Resonance Raman characterization of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> reaction centers with lysine mutations near the accessory bacteriochlorophylls

Lysine residues have been introduced into Rhodobacter capsulatus reaction centers at M-polypeptide position 201 and at L-polypeptide position 178. These positions are in the proximity of ring V of the accessory bacterochlorophylls B_A and B_B, respectively. Resonance Raman studies indicate that the introduction of a Lys residue at either position M201 or L178 results in structural perturbations to the BChl cofactors. Lys at L178 directly interacts with B_B, most likely via a hydrogen bond. The hydrogen bonding interaction is consistent with enhanced B branch electron transfer that is observed in RCs from the S(L178)K/G(M201)D/L(M212)H triple mutant versus the G(M201)D/L(M212)H double mutant (C. Kirmaier et al. (1999) Biochemistry 38 11516–11530). In contrast, the introduction of a Lys at M201 does not result in hydrogen bonding to the B_A cofactor, in contrast to the introduction of a His at M201 (L. Chen et al. (2004) J Phys Chem 3 108: 0457–10464). Accordingly, the alkyl ammonium head group of the side chain of the Lys at M201 residue appears to be distant from B_A.     

Manipulating the direction of electron transfer in the bacterial reaction center by swapping Phe for Tyr near BChl(M) (L181) and Tyr for Phe near BChl(L) (M208)

We have investigated the primary charge separation processes in Rb. capsulatus reaction centers (RCs) bearing the mutations Phe(L181) --> Tyr, Tyr(M208) --> Phe, and Leu(M212) --> His. In the YFH mutant, decay of the excited primary electron donor P occurs with an 11 +/- 2 ps time constant and is trifurcated to give (1) internal conversion to the ground state ( approximately 10% yield), (2) charge separation to the L side of the RC ( approximately 60% yield), and (3) electron transfer to the M-side bacteriopheophytin BPh(M) ( approximately 30% yield). These results relate previous work in which the ionizable residues Lys (at L178) and Asp (at M201) have been used to facilitate charge separation to the M side of the RC, and the widely studied L181 and M208 mutants. One conclusion that comes from this work is that the Tyr (M208) --> Phe and Gly(M201) --> Asp mutations near the L-side bacteriochlorophyll (BChl(L)) raise the free energy of P(+)BChl(L)(-) by comparable amounts. The results also suggest that the free energy of P(+)BChl(M)(-) is lowered more substantially by a Tyr at L181 than a Lys at L178. The results on the YFH mutant further demonstrate that the free energy differences between the L- and M-side charge-separated states play a significant role in the directionality of charge separation in the wild-type RC, and place limits on the contributing role of differential electronic matrix elements on the two sides of the RC.     

High yield of M-side electron transfer in mutants of Rhodobacter capsulatus reaction centers lacking the L-side bacteriopheophytin

We present studies on a series of photosynthetic reaction center (RC) mutants created in the background of the Rhodobacter capsulatus D(LL) mutant, in which the D helix of the M subunit has been substituted with that from the L subunit. Previous work on the D(LL) mutant in chromatophore preparations showed that RCs assembled without the bacteriopheophytin H(L) electron acceptor and performed no charge separation following light absorption. We have successfully isolated poly-His-tagged D(LL) RCs by using the detergent Deriphat 160-C and shown that the RCs are devoid of H(L). The excited state of the primary electron donor, P*, is found to have a lifetime of 180 +/- 20 ps and to decay exclusively (>95%) via internal conversion to the ground state, with no evidence for formation of any charge-separated intermediates. By additional mutation in the D(LL) background of two residues that affect the P/P+ oxidation potential and one that facilitates M-side electron transfer, we achieve an unprecedented 70% yield of P+ H(M)-, more than doubling the highest yield of this state achieved previously. This result underscores the importance of the relative free energies of P* and the charge-separated states in governing the rates and yields of electron transfer in bacterial RCs and provides a basis for systematically investigating M-side electron transfer without any competition from the native L-side pathway.     

