe)13
V13 1220 1262 6(C,H)67, v(C,Cb)22
V“, 1151* 1095 u(C,Cb)31, u(C,,-Et)30
A2, um 1603 1600 u'(C,C,)67, u'(C,Cb)18
V20 1397 1409 v'(C,N)29, u'(C,,-Et)24
1121 1308 1281 6'(C,H)53, u'(C.Cb)18
V22 1121 1118 u'(C,N)37, u'(Cb-Et)26
uz3 1022* 1022 v'(C,Cb)26, u'(Cb-Et)20
82, 1223 1475* 1469 u'(C.C,)52, y'(C,C,,)21
V” 1409 1409 v'(C.Cb)47, u'(Cb-Et)26
V30 1159 1157 u'(Cb-Et)49, v'(C.N)28
V31 1019* 1016 6'(C,-C,)25, 6'(C.C,C.)23
Eu V37 1604 1634 u(C,C,)34, u'(C.C.,)24
V38 1557 1588 u(Cbe)56, u(Cb-Et)16
V39 1487 1486 u'(C.C.,)36, u'(C.Cb)17
v.0 1443 1411 y'(C,Cb)30, u'(Cb-Et)24
u,1 1389 1368 y(C.N)56, 6(C.C,)14
17,2 1224 1275 6(CmH)59, u(C,Cb)9
u,3 1127 1133 u'(Cb-Et)35, u'(C,N)33
v“, 1113 1080 u(Cb-Et)29, u(C.Cb)26
ms 993 997 u'(C.C.,)21, 6(C,C,C,)12

 

* not observed as a fundamental
u = stretch, 6

= in-plane deformation

22
Assignments of the resonance Raman active modes are based on

the observed polarization properties of the bands and support
for the assignments comes from comparison with the isotopic
frequency shifts for NiOEP-d, and NiOEP-”N3. The frequencies
marked with an asterisk in Table 1.1 are not observed as
fundamentals but are inferred from.a study of the overtone and
combination resonance Raman bands (59). The potential energy
distribution (P.E.D.) represents the percentage contribution
of a particular type of force constant to the total energy of
the vibrational mode. For example, C41 stretching is calcu-
lated to comprise 60% of the energy for the V; vibrational
mode. On the basis of the P.E.D., modes are classified as ORR
stretching modes (112, 1111), 0,0,, stretching modes (V3, um, um),
C,N stretching modes (u,, v12), CmH bending modes (V13, V21),
etc. (55). The vibrational motion.is:more complicated.than the
P.E.D. would suggest. A better representation of the
vibrational mode is given by the Cartesian displacements of
the atoms in the vibration. Figure 1.8 shows the form of the

uz vibrational mode.

With the vibrational mode assignments for NiOEP it is
possible to interpret the resonance Raman data! for other
metalloporphyrins and hemoproteins. Prior to the normal
coordinate analysis, Spaulding gt _1. (61) had noticed that
the frequency of the anomalously polarized mode in metallo-
porphyrins corresponding to the 1603 cm'1 mode of NiOEP in

CH2C12 solution (now identified as 1’19) exhibited an inverse

23

\ ' I
x .‘ i.
\ "‘.‘ O- I
o ‘\ I, -K’
I \ ’ \
s I \
+- ’ ’ \
--\» ’ \ 6’ -»
\\ ,’ x I
<4 ’ “ "
I
a ‘~~( ‘rfl‘f'
I ‘ r \‘
«tn-~-

Figure 1.8 Atomic displacements in the uz vibrational mode
of NiOEP (from ref. 55)

24
linear correlation with the core-size of the metalloporphyrin.

A similar correlation was observed for two modes labelled u,
and uh of metallotetraphenylporphyrins by Huong and Pommier
(62) who expressed the relationship between the vibrational

frequency and core-size by the equation:

v=K(A-d)

where u is the vibrational frequency in cmq, d is the core-
size of the metalloporphyrin in A, A is the intercept and K
is the slope. The vibrational modes of metalloporphyrins
between 1450 and 1700 cmq'are collectively referred to as the
core-size sensitive modes. The frequencies of the resonance
Raman modes um,ug, um, um and um have been shown to exhibit
a linear correlation with core-size (63-66). The slope or R
value is proportional to the amount of CJI, stretching
character in the P.E.D. of the vibrational mode. Expansion of
the core produces a deformation of the C,CmC, bond angles while
the pyrrole rings maintain their structural integrity (67).
Weakening of the CJ% bonds is reflected by a lowering of the
vibrational frequency. Modes with.(;C, stretching character
(V3, um and um) are more sensitive to core-size changes than
are the (3be stretching modes (u2 and um). Determination of
the core—size sensitivity thus provides a useful means of
assigning vibrational modes in related metalloporphyrin

systems.

25
6. The Application of Resonance Raman Spectroscopy to the

Study of Chlorophyll

From the discussion of the role of chlorophyll in photo-
synthesis, it is apparent that plants have adapted a single
type of chromophore (chlorophyll a) to serve distinctly
different functions in the reaction center and antenna
complexes. The spectral and redox properties of the chloro-
phylls are determined by the specific interaction with their
protein environment. These interactions are possible through
ligation of the central magnesium atom or through the
peripheral substituents on the chlorophyll macrocycle. In
principle, resonance Raman spectroscopy with its ability to
probe the molecular structure of a chromophore within a
protein environment provides an attractive means for the

investigation of chlorophyll-protein interactions in vivo.

The application of resonance Raman spectroscopy to the
study of chlorophylls in yitrg and in the antenna complexes
of higher plants has been pioneered by Lutz (68-76) . The
reaction center chlorophylls in higher plants are more
difficult to study because of their low concentration relative
to the antenna chlorophylls. Improvements in the biochemical
procedures for the isolation of the reaction center protein
complexes (77-79) offer promise that resonance Raman
spectroscopy will be successful in establishing the structures

of the reaction center chlorophylls (79).

26
7. Aim and Sc0pe of this Work

In the work to be presented, three systems of increasing
complexity from synthetic metallo-octaethylchlorins to
chlorophylls to chlorophyll-binding proteins have been
studied. As described in Chapter 3, resonance Raman and IR
data on MOEC and selectively methine-deuterated CuOEC have
been obtained to characterize the vibrational modes of
metallochlorins. TheSe results provide the basis for the
interpretation of the resonance Raman spectra of metallo-
chlorin n cation radicals (Chapter 4) and.metallochlorophylls
(Chapter 5). In turn, it is shown in Chapter 6 how the study
of the isolated chlorophylls in solution can be used to
understand the resonance Raman spectra of chlorophyll-binding

proteins.

CHAPTER 2

MATERIALS AND METHODS

1. Materials

1. Preparation of Chlorins

trans-HZOEC was prepared by the reduction of Fem(OEP)Cl

 

(Aldrich Chemical Company) with sodium metal in iso-amyl
alcohol under nitrogen (80). Chromatography on alumina (Grade
I) with benzene/ether (9:1, v/v) was used to eliminate
residual HZOEP (81) . CuOEC and ZnOEC were prepared from HZOEC
by standard metal-insertion procedures (82) . Chromatography
on silica gel with benzene was used to remove any traces of
unreacted HzOEC. NiOEC was prepared by refluxing nickel
acetate with HZOEC in glacial acetic acid/CHZClz (5:1, v/v)
under nitrogen (83). Chromatography on magnesium oxide with
hexane/benzene ( 9:1, v/v) followed by chromatography on silica

gel with benzene was used for purification.

Methine-deuterated HZOEC-d“ HZOEC-a,B-dz, and HZOEC-
7, 6-d2 were prepared from HZOEC by Asaad Salehi (Michigan State

University). HZOEC-d, was prepared by treatment of HZOEC with

27

28
DZSO,/DZO (9:1, v/v) (84) . Reduction of the DZSO,/DZO ratio to

6:1 (v/v) afforded HZOEC-7,6-d2 (85) . Re-exchange at the 1 and
6 positions of HZOEC-d, with HZSO./HZO (6:1, v/v) produced
HZOEC-oJB-d2 (85) . The proton NMR spectrum (A. Salehi, ref. 85)
of tLa_n_s_-HZOEC shows two peaks at 9.7 and 8.9 ppm, corre-
sponding to the a,fi and 1,6 methine hydrogens, respectively.
Deuteration at the 0,6 methine positions is seen in the
disappearance of the 9.7 ppm peak and at the 1,6 methine
positions by disappearance of the 8.9 ppm peak. In HZOEC-d“
both signals are completely absent. Deuterium substitution is
quantitative for HZOEC-d, and HZOEC-1,6-d2. HZOEC-a,fi-dz showed
90% deuteration at the (1,8 positions and complete proton

recovery at the 1,6 positions.

Oxidation of CuOEC-1,6-d2 with 2,3-dichloro-5,6-
dicyanoquinone in benzene yielded CuOEP-dz (86), which was

purified by chromatography on silica gel with CHZClz.

ii. Preparation of Chlorophyll a and b

Chlorophyll a and b were prepared from fresh spinach
leaves according to the procedure of Omata and Murata (87).
Chromatography on DEAE-Sepharose CL-6B (Pharmacia) with
acetone removed carotenoids and pheophytins. Separation of
chlorophyll a and b was achieved by chromatography on

Sepharose CL-6B with hexane/z-propanol (20:1, v/v) to elute

29
chlorophyll a followed by hexane/Z-propanol (10:1, v/v) to

elute chlorophyll b. All operations were carried out under dim

light in a cold box (10°C) .

iii. Preparation of Metal—Substituted Chlorophyll a

Chlorophylls were extracted from spinach leaves by
treatment with acetone. Dioxane and water were added to
precipitate the chlorophylls which were then collected by
centrifugation (88). The crude chlorophyll extract was
dissolved in ether and 1 N HCl added to convert the
chlorophylls to pheophytins. The ether extract was washed with
water, dried over NaZSO, and then evaporated. Pheophytin a was
separated from pheophytin b by the use of the Girard 'T'
reagent ( ( carboxymethyl) tri-methylammonium chloride hydrazide)
(89). Chromatography on alumina with CH2C12 yielded pure
pheophytin a free of carotenoids (as confirmed by TLC on
alumina). Nickel, copper and zinc-substituted chlorophyll a
were prepared from pheophytin a following the procedure of
Boucher and Katz (90) by using the metal acetate in

chloroform/acetic acid under nitrogen.

30
iv. Isolation of Chlorophyll-Binding Proteins

The light-harvesting chlorophyll a/b protein complex
(LHC) was isolated from pea leaves by the procedure of Burke
gt a1. (21). Thylakoid membranes with a chlorophyll concen-
tration of 0.8 mg/ml were solubilized in 0.78% Triton X-100
and then fractionated on a 0.1 to 1.0 M sucrose gradient. The
LHC fraction was identified as a deep red fluorescent band
when illuminated from behind. This fraction was removed and
aggregated by the addition of MgCl2 (to give a Mg”'
concentration of 7 mM) so that purified LHC could.be collected
by centrifugation. The chlorophyll a/b ratio and total
chlorophyll concentration were determined by the procedure of

Mackinney (91).

LHC, the 28 kDa chlorophyll a-binding protein and the
LEO-depleted Photosystem. II :reaction. center' complex from
spinach. were isolated from. Photosystem II :membranes and
supplied by Demetrios Ghanotakis (University of Michigan)

(92).

31
2. Spectroscopic Techniques

1. Electronic Absorption Spectroscopy

Electronic absorption spectra were recorded for solutions
of metallochlorins, chlorophylls and chlorophyll-binding
proteins on a Perkin Elmer Lambda 5 UV/vis spectrometer.
Spectra were routinely recorded before and after Raman
experiments to ensure that the laser beam had not induced

decomposition of the sample.

ii. Infrared Spectroscopy

Infrared spectra were recorded on a Perkin Elmer model
1750 FTIR spectrometer with 2 cmd'resolution. Spectra in CCl,
solution were obtained by using a 0.2 mm pathlength cell with
KBr windows and.the pure solvent as reference. Samples pressed

into KBr discs were recorded with an air reference.

iii. Resonance Raman Spectroscopy

Resonance Raman spectra were obtained on a computer-
controlled Spex 1401 double monochromator equipped with a
cooled RCA C31034 photomultiplier tube and photon-counting

electronics (93). Data were collected at 1 cmd'intervals with

32
a 1 s dwell time and 5 cm"1 resolution. The measured Raman

frequencies are accurate to 11 cm”. Laser powers employed are
noted in the figure captions. Generally, the lowest power
consistent with maintaining an acceptable signal to noise
ratio was used. Excitation at 406.7 and 413.1 nm was provided
by a Coherent model 90K krypton ion laser: at 441.6 nm by a
Liconix model 4240 He-Cd laser: at 454.5, 457.9, 465.8, 472.7,
476.5, 488.0, 496.5, 501.7 and 514.5 nm by a Spectra Physics
model 165 argon ion laser and at 615.0 nm from Rhodamine 590

in an argon ion pumped Spectra Physics model 375 dye laser.

Collection of the Raman scattered radiation was achieved
with either a 90° scattering or 180° backscattering geometry.
The 90° scattering geometry permits ‘measurement of the
depolarization ratio of the Raman bands. The depolarization
ratio, p is defined as p=I‘L/I.., where IL and I" are the
intensities of light scattered perpendicular and parallel,
respectively, to the polarization of the incident laser beam.
For polarized Raman bands, p<§flt depolarized.bands, p=$flt and
for anomalously polarized bands, p>°/,. The measured
depolarization ratios are accurate to 10.1. Samples were
contained in a spinning cylindrical quartz cell to avoid
photodecomposition. As a rule, the concentration of the sample
was adjusted to give an optical absorbance of 2.0 in a 0.5 mm
pathlength cuvette at the excitation wavelength. The 180°
backscattering geometry was used for concentrated samples.

These could be de-gassed and sealed under argon in quartz EPR

33
tubes. This method was particularly suited to highly fluo-

rescent compounds (eg. ZnOEC). Attachment.of a low temperature
Dewar enabled Raman spectra to be collected at temperatures
down to -140%:(93). Backscattering was also used for samples
pressed into KBr discs. Figure 2.1 shows a diagram of the

apparatus designed and built for use with KBr disc samples.

Translator

__1

“P

 

 

34

 

 

 

 

 

KBr disc
holder
Collection
optics
.L__

 

Backscattering
mirror

 

 

 

 

 

\

Prisms

Laser beam

Figure 2.1 Diagram of the apparatus used for the collection
of Raman spectra of KBr disc samples

CHAPTER 3

VIBRATIONAL PROPERTIES OF METALLOCHLORINS

1. Introduction

The presence of a chlorin macrocycle is a characteristic
structural feature of chlorophylls. Knowledge of the vibra-
tional properties of simpler metallochlorin model compounds
is essential to the successful interpretation of the more
complex resonance Raman spectra of chlorophyll. Resonance
Raman spectroscopic studies of metalloporphyrins and hemo-
proteins (45,52-54) have benefitted greatly from the avail-
ability of a consistent set of vibrational mode assignments
based on the normal coordinate analysis of NiOEP (55,56). In
contrast, metallochlorins have received less attention and a
consensus on their characteristic vibrational properties has

yet to emerge.

Metallo-octaethylchlorins (MOEC), (Figure 1.2(b)) with
their highly symmetric structure are particularly suitable as
metallochlorin model compounds. IR spectra of ZnOEC, CuOEC,
NiOEC, Mg(OEC)py2 (py = pyridine) and Fem(OEC)X (where X = F,

Cl, Br and I) were first reported by Ogoshi g; a;. (94) in

35

36
1975. The spectra were similar to those of the corresponding

metallo-octaethylporphyrin (MOEP) complexes except for the
appearance of new bands in the 1500 to 1700 cm"1 region that
were observed to be metal sensitive. Resonance Raman spectra
of metallochlorins were reported in 1979 by Ozaki gt__;. (95)
for CuOEC, CuOEC-1,6-d2, CuOEC-”N“ NiOEC, Fem(0EC)x (x = F,
C1) and Fem(OEC)Im2 (Im == imidazole) in CHZClz solution with
Q, excitation at 488.0 nm. Based on the isotopic frequency
shifts for the CuOEC complexes, metallochlorin mode assign-
ments were proposed for some the high frequency bands by
direct comparison with NiOEP. These assignments were later
extended to include low frequency modes from a study of iron
OEC and OEP complexes in a variety of spin, oxidation and
ligation states (66,96). Andersson and co-workers reported
resonance Raman spectra of FempPP (pPP = photoprotoporphyrin
IX dimethyl ester) and FemDC (DC == deuterochlorin IX dimethyl
ester) with Soret, Q, and Q, excitation (97) . Mode assignments
were again proposed by comparison with the analogous iron

protoporphyrin and deuteroporphyrin complexes.

As pointed out by Boldt gt gt. (83), such an approach
assumes that the form of the normal modes are unchanged. In
their resonance Raman study of NiOEC, which was supported by
normal coordinate calculations, the latter authors concluded
that the metallochlorin normal modes were substantially
altered. A number of modes in the reduced ring macrocycle

appear to be localized rather than delocalized over the whole

37
macrocycle as occurs in the case of metalloporphyrins. Support

for their assignments came from comparison with the resonance
Raman spectra of Cu diol chlorin-d, (diol chlorin = cis—3' ,4'-
dihydroxy-2,4-dimethyl-deuterochlorin IX dimethyl ester) in

KBr recorded by Andersson gt g_. (98).

In this Chapter, the IR and resonance Raman spectra of
ZnOEC, CuOEC, NiOEC, CuECI (ECI = etiochlorin I) (Figure 3.1)
and selectively methine-deuterated CuOEC complexes obtained
with Soret, Q, and Q, excitation are presented. Both the IR
and resonance Raman spectra are necessary to characterize
fully the metallochlorin vibrational modes. By using the
symmetric MOEC complexes the reliance on a single metal and
the complications introduced.by other peripheral substituents
are avoided. Metal substitution with Zn, Cu and Ni covers a
wide range of core-sizes without introducing the ligand, spin
and oxidation-state effects associated with the Fe complexes.
The amounts of C4;,and CRR stretching character in the core-
size sensitive modes are distinguished by the sensitivity of
the vibrational frequency to metal substitution. Comparison
of the spectra of CuOEC and CuECI identifies those modes with
a contribution from cg; and ca; (5 = substituent) stretching
and C“; bending coordinates as in the case with the analogous
porphyrin species (99). The frequency shifts observed for
CuOEC upon d, as well as oz,fi-dz and 7,6-d2 methine deuteration
confirm the mode assignments and permit an assessment of the

extent of localization for a given mode. The results obtained

38

 

MOEC: R|-R8=C2H5 M=Zn,CU,Ni
MECIo R‘,R3,R5,R7 =CH3 M=CLI
Fi2:‘?¢:"-"’6"'-"e‘Csz
Figure 3.1 Structure and labelling scheme for metallochlorin

model compounds. MOEC == transemetallo-octaethylchlorin and
MECI = trans-metallo-etiochlorin I

 

39
support the concept of mode localization introduced by Boldt

gt _a_;. although the mode composition for several prominent

modes differs from that deduced in their analysis.

