EPR, ENDOR, and TRIPLE Resonance Studies of Modified

Aug 20, 1993 - Max- Volmer- Institut f i r Biophysikalische und Physikalische Chemie, Technische Universitat Berlin, Strasse d. 17. Juni 135, 0-10623 ...
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J. Phys. Chem. 1994, 98, 354-363

354

EPR, ENDOR, and TRIPLE Resonance Studies of Modified Bacteriochlorophyll Cation Radicals H. Kiss,? J. Rautter,+ W. Zweygarbt A. Struck,* H. Scbeer,* and W. Lubitz'J Max- Volmer-Institut f i r Biophysikalische und Physikalische Chemie, Technische Universitat Berlin, Strasse d. 17. Juni 135, 0-10623 Berlin, FRG, and Boranisches Instirut der Ludwig-Maximilian-UniversitatMiinchen, Menzinger Strasse 67, 0-80638 Miinchen. FRG Received: August 20, 1993; In Final Form: October 15. 1993O

A series of substituted bacteriochlorophyll molecules, all used in reconstitution experiments of reaction centers of Rhodobacter sphaeroides (Struck et al. Biochim. Biophys. Acta 1991,1060,262-270), were characterized by EPR, electron-nuclear double (ENDOR), and electron-nuclear-nuclear triple (TRIPLE) resonance spectroscopy in their monomeric radical cation states. Effects of different substituents at position 3 in the porphyrin macrocycle were considered, especially for two "crosslinks" between plant and bacterial chlorophylls. These are 3-vinylbacteriochlorophyllwhere the "bacteria" acetyl group at position 3 was substituted by vinyl and 3-acetylchlorophyllwhere the "plant" vinyl group was substituted by acetyl. In addition, effects of substitutions at position 132were studied. All major hyperfine coupling constants of proton and nitrogen nuclei were elucidated from the spectra and assigned to molecular positions by comparison with the parent radicals. The data were compared with those calculated by an INDO-type program, showing that INDO essentially models the effect of the different substituents correctly.

Introduction Chlorophyll (Chl) and bacteriochlorophyll (BChl) molecules play a multifarious role in plant (oxygenic) and bacterial photosynthesis, respectively. In the light harvesting "antenna" complexes (LHC), they are involved in the absorption of light and the transfer of the excitation energy; in the reaction centers (RCs), they take part in the light-inducedcharge separation and the subsequent electron transport processes.' In addition, (B)Chls have recently become prime candidates in photodynamic therapy because they are excellent singlet oxygen sensitizers, have strong absorptions in the red and near-infrared optical ranges, and are rapidly degradedS2 For a profound understanding of the structure-function relationship of photosynthetic complexes, a knowledge of both the spatial and electronic structure is required. Here, the crystallization and subsequent X-ray structure analysis of the RCs from two purple bacteria, Rhodopseudomonas ( R . )viridis3 and Rhodobacter (R.)sphaeroides? have been of key importance. The latter contains three protein subunits (L, M, and H), a BChl a dimer (D), two monomeric BChl a, two bacteriopheophytins (BPhe) a, two ubiquinones QAand QB, and a nonheme Fe2+.The cofactorsare arranged in two branches which are related to each other by an approximate Crsymmetry axis running through the dimerand theiron. Inspiteofthe high symmetry, electron transfer (ET) proceeds only via one pigment branch? The first ET steps are very fast, highly directional, and require no activation energy; the quantum yield of the process is very close to unity. Apart from the protein, holding the prosthetic groups in the right juxtaposition for optimum ET, the choice of the cofactors is also an important factor in this highly optimized process: Different bacteria may contain different metals, quinones, and pigments in their RCs.5 For example, three major classes of BChl's are found: BChl a (e.g. in R. sphaeroides), BChl b (in R . viridis), and BChl g (in Heliobacterium chlorum).6 The major structural difference between these species lies in the substitution pattern of the bacteriochlorin macrocycle, i.e. in the substitution at positions C-3 and C-8 in rings A and B (Figure la). In contrast

* To whom correspondence should be addressed.

Technische Universitat Berlin. UniversitPlt Munchen. *Abstract published in Aduance ACS Absrracts. December IS, 1993. t

0022-3654194f 2O98-O354$04.50f 0

to the bacterial species, the plant chlorophylls have a dehydrogenated ring B (Figure IC). During the last few years, site-directed mutagenesis has been widely used to alter the amino acid surrounding of the cofactors with the aim of elucidating the role of the protein in the lightinduced charge-separation proces~.~Another supplementary approach to determine the structure-function relationshipof the RC is the specific chemical alteration of the cofactorsthemselves. Removal and replacement has been reported for the twoquinoness and the metalg in the RC of R . sphaeroides. Recently, Scheer et al. reported the first successful exchange of the (monomeric) BChl's and BPhe's in RCs of the same species.'* This paved the way for replacing the native pigments with a variety of other species. Thereby the specific properties of the protein-binding pocket can be probed. Furthermore, pigments with altered spectroscopicand redox properties can be introduced which change the RC function. A first step in this endeavor is the synthesisand characterization of suitable pigment molecules with modified subsitutents. In the past, the chemical and physical properties of chlorophylls and many chlorophyll derivatives have been studied in great detail, whereas those of the bacteriochlorophylls are less well known.' To understand the structure-function relationships of BChl's and Chl's in more detail, it is important to obtain structural links between the different naturally occurring species. One such link is [3-acetyl]-Chla, bearing the 3-acetyl group characteristic for BChl a and b but the unsaturated ring B of the plant chlorophylls (Figure IC). The complementarylink is [3-vinyl]-BChl a, which differs from Chl a by the hydrogenated double bond in ring B and from BChl a by the presence of the vinyl group instead of the acetyl group at position 3 (Figure la). The vinyl and acetyl groups are both conjugated to the r-macrocycle; therefore, a species with a nonconjugated alkyl group will complete the picture: [3-a-hydroxyethyl]-BChla. Another interesting region of the BChl macrocycle is the sterically crowded region between rings D and E. We have, therefore, prepared a species with an opened ring E (Figure 1b) and various compounds with different substituents at position C-132 (Figure la). The latter are also interesting in view of the possible enolization and epimerization at this position and the putative involvement of such species in the primary processes of photosynthesis.Il Futhermore, it should be pointed out that the position of the closest approach between 0 1994 American Chemical Society

The Journal of Physical Chemistry, Vol. 98, No. 1 , 1994 355

Bacteriochlorophyll Cation Radicals

b

a

C

H2C

H2C

dOOCH3

I

I

COOCHJ

COO-phytyl

1-7

COO-phytyl

8

9,lO

Figure 1. (a) Structure of bacteriochlorophylls. Substituents of positions 3 and 132 are given in Table 1. (b) Structure of Mg-bacteriochlorin q trimethyl ester (B-Chlorin). (c) Structure of the plant chlorophyll u (Chl u) and [3-acctyl]-Chlu (substituents in Table 1). In this paper, protons directly attached to the r system are called a-protons, ,%protonsare one bond away from the T system, y-protons are two bonds away, etc.