Relationship between altered structure and photochemistry in mutant reaction centers in which bacteriochlorophyll replaces the photoactive bacteriopheophytin

Qy-excitation resonance Raman (RR) spectra are reported for two mutant reaction centers (RCs) from Rhodobacter capsulatus in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated beta. The pigment change in both mutants is induced via introduction of a histidine residue near the photoactive cofactor. In one mutant, L(M212)H, the histidine is positioned over the core of the cofactor and serves as an axial ligand to the Mg+2 ion. In the other mutant, F(L121)H/F(L97)V, the histidine is positioned over ring V of the cofactor, which is nominally too distant to permit bonding to the Mg+2 ion. The salient observations are as follows: (1) The beta cofactor in F(L121)H/F(L97)V RCs is a five-coordinate BChl molecule. However, there is no evidence for the formation of a Mg-His bond. This bond is either much weaker than in the L(M212)H RCs or completely absent, the latter implying coordination by an alternative ligand. The different axial ligation for beta in the F(L121)H/F(L97)V versus L(M212)H RCs in turn leads to different conformations of the BChl macrocycles. (2) The C9-keto group of beta in F(L121)H/F(L97)V RCs is free of hydrogen bonding interactions, unlike the L(M212)H RCs in which the C9-keto of beta is hydrogen bonded to Glu L104. The interactions between other peripheral substituents of beta and the protein are also different in the F(L121)H/F(L97)V RCs versus L(M212)H RCs. Accordingly, the position and orientation of beta in the protein is different in the two beta-containing RCs. Nonetheless, previous studies have shown that the primary electron transfer reactions are very similar in the two mutants but differ in significant respects compared to wild-type RCs. Collectively, these observations indicate that changes in the conformation of a photoactive tetrapyrrole macrocycle or its interactions with the protein do not necessarily lead to significantly perturbed photochemistry and do not underlie the altered primary events in beta-type RCs.     

Proton and electron transfer in the acceptor quinone complex of Rhodobacter sphaeroides reaction centers: characterization of site-directed mutants of the two ionizable residues, GluL212 and AspL213, in the QB binding site.

Proton and electron transfer events in reaction centers (RCs) from Rhodobacter sphaeroides were investigated by site-directed mutagenesis of glutamic acid at position 212 and aspartic acid at 213 in the secondary quinone (QB) binding domain of the L subunit. These residues were mutated singly to the corresponding amides (mutants L212EQ and L213DN) and together to give the double mutant (L212EQ/L213DN). In the double mutant RCs, the rate of electron transfer from the primary (QA) to the secondary (QB) acceptor quinones is fast (tau approximately 300 microseconds) and is pH independent from pH 5 to 11. The rate of recombination between the oxidized primary donor, P+, and QB- is also pH independent and much slower (tau approximately 10 s) than in the wild type (Wt), indicating a significant stabilization of the QB- semiquinone. In the double mutant, and in L213DN mutant RCs at low pH, the P+QB- decay is suggested to occur significantly via a direct recombination rather than by repopulating the P+QA- state, as in the Wt. Comparison of the behavior of Wt and the three mutant RC types leads to the following conclusions: the pK of AspL213 in the Wt is approximately 4 for the QAQB state (pKQB) and approximately 5 for the QAQB-state (pKQB-); for GluL212, pKQB approximately 9.5 and pKQB- approximately 11. In L213DN mutant RCs, pKQB of GluL212 is less than or equal to 7, indicating that the high pK values of GluL212 in the Wt are due largely to electrostatic interaction with the ionized AspL213 which contributes a shift of at least 2.5 pH units. Transfer of the second electron and all associated proton uptake to form QBH2 is drastically inhibited in double mutant and L213DN mutant RCs. At pH greater than or equal to 8, the rates are at least 10(4)-fold slower than in Wt RCs. In L212EQ mutant RCs the second electron transfer and proton uptake are biphasic. The fast phase of the electron transfer is similar to that of the Wt, but the extent of rapid transfer is pH dependent, revealing the pH dependence of the equilibrium QA(-)QB- in equilibrium with QAQBH-. The estimated limits on the pK values--pKQA-QB-less than or equal to 7.3, pKQAQB2- greater than or equal to 10.4--are similar to those derived earlier for Wt RCs [Kleinfeld et al. (1985) Biochim. Biophys. Acta 809, 291-310] and may pertain to the quinone head group, per se.(ABSTRACT TRUNCATED AT 400 WORDS)     