2. Results

i. Electronic Absorption Spectra

Figure 3.2 shows the electronic absorption spectra of
ZnOEC, CuOEC and NiOEC in benzene solution. The spectra
display the characteristic features of metallochlorins namely,
separate Q, and Q, transitions with the peak absorbance of the
Q,00 band about half that of the Soret absorption band. The
absorption maxima are listed in Table 3.1 together with the
ratio of the Q,00 to Soret oscillator strength, r for each
complex. The oscillator strength ratios were determined by
measuring the areas under the Soret and Q,00 bands for the
absorption spectra plotted on a wavenumber scale. The energies
of the Soret and Q, transitions follow the order Ni > Cu > Zn.
Also, the ratio of the Q,00 to Soret oscillator strength

decreases from N1 to Cu to Zn.

40

 

 

 

 

 

  

  

 

 

 

   

 

 

398
r.
615
400
390

”J
L)
I:
<
00

n: 614
0
U)
DO
<

617

ZnOEC
590
502 533 572| l
350 450 550 1 650

WAVELENGTH, nm

Figure 3.2 Electronic absorption spectra of ZnOEC, CuOEC and
NiOEC in benzene solution

41

Table 3.1 Electronic absorption maxima (nm) for MOEC complexes
in benzene solution

 

 

Soret QxOl ono Qyoz Qyo1 Qyoo 1'
ZnOEC 401 502 538 572 590 617 0.08
CuOEC 400 495 530 571 591 614 0.09
NiOEC 398 491 523 571 590 615 0.13

 

r = nyOO/fSoret.

These observations are in accord with the Gouterman
four-orbital model (41,42). For alkyl-substituted complexes
the a&,orbital is lower in energy than the alu orbital. As the
metal becomes more electropositive, the energy of the an
orbital is raised relative to the am orbital. This effect is
manifested in the electronic absorption spectrmm as a red-
shift of the Soret and Q bands and a decrease in the ratio of
the Q00 to Soret oscillator strength. Table 3.2 summarizes
electronic absorption spectral data for MOEP complexes in
benzene solution. The Soret and Q bands red-shift and the
oscillator strength ratio decreases in the order Ni:>Cu:>Zn.
The oscillator strength ratios for the MOEC complexes are
larger than the respective MOEP complexes reflecting the

allowed nature of the Q,00 transition in metallochlorins.

42

Table 3.2 Electronic absorption maxima (nm) for MOEP complexes
in benzene solution

 

 

Soret Q01 Q00 r
ZnOEP 404 533 569 0.04
CuOEP 400 527 563 0.05
NiOEP 393 518 552 0.07

 

r = fQOO/fSoret

ii. Resonance Raman Spectra of MOEC

The resonance Raman spectra of ZnOEC, CuOEC and NiOEC in
CHZCl2 solution obtained with Soret excitation at 406.7 nm are
shown in Figure 3.3. The vibrational frequencies for these
species and the depolarization ratios for CuOEC are listed in
Table 3.3. Five modes in the frequency region from 1490 to
1700 cm‘1 corresponding to the 1644, 1602, 1584, 1545 and
1507 cm'1 modes of CuOEC are observed to be metal dependent.
Inspection of the polarized resonance Raman spectra of CuOEC
at 406.7 nm (not shown) reveals that the 1545 cm'1 band
consists of a 1547 cm'1 polarized mode and a 1543 cm"1 anoma-

lously polarized mode.

43

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m
to
‘9.
1 1
N
m
.. 10
3‘3 2
v* 9
. n 7 l
h
2 c
' 9
n T
1.0
I N :2
g 1
T to
in
‘ 8 ts w z
I Q T -
d ' 0
N 9
N Q
1 o 1
7 8
m
-m 1’.’
I I
N
'4
l 1.9
.
N
.. O
: a M.
' n
' r~ V CuOEC
o «e
l“ ‘2
‘ N T 1
6 a:
E! Q T t
1 I :3 n
n v
w
. g; '53
{G ..
9 $ I 2
1 '2 9
d i U
m
n
1 n
_ I
g ZnOEC
T rs
1b
9
I n
N
L?
I
1 ‘ 1
l5OO l7OO

 

CNr'

Figure 3.3 Resonance Raman spectra of ZnOEC, CuOEC and NiOEC
in CHZClz solution obtained with Soret excitation at 406.7 nm.
Laser powers: 5, 7 and 20 mW, respectively

44

Table 3.3 Vibrational frequencies (cm”) and Raman depolarization ratios of MOEC

 

 

NiOEC ouoec 2nosc
406.7 498.0 615.0 IR 406.7 p 488.0 p 615.0 p IR 406.7 1R
-- -- -— 1740 -- -- -- 1741 -- ~-
-- -- -- 1712 -- -- -- 1708 -- 1708
1651 1652 1652 1652 1644 0.8dp 1643 0.5p 1644 0.4p 1644 1623 1624
1610 1613 1613 1611 1602 0.6p 1601 1.0ap 1602 0.5p 1602 1597 1596
1599 1592 1599 1591 1594 0.4p 1584 0.7dp 1583 0.7dp 1584 1570 1570
1554 1554 1554 1553 1547 0.3p 1546 0.6p 1548 0.4p 1547 1534 1538
-- -- -- -- 1543 1.6ap -- —- 1543 -- 1529
1517 1517 1517 -- 1507 0.2p 1506 0.3p 1507 0.4p 1504 1491 1491
-- -- 1502 1499 -- -- 1486 0.3p 1484 -- ~-
-- 1483 -- -- -- -- -- -- -- --
1465 1466 1466 1464 1466 0.7dp 1465 1.0ap 1465 0.5p 1464 1463 1466
-- -- -- 1457 -— -- -- —- -- 1457
-— —- -- 1453 -- -- -- 1452 -- 1452
-- —- -- -- —- -- -- 1443 1440 1441
1403 1404 1401 —— 1399 0.6p 1402 0.7dp 1401 0.5p -- 1399 --
-- -- —- 1397 -- -- -- 1396 -- 1394
1392 -- -- -- 1388 0.3p 1391 1392 0.4p -- 1387 --
-- 1383 -- -- -— -- -- 1384 -- 1383
-- —- -- 1378 -- -- -- 1375 -- 1375
1373 1373 1372 -- 1372 0.3p 1372 0.3p 1373 0.3p -- 1369 -—
1363 1363 -- 1365 1361 0.3p 1361 0.3p -- -- 1359 --

 

Table 3.3 (cont'd.)

45

 

 

NiOEC ouosc ZnOEC
406.7 488.0 615.0 IR 406.7 p 498.0 p 615.0 p IR 406.7 IR
1349 1351 -- -- 1352 0.2p 1350 0.3p -- -- 1349 ~-
1316 -- -- 1315 1318 0.6p 1318 1.4ap 1318 0.7dp 1318 1320 1320
1307 1306 1308 -- -- -- 1310 0.6p —- -- --
-— -- -- 1303 —- -- -- 1301 -- 1301
1275 1275 1276 1274 1276 0.3p 1275 0.7dp 1277 0.4p 1273 1275 1272
1266 1264 1266 1269 1266 0.3p 1265 0.8dp 1267 1262 1264 1266
1241 -- 1239 -- 1238 0.4p -- 1238 0.4p -- 1237 --
-- -- -- 1233 -— —- -- 1235 -- 1235
1218 1219 1219 1217 1216 0.6p 1212 0.6p 1215 0.4p 1211 1207 1205
1204 1202 1202 1200 -- 1199 0.3p 1198 0 3p 1197 1198 1194
-— 1180 -- 1180 -- 1182 0.5p -- 1183 -- 1183
1154* 1154* -- -- 1155* 1157* 1154* -- 1158 --
-- -- -- 1144 -- -- -- 1146 -- 1147
1142 -- 1140 -- 1141 0.3p ~- 1141 0.4p —- 1141 ~-
1125 1123 1124 1123 1129 1128 0.5p 1128 0.5p 1128 -- 1132
-- -- -- -- -- -- -— 1119 -- 1118
-- -- -- -- -- -- -- -- -- 1111
-- -- -- 1109 -- -- -- 1107 -- 1106
-- -- -- 1094 -- -- -- 1095 -- 1094
-- -- -- 1084 -- -- -- 1086 -- --
-- -- -- 1063 -- -- -- 1064 -- 1063

 

46

Table 3.3 (cont'd.)

 

 

NiOEC CuOEC ZnOEC
406.7 488.0 615.0 IR 406.7 p 488.0 p 615.0 p IR 406.7 IR
-- 1059 -- 1058 -- -- -- 1058 -- 1058
1028 1027 -- -- 1026 0.3p 1206 0.3p -- -- 1025 --
1022 1022 1020 -- 1021 0.5p 1020 0.4p 1021 0.4p -- 1020 ~-
-- -' -- 1014 -- -- -- 1015 -- 1013
-- -- 994 996 -- -- -- 988 -- 984
-- 960 961 -- -- 960 961 0.4p -- -- --
-- -- -- 957 -- -- -- 956 -- 957
932 932 -- 932 -- 932 932 932 -~ ~-
919 921 922 921 -- -- 923 918 -- 914

-- -- -- -— 909 0. 3p -- --

 

406.7, 488.0 and 615.0 refer to laser wavelengths (nm) used
Q, excitation, respectively.

p - depolarization ratio: p = polarized, dp = depolarized,
ap = anomalously polarized.

* overlapped by Cit-,Cl2 solvent band

for Soret, Q,

and

47
Resonance Raman spectra of CuOEC (Figure 3.4) and NiOEC

(Figure 3.5) in CHzCl2 solution were obtained with Q,
excitation at 488.0 nm and with Q, excitation at 615.0 nm. The
intense fluorescence of ZnOEC prohibited the measurement of
its resonance Raman spectra with visible excitation. The
frequencies are listed in Table 3.3 along with the
depolarization ratios for CuOEC. The 488.0 nm excitation
spectra agree with those reported by Ozaki gt gt. (95). For
CuOEC, bands that are not seen with Soret excitation are
observed at 1199, 960 and 932 cm'1 with Q, and Q, excitation:
at 1182 cm'1 with Q, excitation only: and at 1486, 1310 and 923
cm'1 with Q, excitation only. The 1486 cm'1 band of CuOEC is
metal sensitive, corresponding to the 1502 cm"1 band of NiOEC

observed with Q, excitation.

iii. IR Spectra of MOEC

For a metallochlorin of C2 symmetry, the resonance Raman
active modes are also IR active. The IR spectra of ZnOEC,
CuOEC and NiOEC in CCl, solution are shown in Figure 3.6 and
the vibrational frequencies are listed in'Table 3.3. The total
number of bands observed by IR spectroscopy is greater than
with resonance Raman spectroscopy as a result of IR activity
of the internal vibrations of the ethyl groups. These modes
can be identified by comparison with the IR spectrum of NiOEP

reported by Kincaid gt gt. (100). The two closely spaced modes

48

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

E
9.
l 1
‘ m
n
fi 50
‘ I XeX-GI. nm
. N
N
N
6 7 ‘3
d : 9
. ' e '
N
T 8 v
19 Q'
o ‘- g
= 3
a ' -
m 1
1
n
. * 6
I
‘ o
m
9
. N l
O
3
9 5 Xex-488.0nm
N ' 9
~ -~ ,
9 Sn
. T 9 '1':- :2
Q E
.1 I I 8
$8 “3 n
=g 8 I
II T
o
N
‘ Q
I
q
“A“ I I ‘ ' I I T l
900 H00 I300 I500 I700
CM"

Figure 3.4 Resonance Raman spectra of CuOEC in CHzCl2 solution
obtained with Q, excitation at 488.0 nm and Q, excitation at
615.0 nm. Laser powers: 100 and 35 mW, respectively

49

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8
e 8
1 I g
1
:8 x -615.0 nm
N ex
4 T
8 9 2
. = 9.‘ 9
I ' 6 l
E.
i 1
, v
‘1‘
- |
. to
m
N l
a N
o:
1
1
N
e
o 8
1 v - N
' 2
1
10 "1'5 )‘ex' 488.0 nm
is _
~ 6 n v I
252 10 "' In
T O 3 9
m n
T B (D '
l '2
l
T T l
ISOO I700

 

Figure 3.5 Resonance Raman spectra of NiOEC in CHzCl2 solution
obtained with Q, excitation at 488.0 nm and Q excitation at
615.0 nm. Laser powers: 70 and 40 mW, respect1vely

50

 

ZnOEC

 

0050.

www—

.350' Y 'Izoo' uoov 1000

Moo ,

500

IGOO

1

I700

 

 

 

CM'I

3.6 IR spectra of ZnOEC,

CCl‘

1n

CuOEC and NiOEC

Figure
solution

51
at 1547 and 1543 cm"1 of CuOEC that can be resolved in the

Raman spectrum by depolarization ratio measurement are
apparent in the IR spectrum; similarly the 1534 cmq'resonance
Raman mode of ZnOEC is seen to consist of two modes at 1538

and 1529 cm".

iv. Effects of Methine Deuteration

Figure 3.7 shows the IR spectra of CuOEC-1,6-d2,
CuOEC-a,B-d2 and CuOEC-d, in CCl, solution and Figure 3.8 shows
the resonance Raman spectra of the methine-deuterated CuOEC
complexes obtained with deexcitation at 615.0 nm. Different
patterns of frequency shifts are observed for deuteration at
the 1,6 and 02,3 positions. The 1644, 1602, 1584, 1507 and
1486 cmfl‘modes of CuOEC exhibit frequency shifts upon both a,fi
and 7,6 deuteration that are additive for CuOEC-d,. Only the
1584 and 1507 cm'1 modes show equal frequency shifts upon a,fi
and 1,6 deuteration. The 1644 and 1486 cm’1 modes are more
sensitive to 1,6 deuteration whereas the 1602 cmfl‘mode shows
a large shift upon a,fi deuteration. The 1543 cm"1 mode shows
no shift upon a,fi deuteration but a shift of -17 cmd‘upon 1,6

and d, methine deuteration.

Below 1350 cm“, many of the vibrational modes of CuOEC
are sensitive to methine deuteration. However, the complicated

pattern of frequency shifts observed for CuOEC-a,fi-d2,

52

l :0
In:
I00.
I80.
Iv;
low:
l5:
lvON_
Insu—
Inn“.
It».
Infin—
Ian.
nun!
Inn‘—
«up!
unan—
Invfl
loan.
:00!

 

    

1.8-dz

< CuOEC-

'0,B'02

CuOEC

-
-

  
 

_ CuOEC ' 64

'wfl .

0

' xz‘oo'

v‘v
I400

 

I800

CM“1

3.7 IR spectra of CuOEC-1,6-d2,

and

-d2

CuOEC-a,B

CCl, solution

1n

Figure
CuOEC-d,

53

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

N
9
1 3
‘ In
<3!
I
d CuOEC-y.8-d2
v
q N
9'.
. h
v
9
I
Iv .-
Ja a
' I
q
1 v
N
9
3 ‘3
‘ :- In 9
I g I 8 CUOEC'0.B‘02
Io ' 9
Q .—
. m 0|
' 9
I
" N
10
T
v
N
. Q
" v
v
. 9
CUOEC'd4
w
v
~ 2*
i 1 ‘ I 1 T 1 1
900 |200 I300 1500 I700

CM"

Figure 3.8 Resonance Raman spectra of CuOEC-1,6-dz, CuOEC-
a,fi-d2 and CuOEC-d, in CHzClz solution obtained with Qy
excitation at 615.0 nm. Laser power: 40 mW

54
CuOEC-1,6-d2 and CuOEC-d, does not permit a one-to-one

correlation of these modes in the various CuOEC complexes.
Nevertheless, it is possible to recognize several modes that

clearly show (1,3 or 1,6 localization in this frequency region.

In order to interpret the frequency shifts for the
methine-deuterated copper chlorin complexes, two methine-
deuterated copper porphyrin compounds were prepared. In the
CuOEP-d. species, all four methine carbons are deuterated; in
the CuOEP-d2 complex, only two of the four bridging carbons
are deuterium substituted at adjacent methine positions. These
compounds allow assessment of the effect of full and partial
deuteration on modes that are delocalized throughout the
macrocycle. Resonance Raman spectra of CuOEP, CuOEP-dz and
CuOEP-d, were recorded with Soret excitation at 406.7 nm
(Figure 3.9) and with visible excitation at 514.5 nm (Figure
3.10) . The C,Cm stretching modes V10, 1119 and V3 of CuOEP show
frequency shifts of -13, -22 and -10 cm'1 in CuOEP-d... For
CuOEP-d2, the frequency shifts of the um and V3 modes are half
of those observed for CuOEP-d," The 1119 mode shows a --14 cm'1
shift for CuOEP-d2 but only a further -8 cm’1 shift for
CuOEP-d," The frequencies of the <3be stretching modes V2 and
V11 are not shifted upon (12 or d. methine deuteration (101).
The results for the um and V3 modes indicate that the
vibrational mode is delocalized over the porphyrin macrocycle
and that the mode composition is not altered by methine

deuteration. The V19 mode, although delocalized, is mixed with

55

- l38l

CuOEP

- I59l

 

 

- l38l

   

N

m

'12
I

 

 

 

CuOEP-d4

- l593

 

 

 

 

 

f 1 1 T 1
900 "'00 I500 I500 I700

CM"

Figure 3.9 Resonance Raman spectra of CuOEP, CuOEP--d2 and
CuOEP-d. in CHzCl2 solution obtained with Soret excitation at
406.7 nm. Laser power: 10 mW

56

 

 

 

 
   

 

 

 

 

 

 

 

 

 

 

 

 

2
.. a
I v- “I,
G N ' 0
‘° T
F
- In
'- o
I v
P
l
CuOEP
o:
[x
co
F
co
0
v
T
o
{B a g
‘— ID 1-
P
I CuOEP-d2
“ o
o
In
.. N V
| to
In
P

 

 

 

 

 

 

‘30 co
‘ o I; g
to "' v-
v- I '3‘ I
.. ,_ o
I 1?-
“ so I
N I
. S’-
-1 a v ,_
*- 2?» . 8 I
. I v- V
' T
.1
I 1 I I v ‘
900 1 1 00 1300 1 500 1700

CM’1

Figure 3.10 Resonance Raman spectra of CuOEP, CuOEP-d2 and
CuOEP-d, in CHzCIZ solution obtained with visible excitation at
514.5 nm. Laser power: 50 mW

57
can bending character (56) and most likely changes in mode

composition upon deuteration. Taken together, these results
for partially and fully methine-deuterated metallo-octa-
ethylporphyrins provide a basis for the analysis of the
corresponding metallo-octaethylchlorin complexes. Most impor-
tantly, they show that for a delocalized mode, methine d2
deuteration can be expected to produce roughly half of the d,

shift.

