EPR and ENDOR Investigation of Rhodosemiquinone in Bacterial Reaction Centers Formed by B-Branch Electron Transfer.

In photosynthetic bacteria, light-induced electron transfer takes place in a protein called the reaction center (RC) leading to the reduction of a bound ubiquinone molecule, Q(B), coupled with proton binding from solution. We used electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) to study the magnetic properties of the protonated semiquinone, an intermediate proposed to play a role in proton coupled electron transfer to Q(B). To stabilize the protonated semiquinone state, we used a ubiquinone derivative, rhodoquinone, which as a semiquinone is more easily protonated than ubisemiquinone. To reduce this low-potential quinone we used mutant RCs modified to directly reduce the quinone in the Q(B) site via B-branch electron transfer (Paddock et al. in Biochemistry 44:6920-6928, 2005). EPR and ENDOR signals were observed upon illumination of mutant RCs in the presence of rhodoquinone. The EPR signals had g values characteristic of rhodosemiquinone (g(x) = 2.0057, g(y) = 2.0048, g(z) ∼ 2.0018) at pH 9.5 and were changed at pH 4.5. The ENDOR spectrum showed couplings due to solvent exchangeable protons typical of hydrogen bonds similar to, but different from, those found for ubisemiquinone. This approach should be useful in future magnetic resonance studies of the protonated semiquinone.

Light induced EPR spectra of reaction centers from Rhodobacter sphaeroides at 80K: Evidence for reduction of Q(B) by B-branch electron transfer in native reaction centers.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides capture solar energy by electron transfer from primary donor, D, to quinone acceptor, Q(B,) through the active A-branch of electron acceptors, but not the inactive B-branch. The light induced EPR spectrum from native RCs that had Fe(2+) replaced by Zn(2+) was investigated at cryogenic temperature (80K, 35 GHz). In addition to the light induced signal due to formation of D(+•)Q(A) (-•) observed previously, a small fraction (~5%) of the signal displayed very different characteristics: (1) The signal was absent in RCs in which the Q(B) was displaced by the inhibitor stigmatellin. (2) Its decay time (τ=6 s) was the same as observed for D(+•)Q(B) (-•) in mutant RCs lacking Q(A,) which is significantly slower than for D(+•)Q(A) (-•) (τ=30 ms). (3) Its EPR spectrum was identical to that of D(+•)Q(B) (-•). (4) The quantum efficiency for forming the major component of the signal was the same as that found for mutant RCs lacking Q(A) (Φ =0.2%) and was temperature independent. These results are explained by direct photochemical reduction of Q(B)via B-branch electron transfer in a small fraction of native RCs.

ENDOR spectroscopy reveals light induced movement of the H-bond from Ser-L223 upon forming the semiquinone (Q(B)(-)(*)) in reaction centers from Rhodobacter sphaeroides.

Proton ENDOR spectroscopy was used to monitor local conformational changes in bacterial reaction centers (RC) associated with the electron-transfer reaction DQB --> D+*QB-* using mutant RCs capable of photoreducing QB at cryogenic temperatures. The charge separated state D+*QB-* was studied in mutant RCs formed by either (i) illuminating at low temperature (77 K) a sample frozen in the dark (ground state protein conformation) or (ii) illuminating at room temperature prior to and during freezing (charge separated state protein conformation). The charge recombination rates from the two states differed greatly (>10(6) fold) as shown previously, indicating a structural change (Paddock et al. (2006) Biochemistry 45, 14032-14042). ENDOR spectra of QB-* from both samples (35 GHz, 77 K) showed several H-bond hyperfine couplings that were similar to those for QB-* in native RCs indicating that in all RCs, QB-* was located at the proximal position near the metal site. In contrast, one set of hyperfine couplings were not observed in the dark frozen samples but were observed only in samples frozen under illumination in which the protein can relax prior to freezing. This flexible H-bond was assigned to an interaction between the Ser-L223 hydroxyl and QB-* on the basis of its absence in Ser L223 --> Ala mutant RCs. Thus, part of the protein relaxation, in response to light induced charge separation, involves the formation of an H-bond between the OH group of Ser-L223 and the anionic semiquinone QB-*. These results show the flexibility of the Ser-L223 H-bond, which is essential for its function in proton transfer to reduced QB.