Simultaneous replacement of Asp-L210 and Asp-M17 with Asn increases proton uptake by Glu-L212 upon first electron transfer to QB in reaction centers from Rhodobacter sphaeroides.

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the first electron transfer to the secondary quinone acceptor Q(B) is coupled to the protonation of Glu-L212, located approximately 5 A from the center of Q(B). Upon the second electron transfer to Q(B), Glu-L212 is involved in fast proton delivery to the reduced Q(B). Since Asp-L210 and Asp-M17 play an important role in the proton transfer to the Q(B) site [Paddock, M. L., Adelroth, P., Chang, C., Abresch, E. C., Feher, G., and Okamura, M. Y. (2001) Biochemistry 40, 6893-6902], we investigated the effects of replacing one or both Asp residues with Asn on proton uptake by Glu-L212 using FTIR difference spectroscopy. Upon the first electron transfer to Q(B), the amplitude of the proton uptake by Glu-L212 at pH 8 is increased in the single and double mutant RCs, as is evident from the larger intensity (by 35-55%) of the carboxylic acid band at 1727 cm(-1) in the Q(B)(-)/Q(B) difference spectra of mutant RCs, compared to that at 1728 cm(-1) in native RCs. This implies that the extent of ionization of Glu-L212 in the Q(B) ground state is greater in the mutants than in native RCs and that Asp-M17 and Asp-L210 are at least partially ionized near neutral pH in native RCs. In addition, no changes in the protonation state or the environment of these two residues are detected upon Q(B) reduction. The absence of the 1727 cm(-1) signal in all of the RCs lacking Glu-L212, confirms that the positive band at 1728-1727 cm(-1) probes the protonation of Glu-L212 in native and mutant RCs.     

Investigation into the source of electron transfer asymmetry in bacterial reaction centers

We have investigated the primary photochemistry of two symmetry-related mutants of Rhodobacter sphaeroides in which the histidine residues associated with the central Mg2+ ions of the two bacteriochlorophylls of the dimeric primary electron donor (His-L173 and His-M202) have been changed to leucine, affording bacteriochlorophyll (BChl)/bacteriopheophytin (BPh) heterodimers. Reaction centers (RCs) from the two mutants, (L)H173L and (M)H202L, have remarkably similar spectral and kinetic properties, although they are quite different from those of wild-type RCs. In both mutants, as in wild-type RCs, electron transfer to BPhL and not to BPhM is observed. These results suggest that asymmetry in the charge distribution of the excited BChl dimer (P*) in wild-type RCs (due to differing contributions of the two opposing intradimer charge-transfer states) contributes only modestly to the directionality of electron transfer. The results also suggest that differential orbital overlap of the two BChls of P with the chromophores on the L and M polypeptides does not contribute substantially to preferential electron transfer to BPhL.     

Identification of the proton pathway in bacterial reaction centers: cooperation between Asp-M17 and Asp-L210 facilitates proton transfer to the secondary quinone (QB)