v. Resonance Raman Spectra of CuECI

For metalloporphyrins, the change in peripheral
substituents that occurs upon going from CuOEP to CuEPI
(Figure 3.1) affects those modes with a contribution from Cbe
and CbC, stretching and CbC, bending coordinates (99) . A
similar dependence of the frequency of the analogous modes on
peripheral substituents is expected for the metallochlorins
and provides a means by which these modes may be identified.
Figure 3.11 shows the resonance Raman spectra of CuECI in
CHzClz solution obtained with Soret excitation at 406.7 nm,
Qx excitation at 488.0 nm and Q’ excitation at 615.0 nm. The
1584, 1547 and 1543 cm’1 modes of CuOEC show increases in
frequency of +4, +4 and +3 cm”, respectively, as a result of
the change in peripheral substituents on going to CuECI. In
addition, the 1238, 1215 and 1198 cm‘1 modes of CuOEC also

exhibit increases in frequency. The change in peripheral

58

-124I

-982
.H40
-4270
-4550

)\ e x«615.0nrn

~1220

 

998

 

 

-I205

 

 

 

‘93l

 

 

   

Xe, - 488.0nm

‘flGOI

 

 

 

 

 

'1600

)‘ex- 406.7nm

 

 

-I644

 

 

 

'4000

 

 

I

900 1 I 300 1 I300 1 1500
CM"
Figure 3.11 Resonance Raman spectra of CuECI in CHZClz solution

obtained with Soret excitation at 406.7 nm, Qx excitation at

488.0 nm and Q, excitation at 615.0 nm. Laser powers: 6, 70
and 40 mW, respectively

4—1
I700

59
substituents lowers the frequencies of the 1372, 1361, 1350

and 1277 cm'1 modes of CuOEC by -4, -5, -3 and -‘7 cm“,
respectively, and the 1021 cmd'mode appears to split into two

components at 998 and 982 cm'1 in CuECI.

vi. Solid State Spectra

For their normal coordinate analysis of NiOEC, Boldt g;
_l. (83) obtained resonance Raman spectra of NiOEC pressed
into Nagxh pellets. However, differences occur in the number
and frequencies of the resonance Raman modes of NiOEC in
Nazso, and in CHzClz solution. In order to compare the results
obtained here with those of Boldt gt, al., solid state
resonance Raman spectra were recorded for NiOEC (Figure 3.12)
and CuOEC (Figure 3.13) in KBr pellets. The high frequency
modes of NiOEC and CuOEC are shifted to lower frequencies in
KBr. Additionally, the high frequency bands of NiOEC display
an.altered.patternxof relative intensities with 406.7 nm Soret
excitation for the KBr sample and Boldt's NazSO. sample
compared to NiOEC in CHZClz solution. Resonance Raman spectra
of CuOEC in KBr on the other hand, showed the same pattern of
relative intensities as in 01,012 solution with Soret, Q, and
(g.excitation. The 1572 cmq'band in the NiOEC spectrum appears
to be an artifact of the solid state since its relative

intensity increased as the ratio of NiOEC’to KBr in the pellet

was lowered. No evidence for this mode could be found in the

60

-1019

—1233

J
-1275
- 1548

I
“1144

Au = 615.0 nm

— 1394

  
   

 

I
~1123
~1307

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O
6:
~ :2
to 5 v
.I T ' I;
CD (D
Q 1-
m
-I v- I
u) I
8 7L"=488.0 nm
I}
[s
‘D
53'-
' 3
m 8 lu=406.7 nm
J o a $3 to ‘3
S E g I I '3 U 52
_, 1- v- N v- '
s: ' I
'T
' I ' I v I f 1
900 1100 1300 1500 1700
CM"

Figure 3.12 Resonance Raman spectra of NiOEC in KBr obtained
with Soret excitation at 406.7 nm, Q, excitation at 488.0 nm
and Q, excitation at 615.0 nm. Laser powers: 20, 70 and 50 mW,
respectively

61

1018

 

 

 

 

 

CD
- It. s
In
3; " v-
- N I I
P
m I
v A. = 615.0 nm
- 1- OX
1- 8 n
' 51‘ 3', 8
- ' (‘0 a '-
ow "’ 0" n
[s O | v I n
" N n '- 0
v- V‘ | v-
-I I U

1396

- 1364

- 1539

g
-1459
-1499
1582 C; 1581
1598 -1631

 

I" = 488.0 nm

 

 

 

 

 

 

 

 

 

 

      

 

 

. K“ = 405.7 nm
'I :2 80
n 1‘33
. " I “
g s l I
5 3
‘ I I
.1
d ' I ' T ‘ I 1 1
900 1100 1300 1500 1700
CM'1

\

Figure 3.13 Resonance Raman spectra of CuOEC in KBr obtained
with Soret excitation at 406.7 nm, Q, excitation at 488.0 nm
and Q, excitation at 615.0 nm. Laser powers: 20, 75 and 50 mW,
respectively

62
IR spectrum of NiOEC in KBr or for an analogous mode in the

resonance Raman spectra or IR spectrum of CuOEC in KBr.

3. Discussion

i. C,,CIn and Cbe Modes

Upon reduction of a egg bond in a metalloporphyrin to
form a metallochlorin, the molecular symmetry is lowered from
D“, to C2. For C2 symmetry, the resonance Raman active and IR
active vibrational modes belong to the A and B symmetry
species. The resonance Raman active polarized A18 and
depolarized Bu modes of D“ symmetry are expected to produce
polarized modes of A symmetry in C2. The anomalously polarized
A2, and depolarized le5 modes correlate with depolarized B
modes. The IR activeIEg modes split into A and B. The A and
8 modes are both IR and Raman active. Under C2 symmetry, eight
cg; stretching (4A + 48) and four cg; stretching (2A + 28)
modes are predicted. Four of the ' QC, and two of the CbCb

stretching modes are derived from E,I modes.

In their initial study, Ozaki gt a;. (95) assigned the
resonance Raman modes of CuOEC at 1643 (p), 1584 (dp) (see
102) and 1506 (p) cm'1 as corresponding to the v10 (81,, dp),
V19 (A2,, ap) and V3 (A1,, p) modes of NiOEP, respectively. The

observed polarizations of these three modes with 488.0 nm

63
excitation are consistent with the D“, to C2 symmetry

correlation. The 1546 cm'1 mode was presumed to consist of a
polarized and an anomalously polarized component. The ap mode
that shifts to 1527 cm"1 on 1,6 deuteration was assigned to
the other C4; mode. If this mode is not derived from an Eu
mode it would have to correspond to v28 (82,, dp) of NiOEP
calculated at 1469 cm'1 (55). The 1602 (ap) and 1546 (p) cm'1
modes of CuOEC, which.are unshifted upon 1,6 deuteration, were
assigned as corresponding to u; (A1,,p) and un (Bludp).
However, the anomalous polarization of the 1602 cm'1 mode is
inconsistent with this mode being derived from a porphyrin.Au

mode.

Such a symmetry-lowering approach does not adequately
account for the vibrational mode assignments for the metallo-
chlorins. For these compounds our results indicate that there
is mixing of C,,CIn and Cbe stretching character in the high
frequency modes that is not predicted by direct comparison
with the normal modes of NiOEP. This is apparent in the
frequency region from 1475 to 1700 cm'1 where seven modes,
corresponding to the 1644, 1602, 1584, 1547, 1543, 1507 and
1486 cm’1 modes of CuOEC, are observed to be metal, and hence

core-size, dependent.

64
The relationship between vibrational frequency and core-

size of a metalloporphyrin often follows the empirical

expression (62):

v=K(A-d)

where u is the vibrational frequency in cm”, d is the center
to nitrogen distance or core-size of the metalloporphyrin in
A, and K (cmfl/A) and A (A) are parameters characteristic of
the macrocycle. The physically useful parameter is the slope
or K value, which is proportional to the amount of 0,0,
stretching character in the vibrational mode. Ozaki gt gt.
(66) have previously applied this kind.of analysis to metallo-
chlorins; they achieved core-size variation by working with
iron complexes in different spin and oxidation states. The
iron chlorins, however, are susceptible to the same sorts of
difficulties in such an analysis as are encountered with the
corresponding iron porphyrins (63,65) and for this reason our
analysis has been carried out for metals other than iron. The
correlations between vibrational frequency and core-size for
the high frequency modes of the Ni, Cu and Zn complexes of OEC
and OEP are shown in Figures 3.14 and 3.15. The same
core-sizes are assumed for the porphyrin and chlorin com-
plexes, namely Ni (1.958 A) (60), Cu (2.000 A) (103) and Zn
(2.047 A) (104). The core-size correlation parameters, K and
A, are listed in Table 3.4 for MOEC and in Table 3.5 for MOEP.

From.the chlorin K'values, the 1644, 1602, 1543, 1507 and 1486

65

1660 ..

 

 

 

P

VIBRATIONAL FREQUENCY, cm"1

 

1450 I I . J I . . . 4 I
1 .950 2.050

CORE-SIZE, A

 

Figure 3.14 Core-size correlation for the high frequency modes
of MOEC

66

1 660

 

 

 

 

VIBRATIONAL FREQUENCY, crn'1

 

 

 

1450 . . .
1.950 2.050

CORE-SIZE, A

Figure 3.15 Core-size correlation for the resonance Raman
active (-—-) and IR active (- -) high frequency modes of MOEP

67

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68

Table 3.5 Vibrational frequencies (cmq), isotope shifts (cm“) and core-size
correlation parameters for metalloporphyrin C5; and C“% normal modes

 

 

Au Au Av Kc A

Mode' CuOEP d; d. subst" (cm'1/A) (A) P.E.D.‘

V10 1639 -6 -13 +3 405 6.05 y(c,c_)49, u(c.c.,)17
y, 1591 +1 0 +6 236 8.74 u(c,,c,,)6o, 0(Cb-Et)19
6,, 1584 -14 -22 -- 449 5.53 u'(c_c,)67, v'(C.Cb)18
.1, 1572 0 -3‘1 +6 203 9.73 u(c,,c,,)57, u(c.-I'-:t)16
v3 1505 -5 -1o +2 382 5.94 u(c,c_)41, u(c,c,,)3s
6,, -- -- -- -- --- ---- 6(cbc.)57, va-Et)l6'
.3, 1554‘ -- -— -- 426 5.66 u(c.c_)34, u'(c.c.)24'
.3, 1483‘ -- -- -- 278 7.34 u'(c.c_)36, u'(c,N)17

 

Mode designation and P.E.D. from the normal coordinate analysis of
NiOEP (55).

Au subst: frequency shift on change of peripheral substituents from CuOEP
to CuEPI (99).

calculated for ZnOEP, CuOEP and NiOEP using data from ref. 105.
see ref. 101.
P.E.D. for v37 and u38 have been interchanged (100).

from ref. 100.

69
cm‘1 modes of CuOEC have substantial C,Cm stretching, but for

the 1584 and 1547 cm'1 modes, contribution from the CbCI.
stretching coordinate is more apparent. The 1486 cm'1 mode of
CuOEC shows the highest K value. Significantly, the K values
for the other chlorin C,C, stretching modes are all lower than
the porphyrin K values for the Raman active C,Cm stretching
modes: v10 (K=405), 019 (K=449) ,and 03 (K=382) and the IR active
V38 (K=426) . The chlorin K values are close to the K value for
the porphyrin IR active 039 (K=278) , which has 36% C.C..
stretching character in the calculated P.E.D. for NiOEP. 0f
the two chlorin Cbe stretching modes, the K value of the 1584
cm"1 mode is virtually identical to that of ya (K=236) whereas
the K value of the 1547 cm”1 mode is lower than that of 1111
(K=203) (106) . The different K values that we observe for the
chlorins indicate altered mode compositions compared to the
MOEP complexes: in particular, they suggest that there is
mixing of C,CIn and Cbe stretching coordinates in the high

frequency normal modes.

To characterize further the high-frequency modes we have
examined their sensitivity to methine deuteration and to a
change in peripheral substituents upon going from OEC to ECI.
The frequency shifts observed for CuOEC-d. and CuECI (Table
3.4) can be compared to CuOEP-d, and CuEPI (Table 3.5). In
methine-deuterated CuOEP-d“ the C,,CIll stretching modes um,
um and V3 exhibit frequency shifts of -13, -22 and -10 cm"1

compared to CuOEP but the €be stretching modes V; and V11 are

70
unaffected. In addition, Kincaid gt g_. observed -11 and -7
cm'1 shifts for the cg; stretching modes u” and u” from the
IR spectra of NiOEP and NiOEP-d4, (100). The 1644, 1602, 1584,
1543, 1507 and 1486 cm"1 modes of CuOEC show substantial
frequency shifts of -10, -13, -9, -17, -13 and -10 cm'1 upon
ch methine deuteration. The magnitude of the frequency shifts
indicates that these modes contain CK; stretching character.
Comparison of the resonance Raman spectra of CuOEP with CuEPI
showed that a change in peripheral substituents resulted in
an increase in frequency of the 04x stretching modes 0, and
un by +6 cm"1 (99). For CuOEC, we observe that the 1584, 1547
and 1543 cm’1 modes increase in frequency by +4, +4 and +3
cmfl'in CuECI. These modes are therefore predicted to contain
(:bCb stretching character. The mode characters deduced from
IL methine deuteration and the change in peripheral substit-
uents (Table 3.4) are in good agreement with those predicted
from the K values for MOEC. The results of the core-size
correlation together with the analysis of the wavenumber
shifts incurred upon methine deuteration clearly demonstrate

that there is mixing of the v(C,C,) and u(Cbe) internal

coordinates in the high frequency normal modes.

The above results show that the mode characters observed
for the high-frequency modes of CuOEC cannot be assigned by
direct comparison .with the normal modes of NiOEP. The
resonance Raman spectrum of CuOEC-1,6-dz reported by Ozaki gt

gt. (95) showed no shift of the 1602 cm"1 mode of CuOEC upon

71
1,6 deuteration and led these authors to conclude that the

1602 cm"1 mode corresponded to the porphyrin V2 01,0,> stretching
mode. We observe that the 1602 cm'1 mode shifts to 1589 cm'1
in CuOEC-d, indicating substantial C,CInn stretching character.
To reconcile these deuteration effects the frequency shifts
for CuOEC-1,6~dz, CuOEC-0,3-d2 and CuOEC-d. were compared. As
seen in Figures 3.7 and 3.8 and Table 3.4, the high-frequency
modes exhibit different shifts upon 0,3 and 1,6 deuteration.
The large (~13 cm'l) frequency shift of the 1602 cm'1 mode
results from deuteration at the 0,3 positions but deuteration
at the 1,6 positions has little effect on the frequency of
this mode. 0n the other hand, the 1543 cm'1 mode of CuOEC is
shifted by ~17 cm’1 upon 1,6 deuteration and is unaffected by
deuteration at the 0,3 positions. Both the 1644 and 1486 cm'1
modes of CuOEC show unequal frequency shifts upon 0,3 and 7,6
deuteration with the larger shift resulting from 1,6
deuteration. The frequency shifts upon deuteration that we
observe for CuOEC support the idea of mode localization in
metallochlorins as proposed by Boldt gt _l. (83) from their

study of NiOEC.

In Table 3.4 our results are compared with the
assignments of Boldt gt gt. for NiOEC. Before proceeding to
our analysis, we note that.differences occur in the number and
frequencies of the resonance Raman modes of NiOEC in Nazso.
and in CHzCl2 solution reported here. The 1644, 1608 and 1572

cm'1 modes of NiOEC in NaZSO, pellets are not observed in

72
either the resonance Raman or IR spectra of NiOEC in solution

and appear to be artifacts of the solid state spectra of
NiOEC. These modes are not considered further in this

analysis.

The 1648, 1614 and 1512 cm"1 modes of NiOEC in Nazso, were
assigned by Boldt gt at. to C3;,stretching modes that are 1,6
localized, 0,3 localized, and delocalized, respectively. In
CuOEC, we observe the analogous modes at 1644, 1602 and 1507
cm'l. From their methine d, shifts and apparent insensitivity
to changes in peripheral substituents at the c; positions,
these modes are assigned to 0.0, stretching vibrations in
agreement with Boldt gt g_. Furthermore, the observation of
equal and additive frequency shifts upon 0,3 and 1,6
deuteration shows that the 1507 cmq‘mode is delocalized over
the chlorin macrocycle. Our deuteration shifts also confirm
that the 1602 cm'1 mode is 0,3 localized. The 1644 cm’1 mode
shows both 0,3 and 1,6 C$;,stretching character but with a
greater localization in the'1,6 methines than in the 0,3
methines. This is a slight alteration from the assignment of
Boldt gt gl. who represented this mode as a 50/50 in-phase
linear combination of the vm and.u$h modes of NiOEP. Since we
observe frequency shifts upon both 0,3 and 1,6 deuteration,
a more accurate representation of this mode could be derived
from a 50/20 in-phase combination of um and.u,h. The 1644 and
1507 cm'1 modes of CuOEC retain much of the character of the

um and v3 modes of NiOEP.

73
The compositions deduced for the 1584 and 1486 cm"1 modes

of CuOEC do not agree with those proposed by Boldt gt g1. The
1584 cm'1 mode exhibits mixed Cbcb and 0,0, stretching character
as judged by the intermediate K value, the +4 cm’1 shift upon
change of peripheral substituents and the ~9 cm"1 shift upon
d. methine deuteration. The equal ~4 cm"1 shifts upon 0,3 and
1,6 deuteration indicate a delocalized mode. Boldt gt 3]..
assigned the analogous 1590 cm'1 mode of NiOEC in Nazso. to
C,C,,, stretching localized in the 0,3 methines. Disagreement
is also found for the 1492 cm'1 mode of NiOEC in Nazso. which
was assigned by Boldt gt g1. to C,C,, stretching localized in
rings I, II and III. We observe the corresponding mode at 1486
cm'1 for CuOEC in CH2C12 solution with Q, excitation. The high
K value and ~10 cm'1 shift upon d. methine deuteration indicate
0,0. stretching rather than 0.0,, stretching character. The
frequency shifts upon 0,3 and 1,6 deuteration show a greater

1,6 localization for this mode.