TABLE 1: Investigated Bacteriochlorophylls (Bchl) and cblorophylls (Chl) and Their Derivatives substituents*

name

IabelO

Ri

Rz

R3 H

figure la

1 COCH3 BChl u H la COCHa 2 pyro-BChl u H la 3 CHCHz [3-vinyl]-BChlu H la CHCH2 4 [3-vinyl]-pyro-BChlu H la CHOHCHa [3-(a-hydroxyethyl)]-BChl d 5 OH la COCHo 6 132-hydroxy-BChld OH la [3-vinyl]-l32-hydroxy-BChld 7 CHCH2 lb 8 Mg-bacteriochlorin e7 trimethyl ester H IC 9 CHCH2 Chl u H IC COCH3 [3-acctyl]-Chl u 10 * The label (1-10) is used in the text and in the figures. The positions of substituents R1, R2, and R3 are given in the corresponding Figure la or IC. e Stereochemistry at 31 undefined. d 132-Epimer mixture, >90% has stereochemistry as shown.

the pigments in the RC is in the vicinity of rings A and C/E. It is, therefore, important to investigate the local effects imposed by specific substituents on the spatial and electronic structure in these rings. Several (bacterio)chlorophyll derivatives have been prepared recently and characterized by Struck et a1.I0 The method of choice to investigate the electronic structure of these species in their oxidized or reduced radical states is EPR-based hyperfine spectroscopy which can resolve the hyperfine coupling constants (hfc's) between the unpaired electron and the different magnetic nuclei (LH, 14N,etc.) in the molecule. Here, we apply electronnuclear double (ENDOR) and electron-nuclear-nuclear TRIPLE resonancespectroscopyin liquid solution.12 Thereby, effects from anisotropic broadening are avoided and sharp resonance lines are obtained from which the isotropic hfc's (including their signs) for all hf-coupled nuclei can be directly obtained. Assignment of the measured hfc's by various chemical and spectroscopictechniques to specific molecular p i t i o n s yields a map of the electron spin density distribution over the molecule. In the cation radical, the electronic structure of the highest occupied molecular orbital (HOMO) of the (B)Chl's is probed, Since the HOMO is of special interest and since the parent compounds BChl u+* and Chl (I+* l4lL5 are quite well understood, we have focused our attention on the cation rudiculs of the modified pigments. Furthermore, characterization of (modified) Chl u+* species is highly relevant for the interpretation of theEPR/ENDOR spectra of the primary donor cation radicals P& and Pzio in plant photosystems I and 11, respectively (for reviews see refs 16 and 17). Thevalence electron density distribution can also be calculated for a given molecular structure by molecular orbital (MO) theory. Various methods were applied to the important class of the I33l4

(bacterio)chlorophylIsin the past.18 A very promising approach to calculate the electron spin densities is provided by the semiempirical all-valence-electron SCF method RHF-INDO/ SP introduced by Plato et al.,l8.*9 which is based on the wellknown INDO method of Pople and Beveridge.m Here, we apply this method to calculate the spin density distributions for the cation radicals of BChl (I,Chl u, and several of their derivatives, and we compare the theoretical values with the experimental ones. This yields insight into the reliability of such calculations predicting orbital distributions for large biologically relevant molecules in vitro and in their native protein environment.

Materials and Methods Bacteriochlorophyll u (BChl a) (1). was isolated from R. sphueroides and purified as described in ref 10. Chlorophyll u (Chl a) (9), was obtained from Sigma and used without further purification. The following pigments with modified substituents were synthesized10 from BChl u (see Table 1): pyro-BChl u (2) [3-vinyl]-BChl u (3) [3-vinyl]-l32-hydroxy-BChlu (7), Mgbacteriochlorin e7 trimethyl ester (8), and [3-acetyl]-Chl u (10). For synthesisof 10, seeref 2 1. All pigment molecules were purified to absorption spectral and chromatographic purity and characterized by their optical absorption, 1H NMR, and FAB-MS spectra; for details seeref 1Oa-d. Figure la-cshows thestructures of all molecules investigated in this paper, and the different substituents are listed in Table 1. For better reading, the compounds are abbreviated in all tables in a mnemonic way: pyro-BChluasp-BChlu, [3-vinyl]-BChluas 3v-BChlu, [34nyl]pyro-BChl a as 3v-p-BChl u, [3-((~-hydroxyethyl)]-BChlu as 3he-BChl u, 132-hydroxy-BChlu as 132h-BChl (I,[3-~inyl]-13~hydroxy-BChl u as 3v-132h-BChl u, Mg-bacteriochlorin e7 trimethyl ester as B-Chlorin, and [3-acetyl]-Chl u as 3ac-Chl u.

KBss et al.

356 The Journal of Physical Chemistry, Vol. 98, No. 1 , 1994

a

A

I

I

0.340

0.342

3+’

0.344

a two-spin system S = l/2, I = l/2, with arbitrary hf coupling tensors and includes the hf enhancement effect.12 Several ENDOR spectra were taken fromeach compound. In somecases, additional lines appeared during the measurements. These lines are caused by degradation products. They could be clearly distinguished from the other lines by their evolution in time. Molecular orbital (MO) calculations were performed using the latest version of the RHF-INDO/SP program developed by Plato et al.18 It is based on the well-known intermediate neglect of differential overlap (INDO) scheme of Pople and BeveridgeZO but uses Dewar’s ‘half-electron method”28 in the framework of a restricted HartreeFock (RHF) approach with a subsequent treatment of spin polarization (SP) effects. The method has been described recently in detail.18

0.348

Bm

Figure 2. EPR spectrum (a) and computer simulation (b) of [3-vinyl]BChl a+* in CH~CI~ICHJOH (6:l) at 240 K (2 X 10-4 M),chemically

oxidized with iodine. Experimental conditions: Bruker TEl02 cavity, modulation amplitude 0.03 mT, modulation frequency 100 kHz, microwave power 0.79 mW, receiver gain 2 X lo5,time constant 320 ms, scan time 8 X 200 s. The simulation employed the experimentally determined hfc’s and assignments for 3+‘ given in Tables 2 and 4.