The reaction center (RC) from Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, Q(B) (the secondary quinone electron acceptor), to form quinol, Q(B)H2. Asp-L210 and Asp-M17 have been proposed to be components of the pathway for proton transfer [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. To test the importance of these residues for efficient proton transfer, the rates of the proton-coupled electron-transfer reaction k(AB)(2) (Q(A-*)Q(B-*) + H+ <==>Q(A-*)Q(B)H* --> Q(A)Q(B)H-) and its associated proton uptake were measured in native and mutant RCs, lacking one or both Asp residues. In the double mutant RCs, the k(AB)(2) reaction and its associated proton uptake were approximately 300-fold slower than in native RCs (pH 8). In contrast, single mutant RCs displayed reaction rates that were < or =3-fold slower than native (pH 8). In addition, the rate-limiting step of k(AB)(2) was changed from electron transfer (native and single mutants) to proton transfer (double mutant) as shown from the lack of a dependence of the observed rate on the driving force for electron transfer in the double mutant RCs compared to the native or single mutants. This implies that the rate of the proton-transfer step was reduced (> or =10(3)-fold) upon replacement of both Asp-L210 and Asp-M17 with Asn. Similar, but less drastic, differences were observed for k(AB)(1), which at pH > or =8 is coupled to the protonation of Glu-L212 [(Q(A-*)Q(B))-Glu- + H+ --> (Q(A)Q(B-*)-GluH]. These results show that the pathway for proton transfer from solution to reduced Q(B) involves both Asp-L210 and Asp-M17, which provide parallel branches to the proton-transfer pathway and through their electrostatic interaction have a cooperative effect on the proton-transfer rate. A possible mechanism for the cooperativity is discussed.     

High yield of B-branch electron transfer in a quadruple reaction center mutant of the photosynthetic bacterium Rhodobacter sphaeroides

A new reaction center (RC) quadruple mutant, called LDHW, of Rhodobacter sphaeroides is described. This mutant was constructed to obtain a high yield of B-branch electron transfer and to study P(+)Q(B)(-) formation via the B-branch. The A-branch of the mutant RC contains two monomer bacteriochlorophylls, B(A) and beta, as a result of the H mutation L(M214)H. The latter bacteriochlorophyll replaces bacteriopheophytin H(A) of wild-type RCs. As a result of the W mutation A(M260)W, the A-branch does not contain the ubiquinone Q(A); this facilitates the study of P(+)Q(B)(-) formation. Furthermore, the D mutation G(M203)D introduces an aspartic acid residue near B(A). Together these mutations impede electron transfer through the A-branch. The B-branch contains two bacteriopheophytins, Phi(B) and H(B), and a ubiquinone, Q(B.) Phi(B) replaces the monomer bacteriochlorophyll B(B) as a result of the L mutation H(M182)L. In the LDHW mutant we find 35-45% B-branch electron transfer, the highest yield reported so far. Transient absorption spectroscopy at 10 K, where the absorption bands due to the Q(X) transitions of Phi(B) and H(B) are well resolved, shows simultaneous bleachings of both absorption bands. Although photoreduction of the bacteriopheophytins occurs with a high yield, no significant (approximately 1%) P(+)Q(B)(-) formation was found.     

Determination of the rate and yield of B-side quinone reduction in Rhodobacter capsulatus reaction centers

In the native purple bacterial reaction center (RC), light-driven charge separation utilizes only the A-side cofactors, with the symmetry related B-side inactive. The process is initiated by electron transfer from the excited primary donor (P*) to the A-side bacteriopheophytin (P* --> P+ H(A)-) in approximately 3 ps. This is followed by electron transfer to the A-side quinone (P+ H(A)- --> P+ Q(A)-) in approximately 200 ps, with an overall quantum yield of approximately 100%. Using nanosecond flash photolysis and RCs from the Rhodobacter capsulatus F(L181)Y/Y(M208)F/L(M212)H mutant (designated YFH), we have probed the decay pathways of the analogous B-side state P+ H(B)-. The rate of the P+ H(B)- --> ground-state charge-recombination process is found to be (3.0 +/- 0.8 ns)(-1), which is much faster than the analogous (10-20 ns)(-1) rate of P+ H(A)- --> ground state. The rate of P+ H(B)- --> P+ Q(B)- electron transfer is determined to be (3.9 +/- 0.9 ns)(-1), which is about a factor of 20 slower than the analogous A-side process P+ H(A)- --> P+ Q(A)-. The yield of P+ H(B)- --> P+ Q(B)- electron-transfer calculated from these rate constants is 44%. This value, when combined with the known 30% yield of P+ H(B)- from P in YFH RCs, gives an overall yield of 13% for B-side charge separation P* --> P+ H(B)- --> P+ Q(B)- in this mutant. We determine essentially the same value (15%) by comparing the P-bleaching amplitude at approximately 1 ms in YFH and wild-type RCs.     