For CuOEC, we assign the 1547 cm’1 mode to (3be stretching
and the 1543 cm'1 mode to a mixture of Cbe stretching and 1,6
localized C.C, stretching. The observation of these two modes
in CuOEC is interesting since the normal coordinate analysis
of Boldt gt g1. predicts four modes in this region at 1561,
1557, 1540 and 1532 cm". Two of the modes (calculated at 1561
and 1557 cm“) are predicted to have (2be stretching character
while the calculated 1540 cm’1 mode is predicted to have C,Cb

stretching character. In their resonance Raman spectra of

74
NiOEC in Nazso, only a single mode at 1546 cm'1 was observed

and this mode did not shift in NiOEC-1,6~d2. This 1546 cm'1
mode of NiOEC was assigned by Boldt gt g1. to C,,Cb and 0,3
localized C,C_, stretching calculated at 1532 cm". The Cbcb
stretching character seen in the 1547 and 1543 cm'1 modes
supports the normal coordinate analysis although the 0,0,
stretching character in the 1543 cm’1 mode has 1,6 localization

and not 0,3 localization.

ii. C,N Modes

Under C2 symmetry, six C.N stretching modes would be
expected for a metallochlorin (107). The resonance Raman
spectrum of CuOEC-”N, recorded by Ozaki gt gl. (95) showed
that the 1402, 1372, 1361, 1318, 1157 (see 108) and 1128 cm’1
modes of CuOEC shifted by ~1, ~6, ~5, ~5, ~9 and ~19 cm",
respectively, upon ”N, substitution. These modes are therefore
assigned to C,N stretching. In .Table 3.6, we have summarized
the Raman active modes observed for CuOEC and NiOEC in CHZClz
solution and for NiOEC in Nazso, along with the assignments
proposed by Boldt gt g1. (83) from their normal coordinate
analysis. In this region, solution spectra gave somewhat
better resolution than pellet data as the 1367 cm'1 mode of
NiOEC in Nazso. (and in our spectra in KBr, Figure 3.12) has
two components in CHZCl2 solution at 1373 and 1363 cm".

Similarly, for CuOEC in KBr (Figure 3.13) , we observe a single

Table 3.6 Vibrational frequencies (cm”) and isotope shifts
for metallochlorin C,N modes

Ian")

 

Normal coordinate analysis

 

Au Au results for NiOECb

NiOEC CuOEC 15N." subst. No. Obs. Calc. Assignment

1404 1402 -1 o 14 1402 1489 uC,Cb(I,II,III),
uC,N(I,III,IV)

1373 1372 ~6 -4 --

1363 1361 -5 -5 17 1367 1339 uC,N(I,II,III,IV),
VCbe(I,II,III)

1306 1318 -5 -3 20 1308 1301 6C,H(0,3),
uC,Cb(I,III)

1154 1157 -9 -3 27 1152 1147 quEt(I,II,III),
VC,N

1123 1128 -19 -3 29 1124 1083 quEt(I,II,III),

SC,C,N (IV)

 

‘ from ref. 95.

b from ref. 83.

76
1364 cm’1 mode that corresponds to the 1372 and 1361 cm'1 bands

of CuOEC in solution.

The normal coordinate analysis of NiOEC indicates that
there is mixing of the C,N character with C,Cb, Cbe and CbEt
stretching coordinates. The involvement of the Cb atoms in
these vibrational modes can be seen from the sensitivity of
four of the modes to a change of peripheral substituents in
CuECI (Table 3.6). The 1308 and 1124 cm'1 modes of NiOEC in
Nazso, were assigned to modes 20 and 29, respectively, but the
P.E.D.'s have no contribution from C,N stretching. In order
to account for the observed 15N), frequency shifts it would
perhaps be better to assign the 1318 cm"1 mode of CuOEC to
mode number 19 (uC,N(I,III,IV), 6C,H(1,6)) and the 1128 cm‘1

mode to number 28 (quEt(I,II,III), vC,N(I,III)).

iii . Cmfi Modes

The normal coordinate calculations of Boldt gt gt. (83)
indicate that the 0,3 and 1,8 hydrogen motions of NiOEC are
mixed with porphyrin skeletal modes and furthermore, that the
1,6 hydrogen motions are mixed with C1,}! deformations on ring
IV. Table 3.7 collects our information on the more prominent
modes that have can contributions. As was found for the
skeletal modes, the 0.11 modes can be divided into three

classes: a) those that are localized to the 0,3 porphyrin-like

77

Table 3.7 Vibrational frequencies (cm'l) for CuOEC normal modes

in the frequency region below 1350 cm'1

 

 

CuOEC 0,3-d2 7,6~d2 d. NiOEP mode #
1. 0,3 localized
1277 -- 1275 -- --
1215 946 1215 946 um
-- 1325 -~ 1322 uu
-- 1178 -- 1180 u“
2. 1,6 localized
1238 -~ 1224 ~-
1198 ~-
-- 1173
1141 1140 914 914

 

78
part of the molecule, b) those that are 1,6 localized, and c)

those that are substantially delocalized throughout the

macrocycle.

The 0,3 CmH deformations show clear porphyrin-like
behavior. Comparison of the spectra of CuOEC and CuOEC-0,3-d2
(Figure 3.8) reveals that the mode at 1215 cmd'shifts to 946
cmd'and an intense band at 1178 cmd'appears upon deuteration.
This behavior is analogous to that exhibited by the um and v“
modes of NiOEP (55,56,59). Moreover, the appearance of the
1325 cm’1 mode in the Cu0]i:C-0,3~d2 spectrum reflects that of
V12 in NiOEP, which is usually not observed in the natural
abundance spectrum (55). Boldt. gt, gt. predicted. an 0,3
localized mode at 1277 cm'1 and the spectra in Figure 3.8
confirm this. However, this vibration does not have a
corresponding metalloporphyrin normal mode. The vibrational
coordinates of the above four modes are 0,3 localized as they
are unperturbed by 1,6-d2 deuteration and their frequencies in
the 0,3-d.2 species persist virtually unchanged in CuOEC-d“
Some indication that the 0,3 CtH bending deformations
contribute to more delocalized modes can be seen in the
behavior of the cluster of modes in the 1190-1250 cmd'region,
which shows complicated intensity and frequency shifts in all

three of the deuterated complexes relative to CuOEC.

The 1,6 CIll deformations were suggested by Boldt gt gt. to

be extensively mixed with ring modes and thus to exhibit

79
complex behavior. Nonetheless, the data of Figure 3.8 provide

several key insights into the characteristics of these
motions. Upon 1,6-d2 deuteration the mode at 1141 cm'1
disappears and new modes at 914 and 1173 cmd'become apparent.
The 1238 cm'1 mode apparently downshifts to 1224 cm”1 upon the
same substitution. The behavior of the 1141 and 914 cmfl’modes
indicates that these motions are strongly localized as their
frequencies carry through essentially unchanged in the 0,3-d2
and d, species, respectively. A judgement as to the extent of
localization of the 1173 cmq'mode is rendered difficult since
this region is obscured by the strong 0,3 localized 1180 cm’1
mode in CuOEC-0,3-d2 and CuOEC-d.. Soret excitation data (not

shown), however, suggest that.this mode is also 1,6 localized.

iv. Ethyl Group Vibrations

The IR spectrum of CuOEC shows seven modes at 1464, 1452,
1375, 1064, 1058, 1015 and 956 cm'1 that are insensitive to
metal substitution, methine deuteration and a change in
peripheral substituents and are therefore assigned to ethyl
group vibrations. These modes are not observed in the
resonance Raman spectra except for the 1464 cmd'mode that is
present in nearly all of the resonance Raman spectra of the
chlorin complexes that we have examined. The chlorin
macrocycle does not influence the internal vibrations of the

ethyl groups since the frequencies are all close to the values

80
l. (100) from the IR spectrum of NiOEP

reported by Kincaid gt

in an argon matrix.

4. Conclusions

From our analysis of the resonance Raman and IR spectra
of the MOEC complexes we have shown that the high frequency
vibrational modes of metallochlorins cannot be assigned by
direct comparison with the normal modes of metalloporphyrins.
The normal coordinate analysis of NiOEC performed by Boldt gt
gt. therefore represents the starting point for the descrip~
tion of the vibrational modes of metallochlorins. In the work
presented here, the mode compositions deduced for metallo~
chlorins by metal substitution, methine deuteration and a
change in the ‘peripheral substituents of OEC Ihave been
compared with the assignments proposed by Boldt gt gt. The
overall agreement is fairly good although several mode
assignments need to be modified in the normal coordinate

analysis.

The agreement between our results and the normal
coordinate analysis of Boldt gt gt. is especially gratifying
considering that it is the first normal coordinate treatment
of a metallochlorin. The wealth of information obtained from
the resonance Raman spectra of metalloporphyrins and

hemoproteins has been possible largely because of the accepted

81
set of mode assignments based on the normal coordinate

analysis of NiOEP published by Abe gt gt. in 1978 (55).
However, several other groups have also performed normal
coordinate treatments for metalloporphyrins (109-116): the
first, by Ogoshi gt gt. (109) appearing in 1972. Even now
there is not complete agreement on metalloporphyrin normal
mode assignments, especially for the IR active Eu modes.
Recent years have seen new normal coordinate analyses that
have attempted to include the peripheral substituents in the
calculations (115,116). Similarly, futher normal coordinate
treatments for metallochlorins can be expected. We hope that
our results for CuOEC and the methine-deuterated CuOEC
complexes will provide the basis for a new normal coordinate
analysis to improve the vibrational mode assignments for

metallochlorins.

CHAPTER 4

VIBRATIONAL PROPERTIES OF METALLOCHLORIN fl CATION RADICALS

1. Introduction

In photosynthesis, the primary charge separation event
occurs at the reaction center chlorophylls P~700 of
Photosystem I (4) and P~680 of Photosystem II (5). In this
process, an electron is removed from the II system of the
chlorophyll macrocycle resulting in the formation of a
chlorophyll 1r cation radical. P~700 is thought to have a
dimeric structure in the ground neutral state but in the
oxidized state, P~7OO+ the unpaired electron appears to be
localized, on one of the. two chlorophylls (11-14). Lutz
compared the resonance Raman spectra of the bacterio-
chlorophyll a cation radical in solution and in the reaction
center, P~870 of Rtodopseugomonas sphaeroides (117). It was
concluded that the positive charge is localized on one of the
two bacteriochlorophylls of P~87O+ on the time scale of the
resonance Raman effect (~107” s). Establishment of the vibra-
tional properties of metallochlorin Ir cation radicals is
essential if resonance Raman spectroscopy is to be used to

characterize the reaction center chlorophylls.

82

83
The vibrational properties of metalloporphyrin t cation

radicals have been characterized by resonance Raman spec-
troscopy in this lab by T. Oertling and A. Salehi (99,
118-120). The results from these studies showed similar
potential energy distributions in the normal modes for both
the neutral and oxidized MOEP species. In other words, the
mode compositions do not change upon oxidation of the
porphyrin macrocycle. The frequencies of the cg; stretching
modes were observed to increase, while those of the C5; and
(aN stretching modes decreased. In collaboration with these
authors, preliminary results of the investigation into the
resonance Raman spectral characteristics of metallochlorin w

cation radicals have been reported (121).

The n cation radicals of metallo-methyloctaethylchlorin
(MMeOEC) (Figure 4.1) were chosen for the initial resonance
Raman study. These compounds do not have vicinal protons on
the reduced ring (122) so that oxidative dehydrogenation back
to the porphyrin is not possible. The resonance Raman spectra
of the Cu, Co and Ni complexes of MeOEC show two modes in the
1500-1520 and 1620-1650 om’1 regions that appeared to be
analogous to the C,C, stretching modes V3 and um, respectively
of metalloporphyrins (121). Upon oxidation of the chlorin
ring, the I’m-like mode shifted by ~10 cm'1 but the v3~like mode
exhibited.a slight increase in frequency. The K values for the
I’m-like mode indicated C,,Cm stretching character in both

MMeOEC (K=385) and MMeOEC” (K=375). The decrease in frequency

84

 

MMeOEC
M = C0, CU, Ni

Figure 4.1 Structure of metallo-methyloctaethylchlorin
(MMeOEC)

85
upon oxidation for the vm~like mode is consistent with the

results of Oertling gt gt. (120) for a C5; mode of MOEP. The
lower’ K 'value and increase in frequency’ upon oxidation
suggested that the ug~like mode of MMeOEC consisted of mixed
C.C, and Cbcb stretching character. In MMeOEC” it was proposed
that the frequency increase from the Cbe character offsets

the frequency decrease from the C5; character (121).

The study of the I cation radicals of MMeOEC ‘was
performed before the analysis of the vibrational mode
characteristics of the MOEC complexes had been completed. To
explore further the vibrational properties of metallochlorin
1f cation radicals, the resonance Raman spectra of the 1r cation
radicals of CuOEC, CuECI (Figure 3.1) and. the 'methine~
deuterated CuOEC complexes (CuOEC-0,3-d2, Cu0EC~—y,6~d2 and
CuOEC-d0 are presented in this Chapter. Comparison of the
spectra of the n cation radicals with those of the neutral
species shows that our initial interpretation requires
modification and that unlike the situation in metallo-
porphyrins, changes in mode composition occur upon oxidation

of metallochlorins.

86
2. Results

Metallochlorins were oxidized chemically at room
temperature by using the AgClO../ CHZClz method described by
Salehi gt g_. (118). The electronic absorption spectrum of
CuOEC“ in CHZCl2 solution is shown in Figure 4.2. The spectrum
displays a split Soret band and a disappearance of the visible

bands.

Resonance Raman spectra of CuOEC, CuECI, CuOEC-0,3-d2,
CuOEC-7,4‘S-d2 and CuOEC-d, and their Ir cation radicals were
obtained with Soret excitation by using the 363.8 nm line from
a Coherent Innova 100-20 Ar“ laser. Spectra were recorded by
T. Oertling on a Spex 1877 triple monochromator equipped with
OMA III electronics. Figure 4.3 shows the resonance Raman
spectra of the neutral copper chlorin complexes in CHZClz
solution. The resonance Raman spectra of the corresponding 1
cation radicals are shown in Figure 4.4. In the high frequency
region, the 1642, 1601, 1582, 1547 and 1507 cmq'modes of CuOEC
are replaced by another set of modes at 1632, 1611, 1605, 1560

and 1511 011'1 in CuOEC“.

87

 

 

 

 

 

389
374
m
o
z
<
m
n:
8 <—— X2
:0
<
478
I l l J
300 400 500 600 700

WAVELENGTH, nm

Figure 4.2 Electronic absorption spectrum of CuOEC+' in CHZCl2
solution

88

five I

 

kex = 363.8 nm

a) Cu trans- OEC

 

 

>._..wzm.rz_ Z<E<m

 

1

j,

I

‘l

T

I

fir

I

900 1000 1100 1200 1300 1400 1500 1600 1700

 

r_

RAMAN SHIFT (cm°‘)

CuOEC-1,6-d2,

CH2C12 solution obtained

in

4.3 Resonance Raman spectra of CuOEC,
CuOEC-d. and CuECI

Figure
with Soret excitation at 363.8 nm

CuOEC-a,p-dz,

89

363.8 nm
&*

hex

A

b) 412 (Y

_?:mZMHZ_

 

  

 

 

Z<2<m

1

900 1000 1100 1200 1300 1400 1500 1600 1700

1

 

RAMAN SHIFT (cm‘)

btained

1011 O

”, CuOEC-1,6-df“,

CHZCI2 solut

1n

+.

ra of CuOEC

aman spect
and CuECI

+.

“, CuOEC-d.

Resonance R
with Soret excitation at 363.8 nm

Figure 4.4
CUOEC-a , B-dz

90
3. Discussion

In their study of metalloporphyrin 1r cation radicals,
Oertling gt al. were able to directly correlate the resonance
Raman modes of MOEP with those of MOEP“. The K values for the
u;, V3, um and un modes in MOEP and MOEP" are very close
indicating similar mode compositions in both the neutral and
oxidized species (120). Assignments of the other high
frequency modes of MOEP“ were achieved by comparison of the
frequency shifts following methine deuteration and a change

of peripheral substituents from OEP to EPI (99).

The HOMO's of metalloporphyrins consist of an amiorbital
and an an orbital (41,42). Figure 4.5(a) shows the electron
density contour maps for the a1“ and a“ orbitals of free base
porphine from the M.O. calculations by Spangler g; al. (123).
In the an, orbital, electron density is located on the C,Cb
bonds whereas in the amlorbital, electron density is centered
on the C; and nitrogen atoms. From these diagrams, Oertling
e; al. predicted that formation of the 2A,, state metallo-
porphyrin.w cation radical (by removal of an electron from1the
an, orbital) should result in decreased frequencies of the
C.C,,, C,Cb and C,N stretching modes and increased frequencies
of the C“; stretching modes (Table 4.1). On the other hand,
formation of the inn state I cation radicals would produce
lower frequencies for the C,C., and C,,,Cb modes and higher

frequencies for the C,Cb and C,N modes. The pattern of

91

a) Free Base Porphine

 

Figure 4.5 Electron density contour maps for the am and a“
orbitals of a) free base porphine and b) free base chlorin
(from ref. 123)

92
frequency shifts .observed for MOEP upon oxidation is

consistent with the zAlu ground state for MOEP”.

Table 4.1 Predicted and observed frequency shifts for
porphyrin normal modes upon oxidation (from ref.99)

 

 

Predicted Observed Ava,
2A1. 21s.,u for MOEP CuOEP
VCaCn " ‘ ‘ V10('5) : V3(’4)
uC.Ch - + " V29 ('14)
uC.N - + - v, (-17)
vaCb + - + u2(+24) , v11(+34)

 

The am and.am,orbitals of free base chlorin (Figure 4.5)
are almost identical to those of porphine except for the
region around ring IV in the an, orbital. Electron density
shifts from the C5; bonds in the an orbital of porphine onto
the 1,6 C,Cm bonds adjacent to ring IV of chlorin. EPR
measurements show that chlorin 1r cation radicals have unpaired
spin density at the C, positions as a result of removal of an
electron from the ah,orbital (124). Therefore, metallochlorin
resonance Raman modes are expected to show the same pattern
of frequency shifts as for MOEP, namely, decreases in
frequency for the C,Cm, C,,Cb and C,N modes and increases in
frequency for the C“% modes. In addition, metallochlorin 1,6.
localized C4;,stretching modes are predicted to show a larger

decrease in frequency compared to metalloporphyrins since the

93
C,C. interactions are non-bonding in the porphyrin an, orbital

but bonding in the ring IV 1,6 methines of the chlorin an

orbital.

If it is assumed that the resonance Raman modes of
CuOEC'h can be directly correlated with those of CuOEC then
the high frequency modes of CuOEC/CuOEC” may be assigned as
in Table 4.2. The 1642 cm" mode of CuOEC decreases in
frequency by 10 cmq’upon oxidation and is therefore predicted
to have C,CIn stretching character. The 1601, 1582 and 1547
cm’1 modes of CuOEC all show substantial frequency shifts of
+10, +23 and +13 cm", respectively, in CuOEC+°. <3be stretching
character is predicted for these three modes. Compared to
CuOEP, the decrease in frequency of the 1642 cm’1 C,C.
stretching mode of CuOEC is larger whereas the frequency
increases for the our stretching modes of CuOEC are smaller.
Using the same argument as for MMeOEC, the slight 4 cm"1
increase in frequency of the 1507 cm"1 mode of CuOEC can be
attributed to mixed C5; and CJ% stretching character (121).
However, for the 1601 and 1507 cm'1 modes of CuOEC the mode
characters predicted from the oxidation shifts do not agree

with the previously determined mode assignments.