Exchange of the native pigments in the reaction centers with 2 and 6 was achieved by the method described in ref 10. The EPR/ENDOR samples were prepared on a high-vacuum line in dim green light. First, water was removed from the material by repetitive (SX) azeotropic codistillation with CH2C12. The BChl u derivatives were dissolved in a mixture of CH2C12/ CH3OH (6:l) to a concentration of 3 X 10-4 M. The cation radicals were generated by oxidation with a 5-fold excess of iodine at about 0 “C to prevent decomposition of the compound^.^^.^^ For Chl u and [3-acetyl]-Chl u, a solvent mixture of CH2C12/ THF (1O:l) and a 10-fold excess of iodine were used.24 The reaction centers were measured in liquid solution. These samples were concentrated to approximately 2.5 X 10-4 M and filled in capillaries (i.d., 1 mm). ENDOR spectra were taken at about 283 K under continuous illumination by R focused 100-W tungsten lamp equipped with a water filter (10-cm path length). EPR and ENDOR measurements were performed on a Bruker ER 200D spectrometer with home-built ENDOR and TRIPLE resonance accessories. A TMIlo-type mw cavity/rf coil arrangement similar to that described earlier25.26 was constructed which provided a high Q factor (-5000) and thereby a high ENDOR sensitivity. The rf coil (0.d. 8 mm, 16 turns, 20-mm height) had an inductance of L = 0.65 pH; it was glued onto the outside of the quartz Dewar in the ENDOR cavity. A maximum rf field BZof 20(25) G,,(rot. frame) at 14(3) MHz was achieved with an rf power input of 500 W. The ENDOR frequency was supplied by an rf synthesizer (Rohde & Schwarz, SMG), which was frequency-modulated (fm) at 12.5 kHz, followed by a 500-W power amplifier (EN1 A-500), rf coil, and a 5 0 4 load (Lucas Weinschel). The spectrometer was controlled by an Atari Mega ST4 computer that wasalsoused for data acquisition and handling. The rf power was leveled to achieve a constant B2 value across the whole ENDOR spectrum. For General TRIPLElZexperiments, a fixed frequency from a second unmodulated rf source (Rohde & Schwarz, SMG) was added with a Minicircuits power combiner to the swept rf. A temperature-control system (Bruker ER 41 11 VT) was used with precooled nitrogen gas passing through the quartz Dewar (0.d. 8 mm, i.d. 5 mm), placed inside the ENDOR cavity. The temperature at the sample could be adjusted between 120and 300 K. ENDOR spectra of the samples were taken in the liquid state at 240 K and also in frozen solution at 120-130 K. For the analysis of the powder ENDOR patterns a simulation program was used which has been developed in our laboratory on the basis of a program described in ref 27. It is able to handle

Results and Discussion

EPR Spectra. The liquid-solution EPR spectra of the cation radicals of all BChl u derivativesgave the typical broad Gaussian lines, with some variation in line widths (1.3-1.4 mT). Nuclear hyperfine structure was only present under high-resolution conditions and was, in most cases, similar to that found for the parent radical BChl u+*.13,23 All BChl u+* cation radicals carrying no acetyl group at position 3 exhibited somewhat different EPR spectra. A good example is provided by 3+*;the respective EPR spectrum is depicted together with its computer simulation in Figure 2. For the simulation, the ‘Hand I4Nhfc’s from ENDOR and their assignments to molecular positions (Table 2 and 4) were used. The agreement between experiment and simulation is very satisfying. Analysis of 1HENDOR Spectra. In Figures 3 and 4 the liquidsolution lH ENDOR spectra of the cation radicals of the various BChl u derivatives are depicted. All measurements were done with the same experimental conditions (see figure captions). According to the ENDOR resonance condition12 =bn

* a/2I

(1) all lines occur in pairs equally spacedaround the respective Larmor frequency, v,. The splittings of the line pairs directly yield the isotropic hfc’s a. In the ‘H ENDOR spectra shown, YIH is 14.7 MHz at a field BOof 0.345 T. Note that the ENDOR intensities of the high-frequencylines aregenerally larger due to the hyperfine enhancement effect.12 An assignment of the methyl proton lines in all spectra is easily possible by inspection of the respective IH ENDOR spectra in frozen solution.29 In such a powder-type spectrum only the resonances from the methyl protons give rise to quite narrow and intense lines due to their almost axially symmetric tensors and their low hf anisotropy which is caused by the free rotation of the methyl groups even in solid matrices (170K). Such frozen solution spectra have been obtained for all radicals studied in this paper primarily with the aim of identifying the methyl proton resonances. A typical spectrum is depicted in Figure 5 together with a computer simulation of the powder pattern. The assignments of the other proton hfc’s rely on a comparison with the fully analyzed spectrum of the parent radical cation BChl u+* l4 and on the MO calculations.18 Figures 3 and 4 show a comparison of the ‘H ENDOR spectra of the various radical cations. The isotropic hfc’s are collected in Table 2. The fully analyzedI4 spectrum of 1+*is taken as a reference point for the discussion of the other spectra: The six largest hfc’s are marked in the spectra (line pairs 1,1’-6,6’). They are assigned to the &protons in rings B and D at positions 7.8, 17, and 18 (1,1’-4,4’) and to the protons of the methyl groups attached toringsAandC,positions21and 121(5,5’4,6’).Smaller hfc’s areobtainedfrom thea-protons (positions 5,10,20) attached to the methine bridges, from the B-proton at position 132,and from some yprotons at rings B and D. 4”R

Bacteriochlorophyll Cation Radicals

Experimental 'H Hfc's [MHz] from Various Bcbl a Type Radical Cations'

TABLE 2 carbon positione 8 7 17 18 121 21 132

The Journal of Physical Chemistry, Vol. 98, No. 1, 1994 357

BChl (I+'

5

(l+*) +16.48 +13.59 +13.06 +11.63 +9.60 +4.84 -1.66 +2.35 1.29 [+1.29]

+

10' 201 3' 32 71

-0.15

p B C h l ~ * + 3v-BChl~" (2+') (3+9 +16.36 +16.60 +13.61 +13.31 +13.08 +13.03 +11.39 +11.13 +10.26 +9.02 +4.78 +2.61 -1.90 -2.03 2.15 +2.15 +1.20 +1.10 [+1.lo] [+1.20] +1.89 -0.95 0.17 -0.21

compoundb 3 ~ - p B C h l ~ + * 3hc-BChl u+* (4+9 (5+') +16.46 +16.60 +13.48 +13.18 +12.97 +12.98 +11.00 +10.95 +9.64 +10.22 +2.61 +2.95 2.11* 2.2w [2.11] [2.20] +1.21 1.64 [+1.21] [ 1.641 1.85 -0.94 0.13 0.22

132h-BChl a+*

3v-132h-BChl (I+* (7+7 +16.94 +14.10 +10.17 +8.74 +9.69 +2.68

(a+*) +16.77 +14.67 +9.95 +9.21 +8.90 +5.07 +2.45 +1.70 [+ 1.701

+2.42

+1.23

[+1.23] +2.21 -1 -04 -0.14 ~

0.17

B-chlorin+*

@+'I +15.49 +14.61 +9.08 +7.68 +5.50

+5.10 -1.43d 2.41 +1.82 [+1.82]

~~

0.19

81

17l -0.59 -0.57 -0.80 -0.56 0.52 -0.57 -0.58 -0.56 181 -0.38 0.45 0 Isotropic 1H hfc's from liquid solution ENDOR and signs from General TRIPLE resonance (see text); for some hfc's the signs could not be determined unambiguously. For abbreviations of radical cation species sce text. For molecular positions sce Figure 1; given are the carbon positions to which the protons are attached. The assignment of the 'H hfc's is based on the known assignment of BChl u+* (see ref 14);.CH3 gr0u.p (pos 122, 22) were identified by comparison with solid-state spectra. Hfc for CH2 group at position 15. This hfc could also belong to the methin proton at position 5 since the sign could not be determined. /The hfc assigned to position 10 could also belong to position 20 (see Table 5). I

I

I

Q'

3+'

6

I

I

I

I

I

5 3 I4 2

8*

2

5' 48 6'

2'

3'