Fourier transform infrared evidence of proton uptake by glutamate L212 upon reduction of the secondary quinone QB in the photosynthetic reaction center from Rhodobacter capsulatus

The photoreduction of the secondary quinone Q(B) in native reaction centers (RCs) of Rhodobacter capsulatus and in RCs from the GluL212 --> Gln and GluL212 --> Ala mutants has been investigated at pH 7 in (1)H(2)O and (2)H(2)O by light-induced Fourier transform infrared (FTIR) difference spectroscopy. The Q(B)(-)/Q(B) FTIR difference spectra reflect changes of quinone-protein interactions and of protonation state of carboxylic acid groups as well as reorganization of the protein upon electron transfer. Comparison of Q(B)(-)/Q(B) spectra of native and mutant RCs indicates that the interactions between Q(B) or Q(B)(-) and the protein are similar in all RCs. A differential signal at approximately 1650/1640 cm(-1), which is common to all the spectra, is associated with a movement of a peptide carbonyl or a side chain following Q(B) reduction. On the other hand, Q(B)(-)/Q(B) spectra of native and mutant RCs display several differences, notably between 1700 and 1650 cm(-1) (amide I and side chains), between 1570 and 1530 cm(-1) (amide II), and at 1728-1730 cm(-1) (protonated carboxylic acid groups). In particular, the latter region in native RCs is characterized by a main positive band at 1728 cm(-1) and a negative signal at 1739 cm(-1). In the L212 mutants, the amplitude of the positive band is strongly decreased leading to a differential signal at 1739/1730 cm(-1) that is insensitive to (1)H/(2)H isotopic exchange. In native RCs, only the 1728 cm(-1) band is affected in (2)H(2)O while the 1739 cm(-1) signal is not. The effects of the mutations and of (1)H/(2)H exchange on the Q(B)(-)/Q(B) spectra concur in the attribution of the 1728 cm(-1) band in native RCs to (partial) proton uptake by GluL212 upon the first electron transfer to Q(B), as previously observed in Rhodobacter sphaeroides RCs [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., M?ntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732]. More generally, strong homologies of the Q(B) to Q(B)(-) transition in the RCs from Rb. sphaeroides and Rb. capsulatus are detected by differential FTIR spectroscopy. The FTIR data are discussed in relation with the results from global proton uptake measurements and electrogenic events concomitant with the reduction of Q(B) and with a model of the Q(B) turnover in Rb. sphaeroides RCs [Mulkidjanian, A. Y. (1999) FEBS Lett. 463, 199-204].     

Characterization of the bonding interactions of Q(B) upon photoreduction via A-branch or B-branch electron transfer in mutant reaction centers from Rhodobacter sphaeroides

In Rhodobacter sphaeroides reaction centers (RCs) containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the full-length of the A-branch of cofactors is prevented by the loss of the Q(A) ubiquinone, but it is possible to generate the radical pair P(+)H(A)(-) by A-branch electron transfer or the radical pair P(+)Q(B)(-) by B-branch electron transfer. In the present study, FTIR spectroscopy was used to provide direct evidence for the complete absence of the Q(A) ubiquinone in mutant RCs with the AM260W mutation. Light-induced FTIR difference spectroscopy of isolated RCs was also used to probe the neutral Q(B) and the semiquinone Q(B)(-) states in two B-branch active mutants, a double AM260W-LM214H mutant, denoted WH, and a quadruple mutant, denoted WAAH, in which the AM260W, LM214H, and EL212A-DL213A mutations were combined. The data were compared to those obtained with wild-type (Wt) RCs and the double EL212A-DL213A (denoted AA) mutant which exhibit the usual A-branch electron transfer to Q(B). The Q(B)(-)/Q(B) spectrum of the WH mutant is very close to that of Wt RCs indicating similar bonding interactions of Q(B) and Q(B)(-) with the protein in both RCs. The Q(B)(-)/Q(B) spectra of the AA and WAAH mutants are also closely related to one another, but are very different to that of the Wt complex. Isotope-edited IR fingerprint spectra were obtained for the AA and WAAH mutants reconstituted with site-specific (13)C-labeled ubiquinone. Whilst perturbations of the interactions of the semiquinone Q(B)(-) with the protein are observed in the AA and WAAH mutants, the FTIR data show that the bonding interaction of neutral Q(B) in these two mutants are essentially the same as those for Wt RCs. Therefore, it is concluded that Q(B) occupies the same binding position proximal to the non-heme iron prior to reduction by either A-branch or B-branch electron transfer.     