94
Table 4.2 Mode characters predicted for CuOEC/CuOEC”

 

 

CuOEC CuOEC“ Predicted Assignment.
character

1642 1632 vC,,Cm VC.Cm(1,6), vC.Cn(a,fi)

1601 1611 quCb uc,c.,(a,,e)

1582 1605 VCbe VCbe, uc,c,(a,p,1,6)

1547 1560 quCb vaCb

1507 1511 uc,c., yokeh uC.C.(a,fi,—y,6)

 

* From Table 3.4

The frequency shifts resulting from selective methine
deuteration and a change in peripheral substituents of the x
cation radicals may be compared to those of the neutral
species. A shift to lower frequency upon methine deuteration
is indicative of C5;,stretching character in a given mode and
the use of selective methine deuteration at the a,B and 1,6
positions reveals the extent of mode localization. Cbe
stretching character is indicated by an increase in frequency
for a given mode upon the change of peripheral substituents
from OEC to ECI. Table 4.3 summarizes the shifts for the high
frequency resonance Raman modes of CuOEC and CuOEC". The
1642/1632 cm'1 pair of modes of CuOEC/ CuOEC“ show a change
from C4;,stretching with an unequal localization in the 1,6
and a,fi methines in the neutral species to mixed 1,6 localized
C,C., and Cbe stretching upon oxidation. Similarly, the 1582/

1605 and 1507/1511 cm'1 pairs show reduced a,fi localized CJ%

95

Table 4. 3 Vibrational frequencies (cm ) and isotope shifts
(cm ) for the high frequency resonance Raman modes of CuOEC
and CuOEC

 

CuOEC CuOEC“ Au Au Au Au Assignment
a,fi 1,6 d, subst

 

1642 -4 -6 -10 +1 VC.C.(7,6), VC.C.(a,B)
1632 -2 -5 -7 +4 VC.C.(1,6), quCb
1601 -11 +1 -15 +1 uC.C.(a,B)
1611 +1 +1 0 +5 quCb
1582 -1 -1 -3 . +4 quCb, uC.Cm(a,fl,1,6)
1605 O -4 -3 +3 quCb, VC.C,,(1,6)
1547 —1 -1 -4 +3 vaCb
1560 -6 -9 -17 +2 vC.C.(a,fl,1,6), vaCb
1507 -5 -6 -11 -1 VC.C.(a,B,1,6)
1511 -3 -6 '10 +1 vC.C.,(7,6), vC.Cu(a,fl),

V Cbe

 

96
stretching character with an increased contribution from C4%

stretching. Substantial changes in mode composition occur for
the 1601/1611 and 1547/1560 cm'1 pair so that it is unlikely
that a direct correlation exists between these modes in the
neutral and oxidized species. The 1601 cm'1 mode of CuOEC is
an (2,13 localized C,C,, stretching mode for which a frequency
shift of approximately -5 cm'1 would be predicted in the 1r
cation by analogy with the results for MOEP. If the 1601 cm'1
mode is compared to the 1560 cm"1 mode of CuOEC" then the same
trend of reduction of (1,3 localized C,C_ character and gain of
(3be character is maintained but the -40 cm’1 shift is much
larger than any shift previously observed. This would leave
the 1547 cm'1 CbCb stretching mode of CuOEC to correlate with
the 1611 cm'1 Cbe mode of CuOEC'“: an increase of +64 cm".
However, what is probably more important here is not direct
correlation of modes but the overall pattern of increased 1,6

localized C,Cm and ‘3be character in CuOEC“.

97
4. Conclusions

Significant differences exist between the vibrational
properties of metallochlorins and metalloporphyrins. Reduction
of a cg;,bond in a metalloporphyrin to form a metallochlorin
has a far greater effect than a mere lowering of molecular
symmetry. Unlike metalloporphyrins, the neutral MOEC complexes
show mode localization and mixing of C,CIln and Cbe stretching
character. The structural changes in metallochlorins prevent
a direct correlation of the normal modes of MOEP with those
of MOEC. Now, in the metallochlorin 1r cation radicals, changes

in mode composition occur upon ring oxidation.

From their study of the 1r cation radicals of MOEP,
Oertling it _1. concluded that the mode compositions of
metalloporphyrins did not change upon oxidation. These authors
also concluded that there were no significant changes in ring
geometry for metalloporphyrins and their a cation radicals in
solution (125). It is possible that the altered mode
compositions of CuOEC“ result from1 a further* change in
molecular geometry. Unfortunately, x-ray crystal structures
of metallochlorin 1r cation radicals are not available to
support this idea. In addition, with a crystal structure it
would be possible to perform a normal coordinate analysis to
assign the vibrational modes of metallochlorin 1r cation

radicals.

98
The preponderance of 1,6 localized C,C,II stretching modes

in the chlorin 1r cation radicals is puzzling. The 11.0.
diagrams of the an, orbitals of porphine and chlorin would
suggest similar behavior for the a,fi localized C5; modes in
chlorin and delocalized porphyrin Q5; modes but there are no
a,fi localized modes observed in CuOEC“. Chlorin 1r cation
radicals are presumed to have an orbital character but the
radicals may result from a mixture of the a,“ and a2“ orbitals.
Further EPR and ENDOR studies of metallochlorin 1r cation
radicals would help to establish the distribution of spin

density in these species.

CHAPTER 5

VIBRATIONAL PROPERTIES OF METALLOCHLOROPHYLLS

1. Introduction

The study of the IR and resonance Raman spectra of
metallo—octaethylchlorin model compounds is part of a broader
effort to interpret the spectra of naturally-occurring
metallochlorins. Chlorophylls are metallochlorins that play
a crucial role in the process of photosynthesis in higher
plants. The structural complexities introduced into the
chlorophyll molecule by the chlorin macrocycle, the additional
ring V and the peripheral substituents require the use of the
simpler symmetric MOEC complexes in order to understand the

resonance Raman spectra of chlorophyll.

The first resonance Raman spectra of chlorophyll a and
chlorophyll b were reported by Lutz in 1972 (68). This is the
same year in which resonance Raman spectra of several
hemoglobin derivatives were published (131-133). Throughout
the 1970's and 80's resonance Raman studies by many groups
have greatly advanced the area of metalloporphyrin and

hemoprotein chemistry. However, it was not until 1979 that the

99

100
first resonance Raman spectra of metallochlorin model

compounds were reported (95) and 1986 before serious attempts
were made to determine the characteristics of the vibrational
modes of the chlorophylls (83,134,135). Table 5.1 lists the
chlorophylls and chlorophyll-derivatives for which resonance
Raman spectra have been reported. In addition to the
chlorophylls listed in Table 5.1, resonance Raman spectra of
the chlorophyll a cation radical (136), bacteriochlorophyll
a (117,137—140) and its cation radical (117,138,139), and

chlorophylls c1 + c2 (141) have been reported.

Approximately 50 modes are observed in the 50-1750 cm'1
region in the resonance Raman spectra of chlorophyll with
Soret excitation. By' comparison *with. the IR. spectra of
chlorophyll a and chlorophyll b, Lutz identified the resonance
Raman bands for the keto carbonyl stretching vibration of both
molecules at 1690 cm'1 and the formyl carbonyl stretching
vibration of chlorophyll b at 1670 cm"1 (70). No evidence for
a, vinyl stretching' mode was found: the resonance Raman
spectrum of chlorophyll d (where the vinyl group has been
oxidized to a formyl group) showed no difference between those
of chlorophyll a and chlorophyll b in the 1620 cm'1 region
(71). Resonance Raman modes involving motion of the central
magnesium and pyrrole nitrogen atoms in chlorOphyll a and
chlorophyll b were identified by Lutz _e_1_:_ al. in the low
frequency region around 300 cm'1 by 26Mg and 15N isotopic

substitution (72). However, the majority of the vibrational

101

Table 5.1 Chlorophylls and chlorophyll-derivatives for which
resonance Raman spectra have been reported

 

 

Chlorophyll M R,‘ R,‘ Rm" Rn" ref.
chl a Mg CH=CH2 CH, cozcn, C,,H,, 76,*
pheo a H2 CH=CH2 CH, co,CH, C,,H,, 76
chl b Mg CH=CH2 CHo cozcn, C,,H,, 76, *
pheo b H2 CH=CH2 CHo co,CH, c,,H,, 76
chl d Mg CHo CH, co,CH, con,, 76
pheo d H, CH0 CH, co,CH, con,, 76
pyrochl a Mg CH=CH2 CH, H €2,113, 76
2-acetylchl a Mg CH,CO CH, COZCH, C,,H,, 76
3-oximechl b Mg CH=CH2 C=NOH COZCH, CZOH“ 76
M chl a Cu CH=CH2 CH, co,CH, C,,H,, 134-5,*
Ni 134-5,*
Zn 134-5,*
A9 135
Ni MPPh Ni C,H, CH, H CH, 83
Ni PPh Ni CH=CH2 CH, H CH, 83

 

‘ Rm,lg, Rm and Rh are the substituents at the 2, 3, 10 and
7c carbon atoms, respectively, of chlorophyll
(see Figure 1.1).

* This work.

102
modes involving the chlorophyll macrocycle were not assigned.

Metal substitution of chlorophyll a has been used to
identify the core-size sensitive modes (134,135). The results
of these studies support the observation that the frequencies
of three bands in the 1510-1620 cm'1 region of the resonance
Raman spectrum of chlorophyll a could be used to determine the
coordination number of the Mg atom (142) . In various solvents,
bands of chlorophyll a at 1527-1529, 1551-1554 and 1606-1612
cm'1 for S-coordinated Mg shifted to lower frequency at
1518-1521, 1545-1548 and 1596-1599 cm", respectively, for

6-coordinated Mg.

A second approach to the characterization of the
vibrational modes of chlorophyll was undertaken by Boldt 35
al. who examined a series of nickel chlorins in which the
structural complexities of the chlorophyll molecule are added
in a stepwise fashion (83). Resonance Raman spectra were
recorded for NiOEC, NiDMPPh (DMPPh == 9-deoxo—methylmeso-
pyropheophorbide a), NiMPPh (MPPh = methylmesopyropheobide a)
and NiPPh (PPh 8 methylpyropheophorbide a). In this series of
molecules, the chlorin macrocycle, the ring V, the 9-keto
carbonyl and the 2-vinyl group are systematically introduced.
Normal coordinate calculations showed that the C.C,, and Cbe
stretching modes increase in frequency upon addition of ring
V and that many of the modes below 1500 cm'1 retained the same

character as in NiOEC.

103
In this Chapter, the resonance Raman spectra obtained

with Soret excitation of chlorophyll a as well as Zn, Cu and
Ni-substituted chlorophyll a are presented. The MOEC analysis
provides the basis for the interpretation of the resonance
Raman spectra of the metallochlorophylls (M chl a). Comparison
of the K values for the high frequency modes reveals changes
in mode composition on going from MOEC to chlorophyll. The
identification of the core-size sensitive modes of M chl a
provides a means of distinguishing between 5 and 6-coor-

dination of the Mg of chlorophyll a in different solvents and

in yivo.
2. Results

1. Electronic Absorption Spectra

The electronic absorption spectra of chlorophyll a and
Zn, Cu and Ni-substituted chlorophyll a in diethyl ether
solution are shown in Figure 5.1. The absorption maxima are
listed in Table 5.2 together with the ratio of the Qyou to
Soret oscillator strength, r for each complex. The energies
of the transitions follow the order Ni > Cu > Zn z Mg which is
the same trend observed for the MOEC complexes. Conjugation
of the keto carbonyl on ring V causes a further red-shift of
the absorption bands compared to the respective MOEC complex.

The oscillator strength ratios, although much higher than for

104

 

 

 

 

 

 

 

 

 

 

_ 392418
647
37
422
399
Cu
423
8 603
Z
3‘, 405 503 546 65,
a:
O
U)
m
< 376
Zn
430
411
383
Mg
1 L J
350 450 550 650 750

WAVELENGTH, nm

Figure 5.1 Electronic absorption spectra of chlorophyll a and
Zn, Cu and Ni-substituted chlorophyll a in diethyl ether
solution

105
MOEC, also follow the order Ni > Cu > Zn zMg. Two 7) bands are

observed on the high energy side of the Soret band. These are
forbidden in unsubstituted porphyrins but become allowed when
there is a conjugated carbonyl substituent on a porphyrin or

reduced porphyrin.

Table 5.2 Electronic absorption maxima (nm) for metal-
substituted chlorophyll a in diethyl ether solution

 

 

Metal '72 711 Soret 0:01 ono Qyoi Qyoo 1'

Mg 380 409 428 531 575 614 660 0.28
Zn 376 405 423 518 562 606 654 0.25
CD 399 422 503 546 603 650 0.40
N1 371 392 418 496 536 590 647 0.53

 

r = nyOO/fSoret

The electronic absorption spectra of chlorophyll a in
acetone, methanol and pyridine solution are shown in Figure
5.2 and the absorption maxima are listed in Table 5.3. The
spectra of chlorophyll.a in acetone and diethyl ether solution
are characteristic of 5-coordinate Mg. In pyridine solution,
the Mg of chlorophyll a is 6-coordinate and under these
conditions, the Qx00 band shifts to 640 nm. The Mg of chloro-
phyll a is also 6-coordinate in methanol solution but there
is no shift of the Qx00 band. However, the Soret band is lower

in intensity and broadened.

106

 

 

 

 

 

 

 

 

 

 

 

 

_ 430
662
411
383 Acetone
432 5‘5
418 579
535 666“
m :fim
t)
z
<
m
‘6
m Methanol
m
4
443 618
580
536
671
422 n
396
Pyridine
640
620
. l 1 l n l n l
350 450 550 650 750

WAVELENGTH, nm

Figure 5.2 Electronic absorption spectra of chlorophyll a in
acetone, methanol and pyridine solution

107

Table 5.3 Electronic absorption maxima (nm) for chlorophyll
a in various solvents

 

 

SCI-vent '72 "1 soret Q10]. 0:00 03701 QyOO
Diethyl ether 380 409 428 531 575 614 660
Acetone 383 411 430 535 579 616 662
Methanol 390 418 432 536 580 618 666
Pyridine 396 422 443 --- 640 620 671

 

ii. Resonance Raman Spectra

Figure 5.3 shows the resonance Raman spectra of
chlorophyll a and the metal-substituted chlorophyll a com-
plexes obtained with Soret excitation at 406.7 nm. The
resonance Raman spectrum of chlorophyll a was recorded in
frozen acetone at -125°C. Spectra of Zn, Cu and Ni chl a were
recorded. in. diethyl ether solution. at room 'temperature.
Vibrational frequencies are listed in Table 5.4. Twelve modes
of Cu chl a exhibit a core-size dependency. The core-size
correlation parameters K and A in the relation v = K (A - d)

(62) for these twelve modes in M chl a are also included in

Table 5.4.

108

 

 

 

. , . ‘ v 1 - 1
9 0 1100 1300 1500 1700
CM-1

Figure 5.3 Resonance Raman spectra of chlorophyll a in acetone
solution and Zn, Cu and Ni-substituted chlorophyll a in
diethyl ether solution obtained with Soret excitation at 406.7
nm. Laser powers: 35, 20, 20 and 20 mW, respectively

109

Table 5.4 Resonance Raman frequencies (cmq) and core-size
correlation parameters for metal-substituted
chlorophyll a

 

 

Mg Zn Cu Ni K A
<cm“/A) (A)
1613 1615 1641 1657 470 5.49
1553 1561 1581 1596 429 5.68
1532 1539 1554 1570 373 6.17
1492 1499 1510 1521 279 7.42
1436 1439 1451 1463 274 7.31
---- 1358 1361 1364 67 22.2
1348 1348 1351 1353 54 27.0
1331 1334 1336 1339 71 20.7
1225 1224 1230 1240 158 9.81
1186 1187 1191 1196 101 13.9
988 989 991 999 105 11.5
915 918 926 937 217 6.27

 

110
The resonance Raman spectra of chlorophyll a in acetone,

pyridine, methanol and diethyl ether solution at -125%:
obtained with Soret excitation at 441.6 nm.are shown in Figure
5.4. In the region above 1450 cm“, the core-size sensitive
modes of chlorophyll a are observed at 1493, 1529, 1553 and
1616 cmd'in acetone solution. These four modes shift to lower
frequencies in pyridine, methanol and diethyl ether solution.
The keto carbonyl stretching frequency is also observed to be
solvent dependent, occurring at 1681 cm’1 in acetone and
pyridine solution, 1686 cm'1 in diethyl ether solution and

1661 cm’1 in methanol solution (see Chapter 6).

3. Discussion

In Chapter 3, it was shown how metal substitution could
be used to distinguish the amounts of C,C,, and Cbe stretching
character in the core-size sensitive vibrational modes for
metallo-octaethylchlorin (MOEC) complexes. Comparison of the
K values with those for MOEP revealed substantial mixing of
CM; and CHE stretching character in the MOEC normal modes.
Only the 1644 and 1507 cm'1 modes of CuOEC retained similar
character to the porphyrin modes um and u,, respectively.
Although chlorophyll a contains a chlorin macrocycle, the
effects of the additional ring V and altered peripheral
substitution prohibit a direct comparison of the resonance

Raman spectra of chlorophyll a with MOEC.

111

#4
1186
-1290

  
  
 
  
 

Acetone

4w Pyridine
yx
"" a)
3 2
F l
l

 

 

  

 

 

5 Methanol
(D
_ o l
O)
_ N g
.- r- In
m Y-
—. : l
J l
_ Diethyl ether
7 to
Q
_ (D
|
fir T fi‘ 1 ' I ' I
900 1100 1300 1 500 1700
CM‘1

Figure 5.4 Resonance Raman spectra of chlorophyll a in
acetone, pyridine, methanol and diethyl ether solution
obtained with Soret excitation at 441.6 nm. Laser powers: 20,
15, 15 and 15 mW, respectively

112
For CuOEC, seven modes in the region above 1475 cm’1 were

observed to be core-size sensitive. With Soret excitation,
apart from the 1706 cmd'keto carbonyl stretching mode, Cu chl
a shows only four core-size sensitive modes above 1475 cm'1 and
a fifth mode at 1451 cm”; The very large K'values observed for
the 1641, 1581 and 1554 cm‘1 modes of Cu chl a (Table 5.4)
indicate greatly altered mode compositions as a result of the
addition of the fifth ring. For the metallochlorophylls, these
changes in K values cannot be attributed to a simple increase

in the amount of C,C,, stretching character in the normal mode.