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.

Characterization of a highly purified, fully active, crystallizable RC-LH1-PufX core complex from Rhodobacter sphaeroides.

Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1-PufX complex (RC-LH1-PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQA --> D+QA -) in the RC as a function of light intensity. The RC-LH1-PufX complex had a quantum efficiency of 0.95 +/- 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 +/- 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 +/- 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 A. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 A, b = 137.2 A, c = 137.5 A; alpha = 60.0 degrees , beta = 89.95 degrees , gamma =90.02 degrees .

Interactions between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: the cation-pi interaction.

The cation-pi interaction between positively charged and aromatic groups is a common feature of many proteins and protein complexes. The structure of the complex between cytochrome c(2) (cyt c(2)) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides exhibits a cation-pi complex formed between Arg-C32 on cyt c(2) and Tyr-M295 on the RC [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. The importance of the cation-pi interaction for binding and electron transfer was studied by mutating Tyr-M295 and Arg-C32. The first- and second-order rates for electron transfer were not affected by mutating Tyr-M295 to Ala, indicating that the cation-pi complex does not greatly affect the association process or structure of the state active in electron transfer. The dissociation constant K(D) showed a greater increase when Try-M295 was replaced with nonaromatic Ala (3-fold) as opposed to aromatic Phe (1.2-fold), which is characteristic of a cation-pi interaction. Replacement of Arg-C32 with Ala increased K(D) (80-fold) largely due to removal of electrostatic interactions with negatively charged residues on the RC. Replacement with Lys increased K(D) (6-fold), indicating that Lys does not form a cation-pi complex. This specificity for Arg may be due to a solvation effect. Double mutant analysis indicates an interaction energy between Tyr-M295 and Arg-C32 of approximately -24 meV (-0.6 kcal/mol). This energy is surprisingly small considering the widespread occurrence of cation-pi complexes and may be due to the tradeoff between the favorable cation-pi binding energy and the unfavorable desolvation energy needed to bury Arg-C32 in the short-range contact region between the two proteins.

Quinone (QB) reduction by B-branch electron transfer in mutant bacterial reaction centers from Rhodobacter sphaeroides: quantum efficiency and X-ray structure.

The photosynthetic reaction center (RC) from purple bacteria converts light into chemical energy. Although the RC shows two nearly structurally symmetric branches, A and B, light-induced electron transfer in the native RC occurs almost exclusively along the A-branch to a primary quinone electron acceptor Q(A). Subsequent electron and proton transfer to a mobile quinone molecule Q(B) converts it to a quinol, Q(B)H(2). We report the construction and characterization of a series of mutants in Rhodobacter sphaeroides designed to reduce Q(B) via the B-branch. The quantum efficiency to Q(B) via the B-branch Phi(B) ranged from 0.4% in an RC containing the single mutation Ala-M260 --> Trp to 5% in a quintuple mutant which includes in addition three mutations to inhibit transfer along the A-branch (Gly-M203 --> Asp, Tyr-M210 --> Phe, Leu-M214 --> His) and one to promote transfer along the B-branch (Phe-L181 --> Tyr). Comparing the value of 0.4% for Phi(B) obtained in the AW(M260) mutant, which lacks Q(A), to the 100% quantum efficiency for Phi(A) along the A-branch in the native RC, we obtain a ratio for A-branch to B-branch electron transfer of 250:1. We determined the structure of the most effective (quintuple) mutant RC at 2.25 A (R-factor = 19.6%). The Q(A) site did not contain a quinone but was occupied by the side chain of Trp-M260 and a Cl(-). In this structure a nonfunctional quinone was found to occupy a new site near M258 and M268. The implications of this work to trap intermediate states are discussed.

Proton transfer pathways and mechanism in bacterial reaction centers.