Y

YH

5

10

15

20

25

v [MHzl Figure 3. 1H liquid-solution ENDOR spectra of BChl u+* (P), pyre

BChl u*' ( 2 9 , [3-vinyl]-BChl u+* ( 3 9 , and [3-vinyl]-132-hydroxyBChl u+* (7+*). Corresponding low- and high-frequency linea are numbered for the six largst hfc's (1,1'-4,4' indicate the @-protonsat positions 7,8,17, and 18; $5' and 6,6' the CH3 groups at positions 2 and 12. See Figure 1). Experimental conditions: temperature 235-240 K, frequency modulation (fm) depth 80 kHz (for 1+*,3+*) and 100 kHz (for 2+',7+'), time constant 640 ms, scan time 4 X 800 8, microwave power 10 mW, concentration 3 X l ( r M. The comparison between 1+*and the pyro compound 2+* in ' Figure 3 yields no significant change in the six largest couplings, only the methyl proton hfc at 121 is slightly decreased. When

another pyro compound ( 4 9 is compared with the respective parent radical ( 3 9 (Figure 4)' nearly the same shift of this methyl hfc is observed. Obviously, the electronic distribution in ring C experiences the structural change in the attached ring E,

I

I

I

1

5

10

15

20

I

25

v [MHtl Figure 4. IH liquid-solution ENDOR spectra of [3-(a-hydroxyethyl)]BChl u+* (P), [3-vinyl]-BChlu+* ( 3 9 , [3-vinyl]-pyro-BChl(I+* ( 4 9 . and the radical cation of Mg-bacteriochlorine?trimethyl eater (8+*). For line numbers see Figure 3. (Spectrum of 3+*is shown again for a better comparison.) Experimental conditions as in Figure 3 except for fm depth: 80 k H z (5+*,3+*), 100 kHz ( 4 9 6 0 kHz (8+*). Lines resulting from degradation products in 8+* are marked by black dots.

Le. the removal of the carbomethoxy group (Figure la). The uand yproton hfc's remain fairly constant in both radicals. For the two protons at position 132, different hfc's can be expected in 2+*,depending on the degree of planarity of ring E. According to the results from 1+*,the respective hfc's should both have a negative sign. Since no larger negative 'Hhfc is found in the spectrum of 2+*,the hfc of -1.90 MHz is assigned to this position. For radical 4+* an assignment of the respective protons at 132 is

358 The Journul of Physical Chemistry, Vol. 98, No. 1, 1994

A

lb

116

i0 Y

[MHzl

i5

Figure 5. Comparison of lH ENDOR of [3-vinyl]-BChl a+. ( 3 9 in liquid solution at 240 K (a) and in frozen solution at 140 K (b). Experimental conditions: (a) scc Figure 3; (b) fm depth 200 kHz,time constant 640 nu,a n time 10 X 400s, microwave power 12.6 mW. The lines assigned to methyl proton hfc's are indicated by arrows. The solidstate spectrum (b) is shown together with a simulation (dashed line) of the powder patterns for the two methyl proton hfc tensors (see text).

difficult since more hfc's (vinylgroup and methyl at 2') contribute to the group of smaIl hfc's (see Table 2). Replacement of the acetyl group in I+' with a vinyl group (3+9causes a major change of the spectrum. Whereas the four large &proton hfc's remain fairly constant, the two large methyl hfc's are shifted in opposite directions: the 12l-hfc is somewhat increased, but the 2l-hfc is quite drastically decreased. It is important to notice that this is a characteristic effect found in all BChl a species in which the acetyl group is replaced by vinyl (radicals 3+*,4+*, and 7+*; see Figures 3 and 4 and Table 2). Obviously, it is not only the methyl group at position 2 adjacent to the vinyl group which experiences this change but also that at position 12 at the other end of the macrocycle, although to a lesser extent. However, conformations and spin densities of the hydrogenated rings B and D are not changed by the substitution since the respective b-hfc's remain almost constant. In the range of the small couplings,additional hfc's are expected for the vinyl protons. A possible candidate for the proton at position 31 is the +1.89-MHz hfc which is also found in the vinyl species 4+* but is not present in the radicals bearing the acetyl group. A second vinyl hfc (position 32) is assigned to the hfc of approximately -1 MHz, which also appears only in the vinylsubstituted radicals 3+*,4+', and 7+* (see Table 2). Replacement of the acetyl group at position 3 by a nonconjugated alkyl substituent (a-hydroxyethyl) in 5+* results in a change of the two methyl group hfc's, which is very similar to that observed for the vinyl compound 3+' (Table 2). This shows that both substituents have no significant effect on the electron spin distribution in the r system. Obviously, interactions with the neighboring substituents at positions 2 and 5 (Figure la) force the vinyl group into an out-of-plane position (see below) and diminish conjugation between the r systems. This results in a similar effect of the vinyl and hydroxyethyl substituents and in a similar r spin density distribution of the respective radicals I+* and 5+'. An identification of the &protons in the a-hydroxyethyl substituent in the spectrum of 5+* is difficult. These resonances are most probably found in the range of the small hfc's ( 1 3 MHz). An interesting problem is the origin of the quite different hfc's belonging to the methyl protons at positins 2l and 121. Both groups are located in symmetry-equivalent positions at the tetrapyrrol macrocycle and have a keto substitttent as neighbor in all (B)Chl's carrying the 3-acetyl group. One possible reason for the inequivalenceof rings A and C could be the presence of the constrained isocyclic ring E in all Chl's and BChl's. In compound 8 this ring has been opened (Figure lb). The spin density distribution of the respective radical 8+*yields an almost q u a l spin distribution in rings A and C, Le. almost identical methyl hfc's for positions 2l and 12' (Table 2). Similar