The 1641 cm’1 mode of Cu chl a can be correlated with the
1644 cm'1 mode of CuOEC that was assigned to C,C_ stretching
with a greater localization in the 1,6 methines than the 0,8
methines. In M chl a, the K value for this mode increases to
470 from the 317 value observed in MOEC. The increased
core-size sensitivity of this mode is reflected by the fact
that the frequency is higher in Ni chl a compared to NiOEC but
lower in Zn and Cu chl a compared to the respective MOEC
complexes. The large increase in K value may be taken as an
indication of further localization into the 1,6 methines and
that the ring V of chlorophyll places a strain on the 1
methine bridge for the small core-size of Ni. This inter-
pretation is supported by the results of the normal coordinate
analysis by Boldt g; al. for NiPPh (83). The resonance Raman
spectrum of Ni chl a obtained with Soret excitation at 406.7

nm is similar to the spectrum reported by Boldt gt 1],. for

113
NiPPh in NaZSO,. The normal coordinate calculations predict

that the 1658 cmq‘mode of NiPPh (which corresponds to the 1641
cm'1 mode of Cu chl a) does indeed have C,C. stretching
character localized in the 1 methine bridge. Metal substi-
tution shows that this mode correlates with the 1615 cmfl'mode
of chlorophyll a and not the extremely weak 1630 cmfl'mode as
assigned by Boldt gt a1. For the 1451 cm'1 mode of Cu chl a,
an assignment of C4; stretching character is consistent with
the normal coordinate result that the corresponding 1454 cm2'
mode of NiPPh consists of a mixture of a CH,¢deformation on
ring V, 1,6 localized C,C,, stretching and C,C,, stretching
localized in rings I, II and III. It is also interesting to
observe that two other modes, the 1706 cm'1 keto carbonyl
stretching mode and the 1191 cmfl’mode of Cu chl a, exhibit a
metal dependency. The normal coordinate analysis shows that
the keto carbonyl stretching mode is mixed with C,C,_ stretching
character localized in the 7 methine and that the analogue of
the 1191 cm“1 mode at 1188 cm'1 in NiPPh contains a

contribution from C,,,,C10 stretching in ring V.

Difficulties are encountered in trying to reconcile the
core-size sensitivity of the other high frequency modes of
M chl a with the mode assignments proposed by Boldt et al. for
NiPPh. The 1507 cm'1 mode of CuOEC that was assigned to C,C_
stretching delocalized over the macrocycle, correlates with
the 1510 cm'1 mode of Cu chl a. The frequencies of this mode

are higher for the Zn, Cu and Ni chl a complexes compared to

114
MOEC. The K value for M chl a is almost the same as in MOEC,

indicating that there should still be CaCm stretching
character but Boldt g; _l. assign the corresponding mode of
NiPPh at 1522 cm'1 to C,C,, stretching localized in rings I and
III. The 1581 and 1554 cm'1 modes of Cu chl a show no direct
relation to any of the MOEC normal modes but are also
predicted to have substantial C,C,, stretching character based
on their K values. Disagreement is again found since the
analogous modes of NiPPh at 1604 and 1574 cm", respectively,
were assigned by Boldt e_t al. to mixed localized C,C,, and Cbe
stretching character. By relying on only a single metal (i.e.
Ni) it is possible that Boldt gt a1. were not able to assign
these modes correctly. The observation of core-size sensi-
tivity for the high frequency modes does in fact support the
normal coordinate analysis which calculates ten modes from
1467 to 1637 cm'1 that have some degree of C,C_ stretching

character.

Identification of the core-size sensitive modes is
important because the frequencies can be used to determine the
coordination number of the Mg in chlorophyll a. Lutz proposed
that the coordination number of the Mg in chlorophyll a,
chlorophyll b and bacteriochlorophyll a could be diagnosed
based on a Mg-sensitive mode around 300 cm”1 in each of the
three molecules (76). 6-coordination in chlorophyll a was
indicated by a sharp band at 320 cm"1 with a half-bandwidth

of less than 16 cm'1 at 30 K and less than 24 cm'1 at 300 K.

115
S-coordination was indicated by the presence of an additional

shoulder at 302-312 cm’1 with a half-bandwidth of 24 cm"1 or
more at 30 K and 25 cmfl'or more at 300 K. These criteria are
difficult to apply in practice especially for in 2119 systems.
The core-size sensitive modes of chlorophyll a on the other
hand, are easily identifiable and show distinctly different
frequencies for' 5 and 6-coordination. The X-ray’ crystal
structure of ethyl chlorophyllide a dihydrate (37) shows one
water molecule as the ligand to the Mg atom which is 0.39 A
out-of-plane. Conversion to 6-coordination draws the Mg into
the plane of the macrocycle thereby expanding the core. A
lower set of core-size frequencies will then be observed for

6-coordinate chlorophyll a.

Chlorophyll a in acetone solution has a S-coordinate Mg
and displays resonance Raman bands at 1493, 1529, 1553 and
1616 cm'1 (Figure 5.4). In pyridine solution, the Mg is 6-
coordinate and the core-size sensitive bands occur at 1485,
1517, 1542 and 1596 cm”. Previously, the shift of the ono band
in the electronic absorption spectrum of chlorophyll a to 640
nm in pyridine solution was taken to be a characteristic
feature of 6-coordinate Mg (143). However, in methanol solu-
tion, the core-size sensitive modes of chlorophyll a are
observed at 1483, 1516, 1548 and 1597 cm'1 indicating
6-coordination but there is no distinct 640 nm Qx00 absorption
bandJ These results.for chlorophyll a in frozen solution agree

with those of Fujiwara and Tasumi from their resonance Raman

116
study'of'chlorophyll.a in various solvents at:room temperature

(142). One exception is for chlorophyll a in diethyl ether
solution. The room temperature resonance Raman spectrum shows
that the Mg is S-coordinate based on the core-size sensitive
frequencies at 1493, 1529, 1554 and 1607 cm". At lower
temperature, two ether ligands can bind to the Mg so that the
frequencies characteristic of 6-coordination at 1483, 1516,
1542 and 1594 cmfl'are observed in the resonance Raman spectrum

at -125°C.

4. Conclusions

Resonance Raman studies of the chlorophylls have been
dominated by the work of Lutz (68-76). However, a major
limitation to further progress has been the lack of a
consistent set of vibrational mode assignments for chloro-
phyll. The resonance Raman results obtained by Lutz for

chlorophyll in vivo (73-76) are based primarily on a single

 

mode assignment: the keto carbonyl stretching mode. To learn
more about the characteristics of the vibrational modes of
chlorophyll, metal substitution has been utilized to determine

the core-size sensitive modes of chlorophyll a.

Comparison of the frequencies and K values for the
core-size sensitive modes of chlorophyll a with those of OEC

shows that there are substantial changes in mode character

117
between the two types of macrocycle. The observed core-size

sensitivity of the high frequency modes of chlorophyll a is
consistent with the normal coordinate analysis of NiPPh by
Boldt g3, a1. (83). An important result of the normal
coordinate analysis is the prediction of further mode
localization from OEC to chlorophyll. Metal substitution
provides information on the C3; stretching character of the
vibrational modes but does not address the issue of mode
localization. This problem could be approached through the
study of isotopically-substituted chlorophylls or chlorophylls
with different patterns of peripheral substituents. It is
clear that the ring V plays a major role in determining the
mode characters for chlorophyll. Our understanding of these
effects would benefit from data for model porphyrin and

chlorin systems containing an additional ring V.

CHAPTER 6

RESONANCE RAMAN SPECTROSCOPY OF

CHLOROPHYLL-BINDING PROTEINS

1. Introduction

The chlorophylls in plants and bacteriochlorophylls in
photosynthetic bacteria are bound to proteins (20,22). One of
the advantages of resonance Raman spectroscopy in the study
of biological molecules is that this technique may be used to
enhance selectively the vibrational spectrum of a chromophore
within a protein environment. Lutz and co-workers have
obtained resonance Raman spectra of bacteriochlorophyll in the
antenna (144-146) and reaction center (117,137,147,148)
complexes from photosynthetic bacteria. In addition, resonance
Raman spectra of the antenna chlorophylls in chloroplast
preparations and in light-harvesting protein complexes have
been reported by Lutz (68,69,73-76). In photosynthetic
systems, the antenna chlorophylls far outnumber the reaction
center chlorophylls. Isolation of reaction center chlorophyll
protein complexes from higher plants in which the polypeptides
containing the antenna chlorophylls have been stripped away,

offers the posibility of using resonance Raman spectroscopy

118

119
to characterize the structures of P~680 and P-700 as well as

their cation radicals, P-680+ and P-700’.

In the photosynthetic membranes of higher plants the
chlorophylls are localized within three major protein
complexes: a light-harvesting chlorophyll a/b protein complex
(LHC), a Photosystem I complex containing P700 and a
Photosystem II complex containing P680. The PS I and PS II
complexes contain the reaction center chlorophylls plus
antenna chlorophylls. These protein complexes may be isolated

by treatment of thylakoid membranes with mild detergents (22) .

The function of LHC is to absorb light and transfer the
excitation energy to the reaction center chlorophylls. LHC
contains approximately 50% of the chlorophyll in a green plant
with a chlorophyll a/b ratio of 1.2. There are 4 chlorophyll
a, 3-4 chlorophyll b and 1-2 xanthophyll carotenoid molecules
per LHC bound to several polypeptides with molecular weights
in the 20 to 30 kDa range. Electron micrographs of two-
dimensional LHC crystals have been reported (149,150) but
these structures provide no information on the arrangement

and binding of the chlorophylls in the protein.

Considerable effort has been devoted to isolation of the
Photosystem II/oxygen-evolving complex (PS II/OEC) since this
is the site of the water-splitting chemistry (151). In the

thylakoid membrane there are approximately 400 chlorophylls

120
for each PS II. Early preparations yielded complexes con-

taining PS II/OEC and LHC with 225 to 250 chlorophylls per
PS II (152,153). Non-ionic detergents may be used to strip
away the LHC leaving the PS II/OEC core complex with a
chlorophyll/PS II ratio of 60 to 1 (92). A proposed arrange-
ment of the polypeptides and electron transfer components in
this protein complex is shown in Figure 6.1. The PS II/OEC
complex consists of six membrane-bound polypeptides with
molecular weights of 47, 43, 34, 32, 10 and 6 kDa as well as
three extrinsic polypeptides with molecular weights of 33, 23
and 17 kDa. Cytochrome b—559 is bound by the 10 and 6 kDa
polypeptides. The 33, 23 and 17 kDa polypeptides are involved
in manganese binding and oxygen evolution. Namba and Batch
(77) have isolated a PS II reaction center consisting of only
the 34 and 32 kDa polypeptides (also referred to as D-2 and
D-1, respectively) and the cytochrome b-559 polypeptides. This
preparation contains 5 chlorophyll a, 2 pheophytin a, 1 B-
carotene and 1 or 2 cytochrome b-559 molecules per PS II. The
homology between the D-1 and D-2 polypeptides and the L and
M subunits of the reaction center from purple photosynthetic
bacteria suggests that the site of primary charge separation

in PS II is located in D-1 and D-2.

While the arrangement of the polypeptides and electron
transfer components in the PS II reaction center is still
under investigation, procedures have already been developed

for the isolation and crystallization of the reaction center

121

 

‘559

 

 

 

 

 

 

 

 

 

 

Figure 6.1 Proposed. arrangement. of 'the jpolypeptides and
electron transfer components in the PS II/OEC complex
(from ref. 154)

122
complexes from photosynthetic bacteria. Determination of the

X-ray crystal structures of the reaction center complexes from
mogopseudgmonas yiridig (155-158) and W M
(159-161) have provided detailed information on the structures
and orientations of the pigments in the bacterial reaction
center. The reaction center complex from B. viridis contains
three protein subunits labelled H, M and L as well as a
cytochrome subunit. 4 bacteriochlorophyll b, 2 bacteriopheo-
phytin b, 1 menaquinone (QA) , 1 non-heme iron and 1 ubiquinone
(0,) are located in the L and M subunits. The arrangement of
the pigments in the L and M subunits of the reaction center
from 3. vir_1_'dis is shown in Figure 6.2. The primary donor
consists of two bacteriochlorophyll b molecules overlapping
at ring I. There are two branches of bacteriochlorophyll b and
bacteriopheophytin b pigments but electron transfer follows
only the pathway on the QA side. The X-ray crystal structure
also shows that the Mg atoms of the bacteriochlorophylls are
5-coordinate with histidine residues in the L and M subunits
serving as the ligands. The crystal structures of the
bacterial reaction center have been of enormous importance for
understanding the mechanism of primary charge separation in
photosynthesis and for providing a model system to test

theories of electron transfer.

The electronic absorption spectra of the antenna
chlorophyll a molecules in vivo show that the Qy band is

red-shifted and broadened compared to monomeric chlorophyll

123

BChl

 

Figure 6.2 Arrangement of pigments in the L and M subunits of

the reaction center from Rhogopseudomonas yiridis
(from ref. 158)

124
a in solution. Broadening of the absorption band can occur by

a homogeneous or heterogeneous mechanism. Homogeneous broad-
ening could result from the general protein environment of a
chlorophyll a molecule whereas heterogeneous broadening would
involve the creation of several distinct chlorophyll a species
or protein environments. Simply placing a chlorophyll molecule
in a protein environment is not sufficient to account for the
spectral changes however. The spectral properties of a series
of synthetic Mg and Zn chlorophyllide-apomyoglobin complexes
were determined to be within the range observed for solvent
effects (162). A red-shift can be produced if there are
charged amino acid residues present in the chlorophyll a
binding site(s). M.O. calculations predicted shifts of 482 to
740 cm'1 for a point charge placed 3.5 A above various

positions of the chlorophyll macrocycle (163).

The combination of the central magnesium atom acting as
an electron acceptor and the keto carbonyl group acting as an
electron donor allows for a variety of molecular associations
of chlorophyll a with sovent molecules and other chlorophyll
a molecules in solution (164). In polar solvents, chlorophyll
a is monomeric with one or two solvent molecules bound to the
central Mg atom. In non-polar solvents, the carbonyl of one
chlorophyll a molecule can coordinate to- the Mg atom of a
second chlorophyll a thereby forming aggregates. Hydrated
polymers of chlorophyll a result from H-bonding to the keto

carbonyl of chlorophyll a by a water molecule coordinated to

125
the Mg of a second chlorophyll a:

H

Mg---o—H---o—_—-C

It has been proposed that the antenna chlorophylls consist of
aggregates (165) and hydrated polymers (37) . Other suggestions
for chlorophyll a in 2139 are chemically modified chlorophyll
species, e.g. the enol form of chlorophyll a (15) or a pro-
tonated Schiff base linkage of the keto carbonyl (166-169),
although none of these has been isolated from chlorophyll-

binding proteins.

Support for several different forms of chlorophyll in
yiyg comes from the work of French e; 11. (170). These authors
used Gaussian components to curve-fit the red absorption band
(0,) measured at -196°C for chloroplast preparations from a
variety of plants and algae. Four major peaks for chlorophyll
a were observed at average wavelengths of 661.6, 669.6, 677.1
and 683.7 nm and two for chlorophyll b at 640.1 and 649.5 nm.
No structures ‘were jproposed. for’ these components. but a
subsequent review by Brown (171) suggested that chlorophyll

dimers and.aggregates are the most likely forms of chlorophyll
1.01119.

Lutz observed that the resonance Raman spectra of

chlorophyll a and chlorophyll b in higher plant chloroplasts

126
and in solution were similar except for differences in the

carbonyl stretching region from 1630 to 1710 cm’1 and for
magnesium-sensitive modes in the 200 to 350 cm'1 region (68,
69,73-76) . At least five interaction states were distinguished
for the chlorophyll a keto carbonyls and two for the chloro-
phyll b formyl carbonyls in giyg (73-76). Comparison with the
resonance Raman spectra of oligomers and hydrated polymers of
chlorophyll a and chlorophyll b showed that these types of
chlorophyll species do ’not occur in the photosynthetic
membrane. It was concluded that the chlorophylls are monomeric
and bound to proteins by their conjugated carbonyls and

magnesium atoms.

In this Chapter, LHC is used as a model system for the
investigation of the resonance Raman spectroscopic properties
of chlorophyll in 1119. Variation of the excitation wavelength
reveals laser frequencies that may be employed to enhance
selectively the resonance Raman spectra of the chlorophyll a
and chlorophyll b molecules in LHC. The results obtained are
supported by crude excitation profile calculations. The
variety of interactions observed for the Carbonyl groups may
provide a mechanism to broaden and red-shift the electronic
absorption bands of the chlorophylls in LHC. Results are also
presented for a 28 kDa chlorophyll a-binding protein and a

PS II reaction center complex from spinach.

127
2. Results

i. Electronic Absorption Spectra

The electronic absorption spectrum of LHC isolated from
pea leaves is shown in Figure 6.3. The bands at 436 and 677
nm correspond to chlorophyll a and those at 475 and 652 nm to
chlorophyll b. The absorption bands of the chlorophylls in LHC
are red-shifted compared to the absorption maxima of monomeric
chlorophyll a (Soret:430 nm and Q,:662 nm) and chlorophyll b
(Soret:456 nm and Qy:645 nm) in acetone solution (Table 6.1) .
LHC also contains carotenoid molecules that absorb in the 400
to 500 nm region although no specific carotenoid absorption

bands are resolved in the spectrum.

Table 6.1 Electronic absorption maxima (nm) of chlorophyll a
and chlorophyll b

 

 

Chlorophyll a Chlorophyll b
Soret Qy Soret Qy
Acetone 430 662 456 645
LHC (pea) 436 677 475 652
LHC (spinach) 437 676 472 652
28 kDa protein 436 677 --- ---

Reaction center 437 674 --- ---

 

128

 

 

 

 

Chl a
436
’ Chl b
475
Chl a
677
LLI
0
g _
g Chl b
O 652
a)
m
<
1 I 1 I I L
300 400 500 600 700

WAVELENGTH, nm

Figure 6.3 Electronic absorption spectrum of LHC isolated from

pea leaves

129
Figure 6.4 shows the electronic absorption spectra of

LHC, the 28 kDa chlorophyll a-binding protein and the LHC-
depleted Photosystem II reaction center complex isolated from
spinach (92). Table 6.1 lists the absorption maxima of the
chlorophylls in these three protein complexes. The 28 kDa
protein and the reaction center complex show absorption bands
from chlorophyll a but not chlorophyll b. The 468 nm band in

the 28 kDa protein results from absorption by carotenoids.

ii. Resonance Raman Spectra

Resonance Raman spectra of LHC at -125W3‘were recorded
with excitation wavelengths from 406.7 to 514.5 nm. Figures
6.5 and 6.6 show the resonance Raman spectra of LHC obtained
with 413.1, 441.6, 472.7 and 514.5 nm excitation. Vibrational
modes of chlorophyll a, chlorophyll b and carotenoids con-
tribute to these spectra. By comparison with the resonance
Raman spectra of chlorophyll a and chlorophyll b in solution,
it is possible to determine which species contribute to the

resonance Raman spectra of LHC at each excitation wavelength.