The focus of this minireview is to discuss the state of knowledge of the pathways and rates of proton transfer in the bacterial reaction center (RC) from Rhodobacter sphaeroides. Protons involved in the light driven catalytic reduction of a quinone molecule QB to quinol QBH2 travel from the aqueous solution through well defined proton transfer pathways to the oxygen atoms of the quinone. Three main topics are discussed: (1) the pathways for proton transfer involving the residues: His-H126, His-H128, Asp-L210, Asp-M17, Asp-L213, Ser-L223 and Glu-L212, which were determined by a variety of methods including the use of proton uptake inhibiting metal ions (e.g. Zn2+ and Cd2+); (2) the rate constants for proton transfer, obtained from a 'chemical rescue' study was determined to be 2 x 10(5) s(-1) and 2 x 10(4) s(-1) for the proton uptake to Glu-L212 and QB-*, respectively; (3) structural studies of altered proton transfer pathways in revertant RCs that lack the key amino acid Asp-L213 show a series of structural changes that propagate toward L213 potentially allowing Glu-H173 to participate in the proton transfer processes.

Mechanism of proton transfer inhibition by Cd(2+) binding to bacterial reaction centers: determination of the pK(A) of functionally important histidine residues.

The bacterial photosynthetic reaction center (RC) uses light energy to catalyze the reduction of a bound quinone molecule Q(B) to quinol Q(B)H(2). In RCs from Rhodobacter sphaeroides the protons involved in this process come from the cytoplasm and travel through pathways that involve His-H126 and His-H128 located near the proton entry point. In this study, we measured the pH dependence from 4.5 to 8.5 of the binding of the proton transfer inhibitor Cd(2+), which ligates to these surface His in the RC and inhibits proton-coupled electron transfer. At pH <6, the negative slope of the logarithm of the dissociation constant, K(D), versus pH approaches 2, indicating that, upon binding of Cd(2+), two protons are displaced; i.e., the binding is electrostatically compensated. At pH >7, K(D) becomes essentially independent of pH. A theoretical fit to the data over the entire pH range required two protons with pK(A) values of 6.8 and 6.3 (+/-0.5). To assess the contribution of His-H126 and His-H128 to the observed pH dependence, K(D) was measured in mutant RCs that lack the imidazole group of His-H126 or His-H128 (His --> Ala). In both mutant RCs, K(D) was approximately pH independent, showing that Cd(2+) does not displace protons upon binding in the mutant RCs, in contrast to the native RC in which His-H126 and His-H128 are the predominant contributors to the observed pH dependence of K(D). Thus, Cd(2+) inhibits RC function by binding to functionally important histidines.

Determination of proton transfer rates by chemical rescue: application to bacterial reaction centers.

The bacterial reaction center (RC) converts light into chemical energy through the reduction of an internal quinone molecule Q(B) to Q(B)H(2). In the native RC, proton transfer is coupled to electron transfer and is not rate-controlling. Consequently, proton transfer is not directly observable, and its rate was unknown. In this work, we present a method for making proton transfer rate-controlling, which enabled us to determine its rate. The imidazole groups of the His-H126 and His-H128 proton donors, located at the entrance of the transfer pathways, were removed by site-directed mutagenesis (His --> Ala). This resulted in a reduction in the observed proton-coupled electron transfer rate [(Q(A)(-)(*)Q(B))Glu(-) + H(+) --> (Q(A)Q(B)(-)(*))GluH], which became rate-controlled by proton uptake to Glu-L212 [Adelroth, P., et al. (2001) Biochemistry 40, 14538-14546]. The proton uptake rate was enhanced (rescued) in a controlled fashion by the addition of imidazole or other amine-containing acids. From the dependence of the observed rate on acid concentration, an apparent second-order rate constant k((2)) for the "rescue" of the rate was determined. k((2)) is a function of the proton transfer rate and the binding of the acid. The dependence of k((2)) on the acid pK(a) (i.e., the proton driving force) was measured over 9 pK(a) units, resulting in a Brönsted plot that was characteristic of general acid catalysis. The results were fitted to a model that includes the binding (facilitated by electrostatic attraction) of the cationic acid to the RC surface, proton transfer to an intermediate proton acceptor group, and subsequent proton transfer to Glu-L212. A proton transfer rate constant of approximately 10(5) s(-)(1) was determined for transfer from the bound imidazole group to Glu-L212 (over a distance of approximately 20 A). The same method was used to determine a proton transfer rate constant of 2 x 10(4) s(-)(1) for transfer to Q(B)(-)(*). The relatively fast proton transfer rates are explained by the presence of an intermediate acceptor group that breaks the process into sequential proton transfer steps over shorter distances. This study illustrates an approach that could be generally applied to obtain information about the individual rates and energies for proton transfer processes, as well as the pK(a)s of transfer components, in a variety of proton translocating systems.