Ksiss et al. observationshave been reported for the Chl a+*and the respective chlorin radical ~ a t i o n . ~ ~It.is, ~ ' therefore, concluded that in all chlorophyll-type cation radicals the asymmetric spin density distribution (in the HOMO) is caused by the presence of the isocyclic ring E. An interesting feature of the IH ENDOR spectrum of the bacteriochlorin 8+*is the drastic decrease of two of the four large &proton hfc's. Since ring B and its surroundingsare not changed, these two hfc's should belong to ring D (positions 17 and 18). The decrease of the respective hfc's could be explained by the presence of the sterically demanding substituent at position 15 in 8+'. This might influence the geometry of the adjacent hydrogenated ring D (increasing twist angle, see below), whereby both &protons adopt a more in-plane position and the respective hfc's are decreased. A similar effect has been observed earlier in the 132hydroxy-BChl II cation radicaV2which has been reinvestigated in this paper (see data for 6+' in Table2; spectrum not showdX'3). Clearly, the &proton hfc's assigned to ring D (positions 17, 18) are drastically reduced in 6+' compared with I+*. The geometrical changeof ring D isattributed here to thesteric interactionbetween the substituent at carbon 17 and the two substituents at position 132. This steric interaction also influences the methyl hfc at 121 in the neighboring ring C, whereas the hfc's of rings B and A show only minor effects. In the range of the small hfc's, the negative hfc assigned to the 132proton in 1+' is clearly missing in 6+'. The remaining positive and smaller negative hfc's in 6+' are assigned to methine and y-protons. A comparison of the data of radical 7+*,which alsocontains a hydroxy group at position 132,with those of the related radical 3+' shows the same behavior as found for the pair 6+' and I+* (Table 2). Figure 5 compares the lH ENDOR spectrum of 3+' in the liquid state at 240 K with that in frozen solution at 140 K.In the solid state, a pronounced matrix line is observed at the proton Larmor frequency, comprising weakly dipolar-coupled nuclei of the radical and its surroundings, and two broad line pairs with shoulders that are assigned to the freely rotating CH3 groups in positions 12 and 2 (cf. Figure la). A simulation of the powder ENDOR line shape yields the tensor componentsof the two methyl proton hfc tensors. The values are (in MHz) All = 2.1, AZ2= 2.65, A33 = 3.4, and air0 = 2.7 for position 2 and All = 9.0, A22 = 10.1, A33 = 11.5, and am = 10.2 for position 12. The tensors are both not axially symmetric. Furthermore, the anisotropy is different; it is larger for position 2', which is adjacent to the vinyl group. This effect is also found in all radicals carrying the acetyl group. The isotropic hfc's are in good agreement with the data from liquid solution (Table 2). As indicated in Figure 5 (arrows), a spectral comparison yields an unequivocal assignment of the methyl proton resonances in the liquid solution spectrum. In Figure 6,three 'H ENDOR spectra at different temperatures are shown for the Chl a cation radical 9+'. In sharp contrast to the BChl a cation radicals, the ENDOR effect becomes very weak above 200 K. At 195 K the liquid solution spectrum is only partially resolved and shows fairly broad lines (- 150kHz, Figure 6b). A similar spectrum has been described earlier.l4J0t3' At 140 K the sample becomes a soft glass (Figure 6c) in which motional averaging of some hfc anisotropies, e.g. of the methyl protons, is still possible. Furthermore, no matrix line at the proton Larmor frequency VH (14.7 MHz) is observed. At 120 K the sample becomes a polycrystallinesolid powder (Figure 6d). Here, it is possible to distinguish the methyl proton lines from the j3-proton lines as described above (Figure 5). The solid-state spectrum of 9+* is similar to that described earlier by Scheer et a1.15 The measurad isotropic 'H hfc's for 9+' are collected in Table 3, The largest hfc is assigned to the two &protons in the hydrogenated ring D. (Note that in Chl u ring B is unsaturated and has no such @-protons.) The hfc of +7.00 MHz belongs to the methyl group at position 121; whereas, the hfc at -3 MHz

Bacteriochlorophyll Cation Radicals

The Journal of Physical Chemistry, Vol. 98, No. 1, 1994 359

TABLE 3 Experimental and Calculated 1H Hfc%[MHzr for Chl a+ and [&acetyl)-Chl a+* carbon positionb 17

+ c

5

18 121 2' 71 132 5 10 20 31 32

3

experiment Chl a+* 3ac-Chi a+* (9+*) (lo+') (+)9.90 (+)10.24 (+)9.20 (+)7.00 (+)6.95 [3.07] (+)4.75 (+)3.07d (+)2.95 (-) 1.68 1.26* (+)0.53/

8l

17l 18l

5

10

15

20

25

v [MHrl Figure 6. IH ENDOR spectra of Chl a+* (9+') and [3-acetyl]-Chl a+* (lo+'). Corresponding low- and high-frequency lines are numbered, for hfc's and tentative assignments see Table 3. Sample concentration 3 X 10-4 M, solvent CHZClz/THF (lO:l), oxidation with a 10-fold exccss of iodine. (a) lo+' in "glassy state" at 130 K, (b) 9+' in liquid solution at 195 K, (c) 9+* in "glassy state" at 140 K, (d) 9+* in solid state (powder) at 120 K. Note that in 9+' (b-d) lines belonging to sets 1 and 2 and to sets 4 and 5 are superimposed, whereas they are well resolved in lo+*(a). Experimental conditions: fm depth 100 kHz (a,c), 200 kHz (b,d), time constant 640 nu,scan time 4 X 400 s, microwave power 63 mW.

comprises several resonances assigned to the methyl groups at positions 21 and 7l and the CH2 group at 8l according to the analysis of ref 15. The negative hfc is assigned to position 132, and the remaining small hfc might contain contributions from the methine, vinyl, and y-protons (see Table 3). In 10, the vinyl group at position 3 is replaced by an acetyl group. The 1H ENDOR spectrum of the respective cation radical lo+*at 140 K is shown together with the Chl a+* spectra in Figure 6 . lo+*shows an additional line pair which is assigned to the methyl group at position 2 adjacent to the acetyl group. The effect observed here is similar to that found for 1+*and 3+* in the bacterial systems. The assignment given above for the three methyl proton ENDOR line pairs was verified by ENDOR in frozen solution at 120K (spectrum not shown). Furthermore, the resonances of the 8-protons at ring D (positions 17 and 18) are clearly split in lo+*. All isotropic hfc's are collected in Table 3. Analysis of 14NENWR spectra. For all bacterial species the 14N ENDOR spectra have also been obtained. The 14NLarmor frequency at the external field of Bo = 0.345 T is V N = 1.06 MHz. Since the 14N hfc's in BChl-type cation radicals are quite sma11,14,26J4the resonances all occur below 3 MHz. Due to the small 14N transition moments, these ENDOR resonances are difficult to detect in the liquid state, and usually only the highfrequency lines can be observed. An advantage of such spectra is that neither the anisotropy of the hf interaction nor the quadrupolar interaction contributes to the ENDOR line positions, and the isotropic I4N hfc's are directly obtained from the spectra according to eq 1.

0.4Y

[OS31

[0.42]

[3.07] [OS31

[0.42]

calculationc Chl a+* 3ac-Chl a+* (9+9 (lo+') +10.97 +11.00 +9.79 +5.73 +4.94 +4.64 -1.47 +4.36 +5.54 +0.79 +1.71 4.40 +3.05 4.59 4-25

+10.25 +5.77 +6.80 +4.50 -1.56 +4.68 +5.44 +0.48 +2.96 4.25 4-59

,I Experimental data from liquid-solution ENDOR (see Figure 6), calculated values from RHF-INDO/SP using QH = 1420 MHz, signs of hfc's for Chl a+* determined by General TRIPLE resonance (see ref 34), and signs for 3aeChl a+* given in analogyto Chl a+*. For numbering of positions see Figure 1, and for assignment of Chl a+* see refs 15 and 34; the hfc's of 3ac-Chl a+* were assigned accordingly, and the CH3 groups (positions 12,7,2) were identified by comparison with solid-state spectra (see text). In the calculation, rotation angles of the vinyl and acetyl groups of 50 and 75O have been used, respectively, to yield lowest total energy; in both systems a twist angle of 5 . 5 O was employed for ring D (see Figure 8). This hfc is believed to contain couplings from protons at all three positions 2l, 7l, and 8l; see ref 15. * This hfc could also belong to the methine protons, since the sign could not be determined. /The sign of these hfc's could not be determined, they might therefore belong to the methine (5,10,20), vinyl (31,32),and/or y-protons (171,181).

i" i

2

4

3 v [MHzI Figure7. 14Nliquid-solution ENDOR spectra (high-frequencylinesonly) [3-vinyl]-BChla+* (3+*),132-hydroxy-BChla+* (a+*), of BChl a+* (l+*),

and [3-~inyI]-l3~-hydroxy-BChl a+* (7+*). The hfc's arc collected in Table 4. For sample preparation see Figures 3 and 4. Experimental conditions: temperature240 K,fmdepth 100kHz(l+'), 50lcHz(3+',7+'), 150 kHz ( 6 9 , time constant 640 ms, scan time 4 X 400 8, microwave power 5-10 mW. The Larmor frequency VN is 1.06 MHz.