The resonance Raman spectra of chlorophyll a in acetone
solution at -125°C obtained with 413.1 and 441.6 nm excitation
are shown in Figure 6.7. 413.1 nm excitation enhances the high
frequency vibrational modes of chlorophyll a and with 441.6

nm excitation, both the high and low frequency modes are

 

 

 

 

 

 

 

 

P 437
472
676 LHC
389
652
m
0
z
.<. L
a: 437
O
a)
E 413 28 kDa
Reacfion
Center
350 550 650 ‘ 750

50WAVELENGTH, nm

Figure 6.4 Electronic absorption spectra of LHC, the 28 kDa
chlorophyll a-binding protein and the LHC-depleted Photosystem
II reaction center complex isolated from spinach

131

 

 

 

 

 

 

 

 

" (D
N
In
F
.7 ~-
IO
10
._. Y-
[x I
In
F
(v) ..
-i Pm V Aex— 413.1 nm
1m (‘0 V
‘— F
—4 ‘- l g, 1'- 2
I .— T 8
.5 ,0 I co
('3 ID 1-
° N I
0
v- 1-
35
.4 a) CD
[x (D N
O) '- 7‘ IO
—1 h 01 u) v-
| I F
F
I
c-d
V
7 00
F
F
4 I 18,. 441.6 nm
N
33
33 "63
N g I go
‘- q '-
l v- I
I
I 1 V W T 7r I
100 950 1800
CM'1

Figure 6.5 Resonance Raman spectra of LHC at -125°C obtained
with 413.1 and 441.6 nm excitation. Laser powers: 28 and 25
mW, respectively

132

 

 

 

 

 

 

 

 

 

 

 

 

h
7 N
In
P
and
"‘ kex=472.7nm
01
In
1-
o I
o
d T 8 00 8
.. .— 4 w an
5 I. on m I ‘9
u-I '- Q F
I 53 31’! .- I
m N '- I
- h— I I
‘1' 8’ I
In
0’ N
- I i «1
F
N
In
_, 7-
F
g “'8 1ex=514.5 nm
P
.. g ,..
| I
F
N
{3 v
,_ <1-
| ‘-
I K
I j 1 V I
100 950 1800

Figure 6.6 Resonance Raman spectra of LHC at -125°C obtained
with 472.7 and 514.5 nm excitation. Laser power: 45 mW

133

 

 

 

 

 

 

 

 

 

 

 

 

‘0
ST
I
.., O
a,
3
5 I 33
53
d l
‘ A” = 441.6 nm
.. In
In
(0
.I I g?
(‘0 cu, m '-
.. a; $ I 8
.. I.
.1
_I
d 1“ = 413.1 nm
I‘ I ' T I T 1 r ' 1 1 V T
100 500 900 1300 1700
CNV'

Figure 6.7 Resonance Raman spectra of chlorophyll a in acetone
at -125°C obtained with 413.1 and 441.6 nm excitation. Laser
powers: 19 and 22 mW, respectively

134
enhanced. 472.7 nm excitation results in weak enhancement of

the resonance Raman spectrum of chlorophyll a (spectrum not
shown). The resonance Raman spectra of chlorophyll b in frozen
acetone (Figure 6.8) show enhancement of high and low fre-
quency modes with 472.7 nm excitation: enhancement of high
frequency modes with 441.6 nm excitation: and, weak enhance-

ment with 413.1 nm excitation (spectrum not shown).

Carotenoid vibrational modes are strongly enhanced with
all of the excitation wavelengths used. The 514.5 nm exci-
tation resonance Raman spectrum of LHC contains only
carotenoid vibrational modes so that these bands are readily
identified in the resonance Raman.spectra of LHC recorded.with
other excitation wavelengths. Apart from the carotenoids,
413.1 nm excitation results in selective enhancement of the
chlorophyll a vibrational modes in LHC. Chlorophyll b modes
in LHC are selectively enhanced with 472.7 nm excitation.
However, the resonance Raman spectrum of LHC recorded with
441.6 nm.excitation contains bands from both.chlorophyll a and

chlorophyll b.

Resonance Raman spectra of LHC, the 28 kDa chlorophyll
a-binding protein and the LHC-depleted Photosystem II reaction
center complex from spinach were recorded with 406.7, 441.6
and 476.5 nm excitation for solution samples at 43C. Figure
6.9 shows the resonance Raman spectra of the three protein

complexes obtained with 406.7 nm excitation. At this

135

A." s 472.7 nm

 

 

 

 

 

 

l 1 1 l 1 1 l J
—349
— 748
-—921
—1124
‘ —-1189
—1267
in,“
--I.——
-1650

 

 

 

 

 

 

 

 

 

 

 

 

‘ A" = 441.6 nm
1 W U k
. I 1 l 1 I . I . I ' I . 1 . 1 1
‘I 00 500 900 1300 1700
CM’1

Figure 6.8 Resonance Raman spectra of chlorophyll b in acetone
at -125°C obtained with 441.6 and 472.7 nm excitation. Laser
powers: 20 and 40 mW, respectively

136

-1526

 

-1158
-1527

 

 

 

 

 

-1159
-1527

 

 

   

J §
1

1
-1347
--149‘

Reacflon
Center

—-1224

 

-—1107
~1430

—1004

 

 

I
1 500 1 700

Figure 6.9 Resonance Raman spectra of LHC, the 28 kDa protein
and the reaction. center’ compleX' obtained. with 406.7 nm
excitation. Laser power: 20 mW

137
excitation wavelength, the high frequency vibrational modes

of chlorophyll a are enhanced. The vibrational frequencies of
chlorophyll a in the three complexes are nearly identical
except in the carbonyl stretching region above 1650 cm”. In
this region, LHC shows a broad band with a maximum at 1681
cm'1 and a second component at 1664 cm”: the 28 kDa protein
shows a broad band with a maximum at 1686 cm"1 and; the

reaction center complex shows a sharper band at 1690 cmd'and

a second component at 1676 cm”.

The resonance Raman spectra of LHC, the 28 kDa protein
and. the reaction. center complex: obtained ‘with 441.6 nm
excitation are shown in Figure 6.10. The high and low fre-
quency modes of chlorophyll a are enhanced as well as several
high frequency modes of chlorophyll b. In LHC, the modes at
1564, 1631 and 1641 cm'1 correspond to chlorophyll b.
Chlorophyll b vibrational modes are not observed in the

spectra of the 28 kDa and reaction center complex proteins.

Figure 6.11 shows the resonance Raman.spectra of LHC, the
28 kDa protein and the reaction center complex obtained with
476.5 nm excitation. In LHC, chlorophyll b vibrational modes
are observed. The 28 kDa protein and reaction center complex

show only carotenoid bands.

138

— 1525

-—1158

-—1102

-—12.7
—13“
—1439

 

 

 

 

 

-1155

 

 

- 11“

1525 r

 

 

 

   

 

 

 

 

 

4 g ' 28 kDa
I
f
3 a
7 I
h
5 .
Ia
I
' § Reaction
§ ' Center
3 I
* a 5" I
E l '
l
1 1 v u j I f v I 1 r t 1 I I
200 400 600 800 1 000 1 200 1 400 1 600 1 800
CM°1

Figure 6.10 Resonance Raman spectra of LHC, the 28 kDa.protein

and, the reaction. center' complex: obtained. with 441.6 nm
excitation. Laser power: 22 mW

-1523

 

 

 

 

 

 

 

 

 

 

 

 

1’3
l
LHC;
T §
é a: I
I T
. . s,
a a |
I I 3
§
§ I"
| 28 kDa
§
§
T
Reaction
Center
8
3 'I‘
I

1 I v ] r 1 1 ] l I I
200 400 660 800 1 000 1 200 1 400 1600 1 800

CM-1

Figure 6.11 Resonance Raman spectra of LHC, the 28 kDa protein

and. the reaction. center' complex: obtained. with 476.5 nm
excitation. Laser power: 25 mW

140
3. Discussion

i. Selective Enhancement of Chlorophyll a and Chlorophyll b

LHC contains chlorophyll a, chlorophyll b and carotenoid
molecules, all of which have strong electronic transitions in
the region from 400 to 500 nm. With Soret excitation, the
resonance Raman spectrum of LHC is likely to contain contri-
butions from all three chromophores. Unfortunately, it is not
possible to record the resonance Raman spectrum of LHC without
observing the carotenoid bands but by careful choice of the
laser excitation wavelength, selective enhancement of the
vibrational modes of chlorophyll a and chlorophyll b can be
achieved. Comparison of the resonance Raman spectra of chloro-
phyll a and chlorophyll b in solution with those of LHC shows
that with 413.1 nm excitation, the high frequency modes of
chlorophyll a in LHC are enhanced: with 441.6 nm excitation,
the high and low frequency modes of chlorophyll a and some of
the high frequency modes of chlorophyll b are enhanced: with
472.7 nm excitation, the high and low frequency modes of
chlorophyll b are enhanced: and with 514.5 nm.excitation, only
the carotenoid vibrational modes are enhanced. This pattern
of mode enhancements can be explained by considering the
excitation profiles of the chlorophyll a and chlorophyll b

modes in LHC.

141
An excitation profile is a plot of the intensity of a

Raman band as a function of excitation wavelength. Using the
Born-Oppenheimer approximation to separate the electronic and
vibrational wavefunctions allows the expression for the
polarizability tensor, a” (Chapter 1, section 4) to be

expanded and represented as the sum of two terms:

The A and B terms correspond to Franck-Condon and Herzberg—
Teller scattering mechanisms, respectively (45-48). For Soret
excitation, the intensity of a Raman band is proportional to

the square of the A term (172):

I = |A|2 = <qolio> - <qoli1> 2
E00 " hV 'iI‘ E01 -1111 -11"

where <g0| is the lowest vibrational level of the ground
electronic state, E00 is the energy of the Soret transition
and Em = E00 + y, where v is the vibrational frequency for a
particular mode. The incident laser frequency is uL and P is
the damping term or homogeneous linewidth of the Soret excited
state. The first term corresponds to resonance with the lowest
vibrational level of the Soret excited state, |i0>, and the
second term to resonance with the first vibrational level of

the Soret excited state, |i1>.

 

142
In the calculation of the excitation profile, the

Franck-Condon factors <g0| i0> and <gO|i1> are approximated by
the extinction coefficient, 6, for the Soret band maximum.
This simplified expression predicts an excitation profile for
a vibrational mode to have two peaks, one at the 0-0 tran-
sition and one at the 0-1 transition. As the vibrational
frequency approaches the magnitude of the damping term the two

peaks in the excitation profile coalesce.

Calculated excitation profiles for a high frequency 1600
cm'1 mode and a low frequency 300 cm'1 mode of chlorophyll a
and chlorophyll b are shown in Figure 6.12. The extinction
coefficient and linewidth are obtained from the electronic
absorption spectrum of the chlorophylls in acetone solution.
The positions of the Soret maxima are for the chlorophylls in
LHC. The parameters used for chlorophyll a are: e = 101.5
mM’lcm'l, I‘ = 440 cm'1 and Eoo= 22936 cm'1 (= 436 nm): and for
chlorophyll b: e = 148.0 mM'lcm'l, I‘ = 640 cm'1 and E008 21053
cm'1 (= 475 nm) . Figure 6.12 shows two peaks in the excitation
profiles for the 1600 cm”1 mode but only one for the 300 cm'1
mode. The 1600 cm’1 mode of chlorophyll a is enhanced with
413.1 nm or 441.6 nm excitation but not with 472.7 nm or 514.5
nm excitation. The 1600 cm’1 mode of chlorophyll b is enhanced
with 441.6 or 472.7 nm excitation but not with 413.1 nm or
514.5 nm excitation. The low frequency 300 cm'1 mode of chloro-

phyll a is enhanced with 441.6 nm excitation. For chlorophyll

b, the low frequency 300 cm'1 mode is enhanced with 472.7 nm

143

 

 

Chl a Chl b
" f‘\/\
I, \

E “ v = 1600 cm-1
(D
2 \
Ill
I— \
E \
Z
< \ IE.
5 \
<
m

 

 

 

EXCITATION WAVELENGTH, nm

 

 

 

 

 

Chla

>_ v=300cm'1

t

3

DJ Chlb

I'-

E /\

'2' ’ \

s ’ \

n: g «2 I, I2\ In
1- ‘- N ‘6‘
<- 3 / S \\ IT:

I \.L

 

EXCITATION WAVELENGTH, nm

Figure 6.12 Calculated excitation profiles for a 1600 cm"1 mode
and a 300 cm'1 mode of chlorophyll a (—) and chlorophyll b
(- -) in LHC

144
excitation. These calculated excitation profiles provide a

useful means of interpreting the pattern of chlorophyll a and
chlorophyll b vibrational modes observed in LHC with changing

excitation wavelength.

In previous studies by Lutz on the resonance Raman
spectra of chlorophylls in yiyg it was assumed that the 441.6
nm line provided selective enhancement of chlorophyll a modes
and lines from 457.9 nm to 472.7 nm could be used for selec-
tive enhancement of chlorophyll b modes. The results obtained
here clearly show that care must be taken in asserting that
a resonance Raman spectrum corresponds to only chlorophyll a
or chlorophyll b. Selective enhancement for the high and low
frequency modes of chlorophyll b is indeed possible at 472.7
nm. For chlorophyll a however, the low frequency modes may be
selectively enhanced at 441.6 nm and the high frequency modes

at 413.1 nm.

ii. Mg Coordination State

The electronic absorption spectra of chlorophyll a in
solution are characteristic of the coordination state of the
Mg atom. The most pronounced effect is the large red-shift of
the Qx00 Iband of 6-coordinate chlorophyll a in. pyridine
solution compared to 5-coordinate chlorophyll a in acetone

solution (Figure 5.2). One possible mechanism to broaden the

 

145
absorption bands of the chlorophylls in LHC would be to create

a mixture of 5 and 6-coordinate chlorophyll species in the

protein.

In Chapter 5, the core-size sensitive resonance Raman
modes of chlorophyll a were identified by metal substitution
and it was demonstrated that the frequencies of these modes
could be used to diagnose the coordination state of the Mg
atom in chlorophyll a. From these results and those of
Fujiwara and Tasumi ( 142) , the resonance Raman bands of
chlorophyll a at 1527-1529, 1551-1554 and 1606-1612 cm'1
indicate 5-coordination while a set of lower frequency bands
at 1518-1521, 1545-1548 and 1596-1599 cm”‘ indicate 6-
coordination. The two resonance Raman bands of chlorophyll a
at 1551 and 1610 cm'1 in the 413.1 nm excitation spectrum of
LHC are characteristic of S-coordinate Mg. The strong 1526
cm"1 carotenoid band obscures the third core-size sensitive

chlorophyll a mode. There is no evidence for any 6-coordinate

chlorophyll a.

For chlorophyll b, core-size sensitive resonance Raman
modes at 1520-1523, 1564-1566 and 1607 cm'1 were determined
by Fujiwara and Tasumi (142) to be characteristic of
5-coordination. Upon 6-coordination, the 1564-1566 cm"1 mode
splits into two components at 1549-1551 and 1559 cmq'and the
two other core-size sensitive modes shift to lower frequencies

at 1516-1519 and 1594-1596 cm". The 441.6 nm excitation

146
resonance Raman spectrum of LHC shows two chlorophyll b bands

at 1568 and 1607 cm'1 indicating S-coordination. Again, the
third core-size sensitive mode is obscured by the 1526 cm’1
carotenoid band. The 1552 cmd’band in the 441.6 nm excitation
spectrum is a chlorophyll a vibrational mode and not an
indicator of 6-coordinate chlorophyll b. 472.7 nm excitation
clearly shows the 1568 cm"1 5-coordinate chlorophyll b mode

with no contributions from 6-coordinate chlorophyll b (173).

The observation of 5-coordination only for the chloro-
phyll a and chlorophyll b molecules in LHC rules out mixtures
of 5 and 6-coordinate chlorophyll species as a mechanism to
broaden the absorption bands. X-ray crystal structures of the
reaction center from.‘3. ;yi;idis (157,158) and a light-
harvesting bacteriochlorophyll a-protein from the green photo-
synthetic bacterium Prosthegochlori s w (174) show that
all of the bacteriochlorophylls are 5-coordinate. The four
bacteriochlorophyll b molecules in the reaction center complex
and five of the seven bacteriochlorophyll a molecules in the
2. aestuarii complex are ligated by histidine residues from
the protein. Resonance Raman spectroscopy can distinguish
between 5 and 6-coordination for chlorophyll but cannot

identify the ligands.

EL.

 

147
iii. Carbonyl Stretching Region

In agreement with the findings of Lutz, the resonance
Raman spectra of chlorophyll a and chlorophyll b in LHC and
as monomers in acetone solution differ mainly in the carbonyl
stretching region. Chlorophyll a has a conjugated keto
carbonyl at the 9-position (Figure 1.1). In chlorophyll b
there are two conjugated carbonyl groups: a keto carbonyl at
the 9-position and a formyl carbonyl at the 3-position. Table
6.2 lists the bands observed for LHC in the carbonyl

stretching region and their assignments.

Table 6.2 Chlorophyll a and chlorophyll b carbonyl stretching

 

 

frequencies (cm ) in LHC
413.1 441.6 472.7 Assignment
1697 unbound keto chl a
1693 unbound keto chl b
1686 1688 unbound keto chl a
1675 1674 H-bonded keto chl a
1659 1656 H-bonded keto chl a
1635 1636 H-bonded formyl chl b

1626 1627 H-bonded formyl chl b

 

 

148
Resonance Raman spectra of LHC obtained with 413.1 nm

excitation show four chlorophyll a keto carbonyl stretching
modes at 1697, 1686, 1675 and 1659 on“. 441.6 nm excitation
is expected to enhance both chlorophyll a and chlorophyll b
carbonyl stretching modes. The frequencies of the chlorophyll
a keto carbonyl stretching modes at 1688, 1674 and 1656 cm‘1
are close to those observed with 413.1 nm excitation. The two
modes at 1635 and 1626 cm'1 are assigned to chlorophyll b
formyl carbonyl stretching modes. The Soret band maximum of
chlorophyll b in LHC occurs at 475 nm (= 21053 cmfl) so that
the E01 transition for a 1630 cm‘1 mode of chlorophyll b is
expected at 22683 cm’1 or 440.9 nm. Strong enhancement of the
chlorophyll b formyl carbonyl stretching modes is therefore
expected with 441.6 nm excitation. Only chlorophyll b modes
are enhanced in the 472.7 nm excitation spectrum. Using this
excitation wavelength, the two formyl stretching modes at 1636
and 1627 cm"1 are seen as well as the chlorophyll b keto

carbonyl stretching mode at 1693 cm“.

The variety of chlorophyll a and chlorophyll b carbonyl
stretching frequencies in LHC indicates different populations
of chlorophyll molecules in the protein. Four populations of
chlorophyll a and two of chlorophyll b can be distinguished
from the resonance Raman spectra of LHC. The interactions with
the chlorophyll carbonyl groups may account for the broadened

and red-shifted absorption bands in LHC.

TI

 

149
The two modes at 1697 and 1686 cm”1 in LHC most likely

correspond to stretching vibrations of the unbound keto
carbonyls of chlorophyll a molecules within two different
environments in the protein. The frequency of the keto car-
bonyl stretching mode of chlorophyll a is highly dependent on
the solvent. The resonance Raman spectra of chlorophyll a in
acetone, diethyl ether and pyridine solution at -125°C show
the keto carbonyl stretching frequency at 1681, 1686 and 1681
cm", respectively (Figure 5.4) . Fujiwara and Tasumi (142) and
Koyama et al. (175) have reported resonance Raman spectra of
monomeric chlorophyll a in polar solvents at room temperature.
The keto carbonyl stretching frequency ranged from 1680 to
1702 cm'1 with the frequency increasing as the dielectric
constant of the solvent decreased. The two carbonyl stretching
modes at 1697 and 1686 cm'1 are in the frequency range expected

for an unbound keto carbonyl of monomeric chlorophyll a.