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.

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.

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.

Identification of the proton pathway in bacterial reaction centers: both protons associated with reduction of QB to QBH2 share a common entry point.

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, Q(B). This process involves the transfer of two protons from the aqueous solution to the protein-bound Q(B) molecule. The second proton, H(+)(2), is supplied to Q(B) by Glu-L212, an internal residue protonated in response to formation of Q(A)(-) and Q(B)(-). In this work, the pathway for H(+)(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of Q(A) (DQ(A)-->D(+)Q(A)(-)) and Q(B) (DQ(B)-->D(+)Q(B)(-)), studied by using pH-sensitive dyes. The other was the electron transfer k(AB)((1)) (Q(A)(-)Q(B)-->Q(A)Q(B)(-)). At pH 8.5, binding of Zn(2+), Cd(2+), or Ni(2+) reduced the rates of proton uptake upon Q(A)(-) and Q(B)(-) formation as well as k(AB)((1)) by approximately an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D(+)Q(A)(-) is formed 10(5)-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212-->Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn(2+) binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H(+)(2) was thus identified to be the same as for the first proton H(+)(1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Proton and electron transfer in bacterial reaction centers.

The bacterial reaction center couples light-induced electron transfer to proton pumping across the membrane by reactions of a quinone molecule Q(B) that binds two electrons and two protons at the active site. This article reviews recent experimental work on the mechanism of the proton-coupled electron transfer and the pathways for proton transfer to the Q(B) site. The mechanism of the first electron transfer, k((1))(AB), Q(-)(A)Q(B)-->Q(A)Q(-)(B), was shown to be rate limited by conformational gating. The mechanism of the second electron transfer, k((2))(AB), was shown to involve rapid reversible proton transfer to the semiquinone followed by rate-limiting electron transfer, H(+)+Q(-)(A)Q(-)(B) ifQ(-)(A)Q(B)H-->Q(A)(Q(B)H)(-). The pathways for transfer of the first and second protons were elucidated by high-resolution X-ray crystallography as well as kinetic studies showing changes in the rate of proton transfer due to site directed mutations and metal ion binding.

Determination of the binding sites of the proton transfer inhibitors Cd2+ and Zn2+ in bacterial reaction centers.

The reaction center (RC) from Rhodobacter sphaeroides couples light-driven electron transfer to protonation of a bound quinone acceptor molecule, Q(B), within the RC. The binding of Cd(2+) or Zn(2+) has been previously shown to inhibit the rate of reduction and protonation of Q(B). We report here on the metal binding site, determined by x-ray diffraction at 2.5-A resolution, obtained from RC crystals that were soaked in the presence of the metal. The structures were refined to R factors of 23% and 24% for the Cd(2+) and Zn(2+) complexes, respectively. Both metals bind to the same location, coordinating to Asp-H124, His-H126, and His-H128. The rate of electron transfer from Q(A)(-) to Q(B) was measured in the Cd(2+)-soaked crystal and found to be the same as in solution in the presence of Cd(2+). In addition to the changes in the kinetics, a structural effect of Cd(2+) on Glu-H173 was observed. This residue was well resolved in the x-ray structure-i.e., ordered-with Cd(2+) bound to the RC, in contrast to its disordered state in the absence of Cd(2+), which suggests that the mobility of Glu-H173 plays an important role in the rate of reduction of Q(B). The position of the Cd(2+) and Zn(2+) localizes the proton entry into the RC near Asp-H124, His-H126, and His-H128. Based on the location of the metal, likely pathways of proton transfer from the aqueous surface to Q(B) are proposed.

Identification of the proton pathway in bacterial reaction centers: replacement of Asp-M17 and Asp-L210 with asn reduces the proton transfer rate in the presence of Cd2+.