Low-frequency resonance Raman studies of the H(M202)G cavity mutant of bacterial photosynthetic reaction centers

Low-frequency (90–435 cm⁻¹) NIR-excitation (875–900 nm) resonance Raman (RR) studies are reported for the H(M202)G cavity mutant of bacterial photosynthetic reaction centers (RCs) from Rb. sphaeroides that was first described by Goldsmith et al. [(1996) Biochemistry 35: 2421–2428]. In this mutant, the His residue that axially ligates the Mg ion of the M-side bacteriochlorophyll (BChl) of the special pair primary donor (P) is replaced by a non-ligating Gly residue. Regardless, the Mg ion of P_M in the H(M202)G RCs remains pentacoordinates and is presumably ligated by a water molecule, although this axial ligand has not been definitively identified. The low-frequency RR studies of the H(M202)G RCs are accompanied by studies of RCs exchanged with D₂O and incubated with imidazole (Im). The RR studies of the cavity mutant RCs reveal the following: (1) The structure of P_M in the H(M202)G RCs is different from that of the wild-type, consistent with an altered BChl core. (2) A water ligand for P_M in the H(M202)G RCs is generally consistent with the low-frequency RR spectra. The Mg-OH₂ stretching vibration is tentatively assigned to a band at 318 cm⁻¹, a frequency higher than that of the Mg-His stretch of the native pigment (∼ ∼235 cm⁻¹). (3) The BChl core structure of P_M in the cavity mutant is rendered similar (but not identical) to that of the wild-type when the adventitious water axial ligand is replaced by Im. (4) Exchange with D₂O results in more global structural changes, likely involving the protein, which in turn affect the structure of the BChls in P. (5) Assignment of the low-frequency vibrational spectrum of P is generally more complex than originally suggested.     

Protein modifications affecting triplet energy transfer in bacterial photosynthetic reaction centers.

The efficiency of triplet energy transfer from the special pair (P) to the carotenoid (C) in photosynthetic reaction centers (RCs) from a large family of mutant strains has been investigated. The mutants carry substitutions at positions L181 and/or M208 near chlorophyll-based cofactors on the inactive and active sides of the complex, respectively. Light-modulated electron paramagnetic resonance at 10 K, where triplet energy transfer is thermally prohibited, reveals that the mutations do not perturb the electronic distribution of P. At temperatures > or = 70 K, we observe reduced signals from the carotenoid in most of the RCs with L181 substitutions. In particular, triplet transfer efficiency is reduced in all RCs in which a lysine at L181 donates a sixth ligand to the monomeric bacteriochlorophyll B(B). Replacement of the native Tyr at M208 on the active side of the complex with several polar residues increased transfer efficiency. The difference in the efficiencies of transfer in the RCs demonstrates the ability of the protein environment to influence the electronic overlap of the chromophores and thus the thermal barrier for triplet energy transfer.     

Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Balstochloris viridis. Influence of mutations at position M208

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (phi(*-)A) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas) viridis is characterized by 1H-ENDOR as well as optical absorption spectroscopy. The two site-directed mutants YF(M208) and YL(M208), in which tyrosine at position M208 is replaced by phenylalanine and leucine, respectively, are investigated and compared with the wild type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BCh1), B(A), and the BPh, phiA, that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of (phi(*-)A at 160 K indicates that two distinct states of phi(*-)A are present in the wild type and the mutant YF(M208). Based on a comparison with phi(*-)A in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of phiA. Only one phi(*-)A state occurs in the mutant YL(M208). This effect of the leucine residue at position M208 is explained by steric hindrance that locks the acetyl group in one specific position. On the basis of these results, an interpretation of the optical absorption difference spectrum of the state phi(*-)AQ(*-)A is attempted. This state can be accumulated at 100 K and undergoes an irreversible change between 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294-302]. The corresponding absorbance changes in the BCh1 Q(x) and Q(y) regions observed in the wild type also occur in the YF(M208) mutant but not in YL(M208). The observed changes in the wild type and YF(M208) are assigned to RCs in which the 3-acetyl group of phiA changes its orientation. It is concluded that this distinct structural relaxation of phiA can significantly affect the optical properties of B(A) and contribute to the light-induced absorption difference spectra.     

Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light — dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides. A bulk of absorption difference spectra was analyzed in the 940–1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B_A⁻ and β⁻, where β is a bacteriochlorophyll substituting the native bacteriopheophytin H_A) and in the 735–775 nm range (bleaching of the absorption band of the bacteriopheophytin H_B in the B-branch) in the −0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of ∼14 psec at 90 K and ∼18 psec at 293 K, which is accompanied by oscillations with a frequency of ∼150 cm⁻¹. A weak absorption band is found at 1018 nm that is formed ∼100 fsec after excitation of P and reflects the electron transfer from P* to β and/or B_A with accumulation of the P⁺β⁻ and/or P⁺B_A⁻ states. The kinetics of ΔA at 1018 nm contains the oscillations at ∼150 cm⁻¹ and distinct low-frequency oscillations at 20–100 cm⁻¹; also, the amplitude of the oscillations at 150 cm⁻¹ is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm⁻¹ mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The ΔA kinetics at 751 nm reflects the electron transfer to HB with formation of the P⁺H_B⁻ state. The oscillatory part of this kinetics has the form of a single peak with a maximum at ∼50 fsec completely decaying at ∼200 fsec, which might reflect a reversible electron transfer to the B-branch. The results are analyzed in terms of coherent nuclear wave packet motion induced in the P* excited state by femtosecond light pulses, of an influence of the incorporated mutations on the mutual position of the energy levels of charge separated states, and of the role of water HOH55 in the dynamics of the initial electron transfer.     

Trapped conformational states of semiquinone (D+*QB-*) formed by B-branch electron transfer at low temperature in Rhodobacter sphaeroides reaction centers

The reaction center (RC) from Rhodobacter sphaeroides captures light energy by electron transfer between quinones QA and QB, involving a conformational gating step. In this work, conformational states of D+*QB-* were trapped (80 K) and studied using EPR spectroscopy in native and mutant RCs that lack QA in which QB was reduced by the bacteriopheophytin along the B-branch. In mutant RCs frozen in the dark, a light induced EPR signal due to D+*QB-* formed in 30% of the sample with low quantum yield (0.2%-20%) and decayed in 6 s. A small signal with similar characteristics was also observed in native RCs. In contrast, the EPR signal due to D+*QB-* in mutant RCs illuminated while freezing formed in approximately 95% of the sample did not decay (tau >107 s) at 80 K (also observed in the native RC). In all samples, the observed g-values were the same (g = 2.0026), indicating that all active QB-*'s were located in a proximal conformation coupled with the nonheme Fe2+. We propose that before electron transfer at 80 K, the majority (approximately 70%) of QB, structurally located in the distal site, was not stably reducible, whereas the minority (approximately 30%) of active configurations was in the proximal site. The large difference in the lifetimes of the unrelaxed and relaxed D+*QB-* states is attributed to the relaxation of protein residues and internal water molecules that stabilize D+*QB-*. These results demonstrate energetically significant conformational changes involved in stabilizing the D+*QB-* state. The unrelaxed and relaxed states can be considered to be the initial and final states along the reaction coordinate for conformationally gated electron transfer.     