The 413.1 nm excitation spectrum of LHC shows two
additional chlorophyll a keto carbonyl stretching modes at
1675 and 1659 cm". Lutz has proposed that the chlorophyll
carbonyls that are not free are H-bonded to amino acid
residues in the protein (76) . The resonance Raman spectrum of
chlorophyll a in methanol solution (Figure 5.4) shows the
H-bonded keto carbonyl stretching frequency at 1661 cm". The
frequency of this mode increases to 1668 cm”1 in methanol
solution at room temperature (142,175). Koyama gt; :1. observed

that the H-bonded keto carbonyl stretching frequency for

 

150
chlorophyll a in other alcohol solutions ranged from 1670 to

1675 cm”1 and appeared to be independent of the dielectric
constant of the solvent. The 1675 and 1659 cm"1 bands may
therefore be attributed to chlorophyll a H-bonded keto

carbonyl stretching modes with differing H-bond strengths.

H-bonding to the conjugated keto carbonyl of chlorophyll
a can. produce a red-shift. of the electronic absorption
spectrum. Babcock and Callahan (176) determined that H-bonding
to the conjugated formyl group of heme a model compounds
lowered the carbonyl stretching frequency and resulted in a
red—shift of the a (0%) absorption band. The magnitude of the
carbonyl stretching frequency decrease and red-shift both
increase as the strength of the H-bond increases. The low
frequencies of the 1675 and 1659 cm'1 chlorophyll a keto
carbonyl stretching modes and the red-shifted absorption bands
in LHC can be explained by H-bonding. A larger red-shift is
expected for the chlorophyll a population in LHC that has the

1659 cm”1 keto carbonyl stretching frequency.

The same type of analysis can be applied to the chloro-
phyll b carbonyl stretching frequencies in LHC. The 1693 cm‘1
mode in the 472.7 nm excitation is attributed to the unbound
keto carbonyl of chlorophyll b. H—bonded formyl carbonyls are
observed at 1636 and 1627 cm”; Thus there are two populations
of chlorophyll b molecules in LHC with H-bonded formyls of

differing strength but with their keto carbonyls free.

151
iv. 28 kDa and Reaction Center Complex Proteins

Ghanotakis gt _1. have isolated an oxygen-evolving PS II
reaction center core complex from spinach that contains
approximately 60 chlorophylls per PS II (92). Treatment of
PS II membranes with octyl glucopyranoside separates LHC from
the reaction center complex of 9 polypeptides with molecular
weights of 47, 43, 33, 32, 30, 28, 20, 10 and 9 kDa. Solu-
bilization of the reaction center complex with dodecyl fi-D-
maltoside followed by gel-filtration chromatography leaves a
PS II reaction center core complex containing the 47, 43, 33,
32, 30 and 9 kDa polypeptides. The 28 kDa chlorophyll-binding

protein is also isolated in this procedure.

The LHC, 28 kDa protein and LHC-depleted PS II reaction
center complexes isolated by Ghanotakis gt a1. were studied
by resonance Raman spectroscopy. The resonance Raman spectra
of LHC from spinach (Figures 6.9-6.11) are similar to those
of LHC from pea (Figures 6.5 and 6.6). These spectra show the
presence of both chlorophyll a and chlorophyll b in spinach
LHC with 5-coordination of the Mg atoms. Two populations of
chlorophyll b molecules can be distinguished by the stretching
frequencies of their formyl carbonyls at 1631 and 1641 cm'1
in the 441.6 and 476.5 nm excitation spectra. The chlorophyll
a carbonyl stretching region is not resolved since spectra

were recorded at 4°C as compared to -125°C for LHC from pea.

152
Resonance Raman spectra of the 28 kDa and.reaction center

complex proteins recorded with 406.7 and 441.6 nm excitation
show only chlorophyll a and carotenoid vibrational modes. The
reaction center complex preparation still contains 60 chloro-
phylls per PS II. The resonance Raman spectra are dominated
by the antenna chlorophylls so that it 'is not possible to
observe vibrational modes of the reaction center chlorophylls
or the two pheophytin a molecules in each reaction center.
More importantly, the lack of any chlorophyll b vibrational
modes in the 476.5 nm excitation spectra of the 28 kDa protein
and reaction center complex confirms the absence of chloro-
phyll b in these two complexes. Ghanotakis g; a_. suggested
that the 28 kDa protein is either a separate protein
intermediate between LHC and the PS II reaction center complex
or a component of LHC. The 28 kDa protein has a molecular
weight that is typical of LHC polypeptides but these authors
were unable to isolate the 28 kDa protein from LHC. The
absence of chlorophyll b indicates that the 28 kDa protein is

not part of LHC.

y-.f'r'1"l ‘1':

 

‘ .

153
4. Conclusions

LHC contains three types of chromophore: chlorophyll a,
chlorophyll b and carotenoids. The primary function of this
protein.is to absorb light. Comparison‘with the spectra of the
chlorophylls in solution shows that the electronic absorption
bands of LHC are broadened and red-shifted. Broadening of the
absorption bands allows light to be absorbed over a greater
wavelength range thereby increasing the efficiency of light
energy utilization. This is accomplished by the creation of
four separate populations of chlorophyll a and two of chloro-
phyll b in LHC. Different coordination states of the Mg atoms
are not a factor since all of the chlorophylls are 5-
coordinate. H-bonds to the conjugated keto carbonyls are
responsible for creating two of the chlorophyll a populations.
The local protein environments may account for the two other
chlorophyll a populations. The chlorophyll b molecules are
distinguished by the strength of the H-bond to their
conjugated formyl carbonyls. Each.of these species.has its own
distinct absorption spectrum that contributes to the overall
absorption spectrum of LHC. H-bonding to the unconjugated
ester carbonyls may also occur and help to orient the
chlorophyll molecules in the protein but will not influence

the absorption spectrum.

’7

 

154
Determination of the structures of the chlorophylls in

LHC is essential to understanding their function. Once a
photon has been absorbed by LHC, the excitation energy needs
to be transferred to the reaction center. Elucidation of the
mechanism of exciton migration from the antenna chlorophylls
to the reaction center requires knowledge of the arrangements
of the chlorophylls in the protein and the relation of LHC to
the reaction center polypeptides. Help in this area is likely
to come from biochemical procedures for isolating the
individual chlorophyll-binding proteins and.determining their

amino acid sequences.

 

CHAPTER 7

SUGGESTIONS FOR FURTHER WORK

The original goal of this research project was to explore
the vibrational properties of chlorophyll and chlorophyll-
binding proteins using resonance Raman spectroscopy. Resonance
Raman spectra.of chlorophyll a and.chlorophyll b‘were recorded
in solution. and. in ‘the light-harvesting' chlorophyll a/b
protein complex. However, a major obstacle in the inter-
pretation of these spectra was the lack of a set of
vibrational mode assignments for chlorophyll. Attention was
therefore focused on determining the vibrational charac-
teristics of metallochlorins as models for the more complex
chlorophyll. In contrast to the approach taken by other groups
(45,66,95-98), the vibrational modes of metallochlorins could
not be assigned by a direct comparison with the normal modes

of metalloporphyrins.

The starting point for the interpretation of the
resonance Raman and IR spectra of metalloporphyrins is the
normal coordinate analysis of NiOEP performed by Abe gt g1.
(55,56). The set of mode assignments for NiOEP are widely

accepted in resonance Raman studies of metalloporphyrins

155

n are: Mann-mi,
5‘

 

156
although there is still disagreement over the form of the IR

active Eh modes. The NiOEP normal coordinate analysis
attributes modes ya, and was to C.CI and Cbe stretching,
respectively. These assignments are supported by the work of
Willems and Bocian on acetyl and formyl-substituted
deuteroporphyrins (177,178) . Other groups have argued that the
assignments for V37 and V3. should be reversed (65,100,179)
since it is u“ that exhibits the larger core-size dependency
and deuteration shift characteristic of C.C. stretching.
Oertling gt g1. have shown that changing the peripheral
substituents from OEP to EPI can identify those resonance
Raman modes with a contribution from (3be and CbC, stretching
and CbC, bending coordinates (99) . Comparison of the IR spectra
of MOEP and MEPI would be useful for clarifying the E; mode
characters. TheIE; modes for NiOEP have been recalculated by
Abe (compare ref. 56 with 55) but the mode characters for ufl
and v33 were unchanged. A possible source of error in the
determination of the Eutmodes is the use of resonance Raman
frequencies from NiOEP in CIIIZCl2 solution but IR frequencies
from NiOEP in a CsI disk. NiOEP has two crystal structures:
a planar triclinic form (60) and a buckled tetragonal form
(180). We have observed resonance Raman spectra of both forms
of NiOEP in KBr disc samples depending on the ratio of
porphyrin to KBr in the sample. Ideally, resonance Raman and
IR spectra of metalloporphyrins should be measured in the same

medium.

 

     

r..044..d:.t.a_e .— 4.010.443.«Alirelwflreetuou.1.. ...e.....¢.l..h..o. . .. To. :1 a. a .114. ... . 4. I. _. I ..I..

a... .4 - . . It... . acnisrdli.‘

 

157
Numerous factors such as the central metal, axial

ligation, peripheral substitution, macrocycle distortions,
ring oxidation and reduction influence the vibrational
properties of metalloporphyrins in biological systems. Many
of these effects are well understood but one area that
requires further investigation is to determine the conse-
quences of symmetry-lowering. The mode assignments for
metalloporphyrins are based on the normal coordinate analysis
of the highly symmetric NiOEP molecule. Protoheme and heme a
are two examples of naturally-occurring porphyrins that have
conjugated peripheral substituents. The presence of conjugated
substituents eg. vinyl and carbonyl groups, lowers the
molecular symmetry thereby allowing the Eu modes to become
resonance Raman active. Analyses have been reported for
protoporphyrin IX (65,116,179) as well as formyl and acetyl-
substituted deuteroporphyrins (177,178). Further studies of
model compounds are needed to establish precisely how far the
symmetry must be lowered to induce Raman activity of the Eu
modes (and IR activity of the resonance Raman modes).
Determination of excitation profiles would be useful since
Shelnutt has developed expressions for calculating excitation
profiles of metalloporphyrins that include the effects of

symmetry-lowering (181,182).

A special case of symmetry-lowering is the reduction of
a Cbe bond in a metalloporphyrin to form a metallochlorin. We

have seen that the high frequency modes of metallochlorins are

158
simultaneously IR and resonance Raman active and exhibit

mixing of C.C, and Cth stretching character. The normal
coordinate analysis of NiOEC by Boldt gt g1. (83) represented
several modes as linear combinations of normal modes of NiOEP.
For example, the 1648 cm'1 mode of NiOEC in NazSO. was
described as a 50/50 linear in-phase combination of um and
ya,“ It is not clear whether this is simply a convenient
representation of the form of the metallochlorin normal mode
or if it is actually derived by mixing of the metalloporphyrin

v10 mode with the E“l mode V37 as a result of symmetry-lowering.

There are several experiments to complete the charac-
terization of the vibrational modes of metallochlorins. Our
study has concentrated on the modes above 900 cm"1 which leaves
the assignments for the low frequency modes unresolved. Also,
a complication in the assignment of the Cu}! modes is that the
normal coordinate analysis of NiOEC predicts that the a,fi and
7,6-hydrogen motions are mixed with porphyrin skeletal modes
and that the 7,6-hydrogen motions are further mixed with on:
deformations on ring IV. Involvement of the ring IV CbH modes
can be tested by preparing CuOEC with deuterium substitution
on ring IV. Results for this complex as well as the
selectively methine-deuterated CuOEC complexes could then be
utilized in a refinement of the normal coordinate analysis for

MOEC .

.13.. an. 3! .-

 

159
Once the mode assignments for metallochlorins have been

firmly established then the features that are of interest to
the study of metallochlorins in nature e.g. peripheral sub-
stituents and axial ligation can be explored. Excitation
profiles of the intensities and depolarization ratios of the
metallochlorin Raman bands are a topic for investigation.
Ozaki gt g1. (95) measured the depolarization ratios for the
high frequency modes of CuOEC with 488.0 nm excitation and
attempted to correlate the metallochlorin resonance Raman
modes with those of NiOEP. However, the resonance Raman bands
of CuOEC show a wide variation in the depolarization ratio
depending on the excitation wavelength. For example, the 1602
cm'1 mode of CuOEC has a depolarization ratio of 0.6 (p) at
406.7 nm, 1.0 (ap) at 488.0 nm and 0.5 (p) at 615.0 nm.
Excitation profiles and depolarization ratio studies would

help in understanding the resonance Raman scattering mechanism

and symmetry effects in metallochlorins.

Although chlorophyll contains a chlorin macrocycle, the
effects of the peripheral substituents and ring V prevent a
direct correlation of the resonance Raman modes of chlorophyll
with those of MOEC. Boldt gt g1. examined a series of Ni
chlorins in which the structural features of chlorophyll a
were added stepwise (83). However, only a single metal was
utilized. Our results for metal-substituted chlorophyll a show
that ring V plays a major role in determining the vibrational

properties of chlorophyll. The normal modes of metallochlorins

 

160
and metalloporphyrins cannot be directly compared but

characterization of porphyrin model compounds with an
isocyclic ring would help in understanding the effects of ring
V in chlorophyll. Suitable model compounds are protochlo—
rophyll a, which is a porphyrin with the same peripheral
substituents as chlorophyll a (183,184) and the porphyrin
compounds prepared by Kenner and co-workers (185-187). The
peripheral substituents also complicate the determination of
the vibrational mode characters of chlorophyll. It is possible
that vibrational modes of the conjugated carbonyl groups are
mixed with those of the chlorophyll macrocycle. Centeno
observed that several resonance Raman modes of heme a
contained contributions from the conjugated formyl group by
use of O“‘substitution.of the formyl (188). This substitution
was achieved through preparation of the Schiff base of heme
a followed by hydrolysis with Hgf‘. The same approach may be
used for chlorophyll a except that the Schiff base must be
prepared for pyrochorophyll a (189). Reaction of chlorophyll
a with amines to form the Schiff base would instead result in

the rupture of ring V (190,191).

H-bonding to the conjugated keto and formyl carbonyls of
chlorophyll a and. chlorophyll b, respectively, has. been
proposed as a mechanism to account for the broadened and
red-shifted electronic absorption bands of the chlorophylls
in LHC. This conclusion is based on a comparison with the

results obtained for H-bonded heme a model compounds (176).

 

161
To support the LHC results, a further study of H-bonding to

the chlorophyll carbonyls could be carried out. Aggregation
of chlorophyll in H-bonding solvents such as alcohols can be
avoided by using the pheophytins or metal-substituted
chlorophylls.

A final suggestion concerns the manner in which future
resonance Raman studies of metalloporphyrins are conducted.
Resonance Raman work on metalloporphyrins and hemoproteins for
the past decade have relied almost exclusively on the normal
coordinate analysis of NiOEP performed by Abe gt g1. in 1978
(55) . While the mode assignments for NiOEP have benefitted the
study of porphyrins, investigations of other macrocycles have
been limited. Currently, the best approach is that of the
Bocian group. In recent years, these workers have explored a
variety of new systems including metallochlorins (83, 192, 193) ,
chlorophyll model compounds (83), bacteriochlorins (193), a
bacteriochlorophyll a model compound (194) and metallo-
porphyrin anion radicals (195). The resonance Raman spectra
were supported by normal coordinate calculations for each
system thus removing the reliance on the NiOEP mode
assignments. It is anticipated that computer calculations will
assume a much greater role in future resonance Raman studies

of metalloporphyrins and other related systems.

n

 

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The un mode appears as a shoulder on the broad V; band
in the resonance Raman spectra of CuOEP and CuOEP-d2 with
Soret excitation at 406.7 nm. With 363.8 nm excitation,
the spectra of CuOEP and CuOEP-d, obtained by Oertling gt
g1. (99) show an increase in the relative intensity of

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

168

uu compared to u, and no frequency shift upon d, methine
deuteration.

Ozaki gt g1. (95) assigned the 1602 cm1 mode of CuOEC
as dp and the 1584 cm1 mode as ap. These polarizations
should be reversed, i. e. 1602 (ap) and 1584 (dp).

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The K values reported by Ozaki gt 31. (66) for the
FeOEP high frequency resonance Raman modes follow the
same relative order as the K values based on Ni, Cu and
ZnOEP (Table 3.5) but are all higher: um (K=495),

u, (K=322), V19 (K=576), yn (K=288) and v3 (K=414). Using
the same porphyrin core-sizes for the FeOEC complexes and
their previous mode assignments for chlorins, the K
values obtained for the chlorins were: "9m" (K=472),
":12" (K=404), "um" (K=332), "uu" (K=367) and

'93" (K=347). On this basis, the K values for the chlorin
Cbe modes "u," and "um" are higher than the chlorin C.CIII
modes "1119" and "v3". Moreover, the frequencies predicted
for CuOEC and ZnOEC based on the FeOEC core-size
correlation parameters of Ozaki are too low (CuOEC: 1633,
1593,1579,1538 and 1500 cm 1: ZnOEC: 1611, 1574,1564,
1521 and 1484 cm 1).

The six C,N stretching modes are derived from the 9,, tin,
Mm: V222 and u“ modes of NiOEP. The most recent normal
coordinate treatment of the Eu modes of NiOEP by Abe (56)
assigned the P.E.D. of u,5 to C3; stretching and not CJJ
stretching (55).

The 1157 cm'1 mode of CuOEC and 1154 cm'1 mode of NiOEC
are overlapped by the 1154 cm'1 band of CHzClz. The
presence of these modes is confirmed by_ the resonance
Raman spectra in KBr that show an 1155 cm1mode for CuOEC
and an 1153 cm1 mode for NiOEC.

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x-ray crystal structures of the metalloporphyrin
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Lutz claimed to observe both 5 and 6-coordinate
chlorophyll b in LHC based on the relative intensities
of two low frequency bands at 312 and 304 cm1 (75). The
312 cm1 component was attributed to 5-coordinate
chlorophyll b and the 304 cm1 component was attributed
to 6-coordination. Lutz has proposed (76) for chlorophyll
b that a single band at 300- 310 cm 1with a half-bandwidth
of 25 cm1 or less at 30 K and 27 cm1 or less at 300 K
indicates 6-coordination. A S-coordinate Mg is indicated
by the presence of an additional band.at 310- 320 cm 1with
an increase of half-bandwidth to 28- -35 cm1. However,
these criteria are not reliable predictors of the Mg
coordination state. For example, chlorophyll b in acetone
solution at room temperature is predicted by Lutz to have
6-coordination (76) but the frequencies of the core-size
sensitive modes indicate S-coordination (142).

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