Figure 7 shows 14N ENDOR spectra for four selected radicals. For 6+*only two of the four expected 14N hfc's could be resolved, three hfc's could be seen for the parent radical 1+*,and for 7+*, and all four 14N hfc's are clearly distinguished for 3+'. This

Ktiss et al.

360 The Journal of Physical Chemistry, Vol. 98, No. 1, 1994 TABLE 4

Experimental and Calculated I4N Hfc’s [MHz] for Various BChl a-Type Radical Cations’ compound

ring wsitionsb A

C B D A

C B D

BChl a+* (I+*)

pBChl u+* (2+9

3v-BChl a+* (+3+’)

-2.31 -2.31 -3.15 -2.93

-2.25 -2.25 -3.01 -3.01

-2.14 -2.44 -3.18 -2.72

-2.10 -2.55 -3.28 -3.20

-2.18 -2.45 -3.36 -3.20

-1.98 -2.63 -3.20 -3.10

3v-p-BChl a+*

3he-BChl a+* (5+’) Experimental Values -2.23 2.32 -2.67 2.32 -3.23 3.14 -3.23 3.14 Calculated Values -2.01 -2.06 -2.53 -2.60 -3.30 -3.25 -3.13 -3.12 (4+?

132h-BChlu+* (6+’)

3v-132h-BChla+* (7+?

B-chlorin+’ (8+’)

-2.23 -2.23 -3.15 -3.15

-2.33 -2.33 -3.23 -2.95

-2.55 -2.55 -3.33 -3.33

-2.06 -2.56 -3.32 -3.07

-1.92 -2.62 -3.15 -3.03

-2.27 -2.42 -3.25 -3.41

a Experimental isotropic hfc’s from liquid-solution ENDOR, signs from heteronuclear General TRIPLE resonance (see text), and calculated values from RHF-INDO/SP using & = 650 MHz (see ref 19). For labeling of rings A to D see Figure 1; assignments of the experimental hfc’s to specific pyrrol rings are based solely on the theoretical results.

TABLE 5

Calculated lH Hfc’s [MHz] for Various BChl a-Type Radical Cations Using the RHF-INDO/SP Methods compoundb

carbon Dosition 8 ~~

I 17 18 121 21 132 5 10 20 31 32 133 7’ 8l 171 18l

BChl a+* (1+9 +15.72 +13.55 +12.79 +11.81 +9.60 +5.01 -2.62 +3.20 +2.48 +2.65

pBChl a+* (2+9 +15.28 +13.83 +12.87 +12.13 +8.27 +5.15 -7.69 +3.19 +2.69 +2.73 -0.04

3v-BChl a+* (3+9 +15.88 +13.13 +13.00 +11.22 +9.95 +3.69 -2.67 +3.15 +2.50 +2.76 +3.24 -0.48

3v-pBChl u+’ (4+9 +15.54 +13.43 +13.04 +11.55 +8.60 +3.81 -7.84 +3.19 +2.72 +2.82 +3.45 -0.51

3he-BChl a+* (5+’) +15.86 +13.31 +12.98 +11.39 +9.79 +3.64 -2.67 +3.15 +2.54 +2.76 +3.06 +o. 12e

-0.04 -0.31 -0.15 -0.61 -0.24

-0.31 -0.15 -0.62 -0.25

-0.30 -0.15 -0.62 -0.24

-0.30 -0.15 -0.63 -0.25

-0.30 -0.15 -0.62 -0.24

132h-BChl u+* +16.89 +14.34 +10.12 +9.18 +9.84 +5.04 +3.30 +2.46 +2.53 0.04 -0.01 -0.30 -0.16 -0.59 -0.27

3v-132-BChl a+* (7+’) +16.77 +13.75 10.68 +8.70 +10.22 +3.47

+

+3.12 +2.46 +2.74 +3.16 -0.51 +0.04 -0.29 -0.15 -0.61 -0.28

B-chlorin+* (8+’) +15.24 +14.66 +8.63 +7.99 +6.70 +5.55 -2.26‘ +2.99 +2.71 +3.04 -0.17 -0.29 -0.16 -0.54 -0.35

0 The IH hfc’s are obtained from calculated s-spin densities using & = 1420 MHz (see ref 19). b For abbreviations of radical cation species see text. C For molecular positions see Figure 1; the assignment of the IH hfc’s is based on the known assignment of BChl a+* (see ref 14). Hfc for CH2 group at position 15. e Hfc of the proton in the 3a-OH-group: -0.17 MHz.

shows that the different substituents at the macrocycle also influence the nitrogen spin densities. The data for these and all other BChl species (1+*-8+*)are collected in Table 4. Calculation of Hyperfine Coupling Constants. Theoretical isotropic hfc’s were calculated for all BChl a cation radicals from the s-spin densities which were obtained from RHF-INDO/SP calculations.18 For the molecular skeleton mostly standard bond lengths and angles were used, which are given in ref 20 (Tables 4.16,4.17) withsomemodificationsin ring E, whichwerejustified by energy minimization and a comparison with experiments.I8J9 A planar geometry of the porphyrin macrocycle including ring E was assumed. The hydrogenated rings B and D were twisted slightly out-of-plane (see discussion below). The Mg atom was positioned in-plane. Calculations with one or two water ligands attached to the Mg showed only minor changes of the IH and 14N hfc’s, and therefore all our MO calculations were performed without such ligands. When molecular coordinates from X-ray crystalIography36.3’ were employed, the agreement between calculations and experiments was not as good as in the case of our modified standard geometries. Further details on specific structural features of the different molecules are given below. The exact Cartesian coordinates used in the MO calculations are available as supplementary material. The calculated IH hfc’s of the cation radicals of the BChl u derivatives are collected in Table 5. A comparison with the experimental values (Table 2) yields good agreement, in particular for the six largest hfc’s which could be unambiguously assigned in all experiments. These hfc’s are most important for BChl u cation radicals embedded in the RC

pr0tein.1~ The experimental and calculated 14N hfc’s are given in Table 4 together with the experimental values. The result is of similar quality-although an experimental assignment of the 14N resonances to the individual pyrrole rings is not possible. A comparison between experimental and theoretical IH hfc’s is less satisfying for the chlorophyll species 9+’ and lo+*(Table 3). I4N hfc’s have not yet been obtained for thesespecies. In the following, we want to discuss the effect of the various substitutions on the electron spin distribution in the cation radicals. Substitution ut Position 3 in BChl. According to the experiments, the CH3 hfc at position 2 reduces from 4.84 MHz in 1+*to 2.95 MHz in 5+’ and to 2.61 MHz in 3+*. This is predominantly a local effect caused by the substitution of the acetyl group at position 3 in ring A, influencing the adjacent carbon spin density. It is interesting that in both radicals, 3+* and 5+*, also the CH3 hfc at position 12 is increased by -0.6 MHz as compared with l+*.This indicates that the replacement of the conjugated acetyl group has an impact on the entire A system. A chemical manifestation of this long-distance effect has been reported in ref 10d. The p-proton hfc’s depend strongly on the ?r spin densities at positions6,9,16, and 19 and are therefore reliable indicators for the geometry of the rings. The observed trends in the spin density distribution arefully reproduced by the RHF-INDO/SP calculations (cf. radicals 1+*,3+*,and 5+* in Table 5). In the table, the hfc’s are given for an optimized geometry of the rotation angle, CY,for the 3-substituents. This is CY = 75, 50, and 1 5 O for 1+*,3+*,and 5+*, respectively (for definition of angle (Y see Figure 8). It should be noted that