Steady-state FTIR spectra of the photoreduction of QA and QB in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones

In the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides, two ubiquinone molecules, QA and QB, play a pivotal role in the conversion of light energy into chemical free energy by coupling electron transfer to proton uptake. In native RCs, the transfer of an electron from QA to QB takes place in the time range of 5-200 micros. On the basis of time-resolved FTIR step-scan measurements in native RCs, a new and unconventional mechanism has been proposed in which QB- formation precedes QA- oxidation [Remy, A., and Gerwert, K. (2003) Nat. Struct. Biol. 10, 637-644]. The IR signature of the proposed transient intermediary electron acceptor (denoted X) operating between QA and QB has been recently measured by the rapid-scan technique in the DN(L210) mutant RCs, in which the QA to QB electron transfer is slowed 8-fold compared to that in native RCs. This IR signature has been reported as a difference spectrum involving states X+, X, QA, and QA- [Hermes, S., et al. (2006) Biochemistry 45, 13741-13749]. Here, we report the steady-state FTIR difference spectra of the photoreduction of either QA or QB measured in both native and DN(L210) mutant RCs in the presence of potassium ferrocyanide. In these spectra, the CN stretching marker modes of ferrocyanide and ferricyanide allow the extent of the redox reactions to be quantitatively compared and are used for a precise normalization of the QA-/QA and QB-/QB difference spectra. The calculated QA- QB/QA QB- double-difference spectrum in DN(L210) mutant RCs is closely equivalent to the reported QA- X+/QA X spectrum in the rapid-scan measurement. We therefore conclude that species X+ and X are spectrally indistinguishable from QB and QB-, respectively. Further comparison of the QA- QB/QA QB- double-difference spectra in native and DN(L210) RCs also allows the possibility that QB- formation precedes QA- reoxidation to be ruled out for native RCs.     

Identification of the proton pathway in bacterial reaction centers: decrease of proton transfer rate by mutation of surface histidines at H126 and H128 and chemical rescue by imidazole identifies the initial proton donors.

The pathway for proton transfer to Q(B) was studied in the reaction center (RC) from Rhodobacter sphaeroides. The binding of Zn(2+) or Cd(2+) to the RC surface at His-H126, His-H128, and Asp-H124 inhibits the rate of proton transfer to Q(B), suggesting that the His may be important for proton transfer [Paddock, M. L., Graige, M. S., Feher, G. and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188]. To assess directly the role of the histidines, mutant RCs were constructed in which either one or both His were replaced with Ala. In the single His mutant RCs, no significant effects were observed. In contrast, in the double mutant RC at pH 8.5, the observed rates of proton uptake associated with both the first and the second proton-coupled electron-transfer reactions k(AB)(()(1)()) [Q(A)(-)(*)Q(B)-Glu(-) + H(+) --> Q(A)(-)(*)Q(B)-GluH --> Q(A)Q(B)(-)(*)-GluH] and k(AB)(()(2)()) [Q(A)(-)(*)Q(B)(-)(*) + H(+) --> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)], were found to be slowed by factors of approximately 10 and approximately 4, respectively. Evidence that the observed changes in the double mutant RC are due to a reduction in the proton-transfer rate constants are provided by the observations: (i) k(AB)(1) at pH approximately pK(a) of GluH became biphasic, indicating that proton transfer is slower than electron transfer and (ii) k(AB)(2) became independent of the driving force for electron transfer, indicating that proton transfer is the rate-limiting step. These changes were overcome by the addition of exogenous imidazole which acts as a proton donor in place of the imidazole groups of His that were removed in the double mutant RC. Thus, we conclude that His-H126 and His-H128 facilitate proton transfer into the RC, acting as RC-bound proton donors at the entrance of the proton-transfer pathways.