Bacteriochlorophyll Cation Radicals Rotation Twist

-8 n

0 01

2

.-L

c, v)

10

I+'

5-

-~

0-

I-

Figure 8. Side view of a BChl u molecular skeleton showing geometrical changes in structure, which explain the ENDOR results. (i) Twisting of rings B and D out of the molecular plane is defined by the twist angle fl(fl)betweenthe7,8 (17,18)bondandthemolecularplane. Thistwisting is also called "ruffling"(see ref 35). (ii) Rotation of the acetyl group at position 3 out of the molecular plane is defined by angle a. Similar rotations can be performed for the vinyl (3) and a-hydroxyethyl (5) substituents. For the latter, the angle a is given with respect to the plane containing the C-O bond.

calculations using the same angle (75') for all three groups do not change the overall result. However, the spin densities in the respective substituents are influenced by the rotational angle a. For example, the calculated 1H hfc's for positions 31 (32) in vinyl are4.64(-0.03),3.24(-0).48),and0.23(-1.16) MHzforavalues of 75, 50, and ,'O respectively. The MO calculations give essentially the same results for out-of-plane rotation angles f a and f(180' - a ) for both the vinyl and acetyl groups. In the case of the acetyl group (l+*) the angle a has a profound influence on the spin density distribution of the radical cation. For example, the CH3 hfc's in positions 2 and 12 change from 7.14 and 9.37 MHz (a= 0 ' ) to 4.88 and 9.64 MHz ( a = goo), respectively. In contrast, rotation of the vinyl group in 3+*or a-hydroxyethyl group in 5+* has only a minor impact on the orbitaldistribution and the total energyofthesystem. Thisclearly shows the importance of strongly conjugated substituents like acetyl. Rotation of this group will influence both the magnetic resonance and optical properties of the bacteriochlorophylls3* and probably also its reactivity. Thereby, it could act as a fast conformational switch changing the electronic properties of the system. It is important to notice that this group is present in BChl a and b but not in BChl g. The latter is found in heliobacteria which have a RC similar to PS I. Furthermore, all plant photosystems (PS I and 11) contain Chl a (9) which carries no acetyl group. Substitution at Position 132 in BChl. Removal of the carbomethoxy group at position 132 in 1 and 3 leads to the formation of the so-called pyro compounds 2 and 4. In the respective cation radicals this results in a small decrease of the methyl hfc of position 12l, whereas that at position 21 remains unchanged (Table 2). In the experiment there is no significant change of any of the /%protonhfc's in rings D and B. Obviously, the carbomethoxy group at 132has no influence on the geometry of ring D, which should clearly show up in a change of the &proton hfc's (see below). The observed effects on the 'H hfc's are fully reproduced by the MO calculations (Table 5 ) . For the proton at position 132 in 1+*and all other non-pyro BChl radical cations, an hfc of -2.6 to -2.7 MHz is calculated in fair agreement with the experiment. It should be noted that this assignment has been confirmed by H / D exchange in earlier experiment^.^^,'^ In order to reproduce the measured value, a planar ring E and a dihedral angle of 75.5' for the carbomethoxy group and of 42' for the proton were used. This geometry is justified by the larger volume of the former group (steric effect). For the pyro compounds 2+' and 4+* equal angles for the protons at 132(62') gave the best results. Nevertheless, all calculations

in

2'

I

I

12'

2'

I

I

*I.n.o.

7

8 18'17

xty-11g;q

*:*.

c m ,

r

12'

exp. value

I

I

=.- I I-

......

I .U.; ' ' I -- I I I I I

01

I

''IifJI. . 7 18178

I

1

I

I

I

0

5

10

15

20

4~[MHzI Figure 9. Plot of lH hfc's from RHF-INDO/SP calculations using different twist angles for ring D in 1+' and 6+'. Only the large &proton hfc's (positions 7,8,17,18) and CHI proton hfc's (2lJ2') are shown. The CHa couplings and the &proton couplings in ring B remain constant as expected (solid bars), whereas the hfc's in ring D (position 17,18) are changed (open bars). For 1+*and 6+' twist angles of 4.25 and 6.S0, respectively, yield the best agreement with experimental data. Theconcomitantchange in the total energy is small compared with the thermal energy (kT) near room temperature. yielded quite large proton hfc's for these nuclei (see Table 5 ) which are clearly not observed in the spectra (Figures 3 and 4). Various geometrical changes including energy minimization and calculations employing X-ray coordinates36 did not improve the theoretical values.39 Replacement of the proton at position 13*by a hydroxy group profoundly influenced the &proton hfc's at positions 17 and 18, whereas all other couplings remained fairly constant. This change is accounted for in the calculations by a change in the twist angle 8' of ring D (see Figure 8) caused by the additional sterically demanding substituent at position 132in ring E,which is at the same side of the macrocycle as the 17-propionicacid side chain in ring D. The effect of changing this twist angle in the calculation is demonstrated for radicals 1+*and 6+* in Figure 9. Opening of Ring E . The bacteriochlorin radical cation 8+*,in which the constrained ring E has been opened, exhibits almost equal methyl hfc's for positions 2l and 12l (Table 2). This effect is also found in the RHF-INDO/SP calculation, although a slightly larger difference of approximately 1 MHz occurs, which could be attributed to remaining structural changes not accounted for in thecalculations. Furthermore, the experimentallyobserved decrease of the &proton hfc's at positions 17 and 18 in ring D is fully reproduced in the MO calculations by increasing the twist angle from 4.25' in 1+*to 8.5' in 8+'. A similar effect is also found in the 132hydroxy compound 6+* (see above). Substitution at Position 3 in Chl a. Replacement of the vinyl group in 9+*by acetyl in lo+*has an effect on the 1H hfc's of the adjacent CH3 group (position 2'), quite similar to that found in the BChl series (cf. 3+*and l+*).This effect is reproduced by the RHF-INDO/SP calculation (Table 3). The influenceof this substitution on the other hfc's is not very significant either in the experiment or in thecalculation. It must be noted that the overall agreement of experimental and calculated hfc's is less satisfying for the chlorophyll radicals. In particular, the theoretical methine couplings (positions 5 and 10) are much too large and the difference between the calculated methyl hfc's is too small (Table

362 The Journal of Physical Chemistry, Vol. 98, No. 1, 1994 3). The use of geometries resulting from X-ray investigation^^^ of chlorophyllide crystals yielded no better agreement between theoretical and experimental hfc’s. Improved theoretical results might be expected from future calculations performed on a carefully optimized geometry of the Chl system. Nitrogen Positions. The calculation of the I4N hfc’s for all BChl radical cations show two larger hfc’s of >3 MHz for rings B and D and two somewhat smaller hfc’s of 2-2.5 MHz for rings A and C. Substitution on the periphery of the macrocycle does not change this order (Table 4), which allows a tentative assignment of the measured two to four hfc’s to either rings A/C or B/D. The signs of all 14Nhfc’s are negative, indicating very small T spin densities at the nitrogens. The isotropic hfc’s are mainly caused by T-u spin polarization4 resulting from the large T spin densities at the adjacent carbon atoms. For the chlorophyll cation radicals, no 14N hfc’s could be obtained experimentally. The calculated values for 9+* (-1.3, -1.4, -1.8, and -2.9 MHz for rings C, A, D, and B, respectively are generally somewhat smaller than those in 1+*and show one prominent larger value. ENDOR Experiments on the Primary Donor Cation Radical D+’in RCs of R. spbaeroides Reconstituted with Modified Pigments. Reaction centers of R. sphaeroides R-26 in which pigment 3 or 6 has been incorporated were investigated with the aim of identifying whether the bacteriochlorophylls forming the primary donor were exchanged or whether only the other pigments molecules were involved. From the ENDOR experiments of 3+’ and 6+’ discussed above it is clear that pronounced effects in the spectra of D+*can be expected if 3 and 6 replace 1 in the dimer. We have detected the ENDOR spectra of D+*generated by in situ light irradiation of native and the chemically exchanged RCs. The comparison revealed no difference in the spectra (data not shown) within experimental error.41 It is therefore concluded that the dimer, D, remains untouched during the exchange procedure and only the monomeric BChl’s are replaced by the modified species. Since this chemical exchange has no effect on the structure of the primary donor, the BChl a dimer seems to be a fairly stable and structurally well-defined entity.

Conclusions In this paper the cation radicals of various modified bacteriochlorophyll a molecules have been investigated by EPR, ENDOR, and TRIPLE resonance spectroscopy yielding all essential lH and 14N hfc’s. An assignment of these hfc’s to molecular positions resulted in a detailed map of the spin density distribution in these species. The data were compared with MO calculations of the RHF-INDO/SP type. It was found that replacement of the acetyl group in position 3 in BChl a (1) by a vinyl (3) or an alkyl (5) substituent has a profound effect on the spin density distribution. Large changes could also be produced by changing the angle between the acetyl group and the plane of the u macrocycle in the MO calculation. No such effects are observed by rotation of vinyl or alkyl groups. Quitedrastic effects upon acetyl rotation have also been reported for calculations of the optical absorption spectra.36 The acetyl group is present in BChl a and b which are the predominant pigments in a large variety of bacterial species.6 The strong influence of the presence and configuration of this group upon the electronic structure of the BChl’s leads to the question of whether it might play a functional role in the primary photosynthetic processes. One possible function might lie in a fine tuning of the optical absorption properties in the light-harvesting system. In the reaction center, the group could act as a fast electronic switch, which helps the highly efficient unidirectional electron transfer. Furthermore, the group could be of structural importance,e.g. by acting as a hydrogen-bondacceptor or a ligand to the metal centers of neighboring pigments. (It could also be speculated that the group is important for dimer formation.) In

U s s et al. the plant photosystems, Chl a is present, which carries a vinyl instead of the acetyl group. Obviously,nature has found different ways to optimize the photosynthetic processes in these species. It has been shown that the asymmetric spin density distribution of BChl a and Chl a cation radicals is caused by the presence of the isocyclic ring E (Figure 1). The keto group in this ring is spacially fixed and could act as a contact point for hydrogen bonds and other interactions. Ring E is probably important for maintaining the rigid planar structure of the (bacterio)chlorophylls, which may be necessary for incorporation into the protein. In this context it is interesting to mention that species lacking ring E cannot be reconstituted into the sites of monomeric BChl a in RCs of R. sphaeroides.10 This is also true for all pyro compounds lacking the carbomethoxy group, which might therefore also play a structural role for the pigments in the RC. In general, the MO theoretical results obtained for the BChl cation radicals are very satisfying. In the caseof the 132-hydroxy compound 6+*,it could be demonstrated that even fine structural effects on the geometry of the system--g. a twist of the hydrogenated rings-are correctly reproduced. However, it had to be noted that the INDO method in its present parameterization is not very well suited to handle cases of rather unusual geometries, that is, deviations from standard structures (ring E). The results obtained for the cation radicals of the various modified pigments will be important for studies in which these pigments are incorporated into the dimer. So far, this has not yet been achieved, but experiments along these lines are in progress. It is, however, confirmed that the monomeric BChl’s and BPhe’s in the A and B branch of the RC can be selectively exchanged by modified pigments. These species act as electron acceptors (A branch) in the electron-transfer process in which they areconverted to their radical anions. It would be interesting to investigatethe electronic structure of these species by ENDOR in the RC. First it is planned to study the radical anions of the modified pigments in organic solvents. The subsequent investigation of the photoaccumulated state of the intermediateelectron acceptor I- in native and modified RCs should enable us to decide which of the pigments-BChl’s and/or BPhe’s-are involved and to determine the details of their electronic structure in the RC protein.

Acknowledgment. We are grateful to Dr. M. Plato, Freie Universitlt Berlin, for providing the source of his RHF-INDO/ SP program and his advice concerningthe use of this MO method. The ENDOR cavity used was constructed by R. Thanner. J. Schaugg performed part of the RHF-INDO/SP calculations. We thank W. Miiller, W. Pfundstein, and G. Dietrich (Univenitiit Stuttgart) for their skillful work in building the ENDOR cavity and I. Katheder (Miinchen) for expert technical assistance in the preparations. This work was supported by the Deutsche Forschungsgemeinschaft (Sa3 12/TP A4 in Berlin and Sfb 143/TP A9 in Miinchen) and by Fondsder Chemischen Industrie (W.L.). Supplementary Material Available: Cartesian coordinates of all molecular structures employed in the MO calculationsof Tables 2 and 3 (10 pages). Ordering information is given on any current masthead page. References and Notes (1) Chlorophylls; Schecr, H., Ed.;CRC Press: Boca Raton, FL, 1991. (2) Spikes, J. D.; Bommer, J. C. In Chlorophylls;Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1991; pp 1181-1204. (3) Deisenbofer, J.; Michel, H. EMEO J. 1989, 8, 2149-2169 and references cited therein. (4) (a) Feher, 0.;Allen, J. P.; Okamura,M. Y.; Rea, D. C. Nature 1989. 339, 111-116 and references cited therein. (b) Chang, C.-H.; El-Kabbani, 0.;Tiede, D.; Norris, J.; Schiffer, M. Biochemistry 1991, 30, 5352-5360. (5) BIankenship,R. E.;Trost,J.T.; Mancino, L. J. In ThePhotosynthetic Bacterial Reaction Center; Breton. J.. Vermeglio, A., Eds.; Plenum Press: New York, 1988; pp 119-127.

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