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the reduction and protonation of a bound quinone molecule Q(B) (the secondary quinone electron acceptor). We investigated the proton transfer pathway by measuring the proton-coupled electron transfer, k(AB)((2)) [Q(A)Q(B) + H(+) --> Q(A)(Q(B)H)(-)] in native and mutant RCs in the absence and presence of Cd(2+). Previous work has shown that the binding of Cd(2+) decreases k(AB)((2)) in native RCs approximately 100-fold. The preceding paper shows that bound Cd(2+) binds to Asp-H124, His-H126, and His-H128. This region represents the entry point for protons. In this work we investigated the proton transfer pathway connecting the entry point with Q(B) by searching for mutations that greatly affect k(AB)((2)) ( greater, similar10-fold) in the presence of Cd(2+), where k(AB)((2)) is limited by the proton transfer rate (k(H)). Upon mutation of Asp-L210 or Asp-M17 to Asn, k(H) decreased from approximately 60 s(-1) to approximately 7 s(-1), which shows the important role that Asp-L210 and Asp-M17 play in the proton transfer chain. By comparing the rate of proton transfer in the mutants (k(H) approximately 7 s(-1)) with that in native RCs in the absence of Cd(2+) (k(H) >/= 10(4) s(-1)), we conclude that alternate proton transfer pathways, which have been postulated, are at least 10(3)-fold less effective.

Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: implications for the mechanism of electron and proton transfer in proteins.

A proton-activated electron transfer (PAET) mechanism, involving a protonated semiquinone intermediate state, had been proposed for the electron-transfer reaction k(2)AB [Q(A)(-)(*)Q(B)(-)(*) + H(+) <--> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)] in reaction centers (RCs) from Rhodobacter sphaeroides [Graige, M. S., Paddock, M. L., Bruce, M. L., Feher, G., and Okamura, M. Y. (1996) J. Am. Chem. Soc. 118, 9005-9016]. Confirmation of this mechanism by observing the protonated semiquinone (Q(B)H)(*) had not been possible, presumably because of its low pK(a). By replacing the native Q(10) in the Q(B) site with rhodoquinone (RQ), which has a higher pK(a), we were able to observe the (Q(B)H)(*) state. The pH dependence of the semiquinone optical spectrum gave a pK(a) = 7.3 +/- 0.2. At pH < pK(a), the observed rate for the reaction was constant and attributed to the intrinsic electron-transfer rate from Q(A)(-)(*) to the protonated semiquinone (i.e., k(2)AB = k(ET)(RQ) = 2 x 10(4) s(-)(1)). The rate decreased at pH > pK(a) as predicted by the PAET mechanism in which fast reversible proton transfer precedes rate-limiting electron transfer. Consequently, near pH 7, the proton-transfer rate k(H) >> 10(4) s(-)(1). Applying the two step mechanism to RCs containing native Q(10) and taking into account the change in redox potential, we find reasonable values for the fraction of (Q(B)H)(*) congruent with 0.1% (consistent with a pK(a)(Q(10)) of approximately 4.5) and k(ET)(Q(10)) congruent with 10(6) s(-)(1). These results confirm the PAET mechanism in RCs with RQ and give strong support that this mechanism is active in RCs with Q(10) as well.

Identification of the proton pathway in bacterial reaction centers: inhibition of proton transfer by binding of Zn2+ or Cd2+.

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA-*QB-* + H+ --> QA(QBH)-, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD /= 10(2)-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+*QAQB-* --> DQAQB suggests that the binding site is located far (>10 A) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB-* occurs.

Proton uptake by carboxylic acid groups upon photoreduction of the secondary quinone (QB) in bacterial reaction centers from Rhodobacter sphaeroides: FTIR studies on the effects of replacing Glu H173.

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, Glu H173, located approximately 7 A from the center of the secondary quinone acceptor QB, is expected to contribute to proton uptake upon QB- formation in response to the movement of an electron in its vicinity. Steady-state FTIR difference spectroscopy provides a method to monitor proton uptake by carboxylic acids upon photochemical changes. The FTIR spectra corresponding to the photoreduction of QB were obtained at pH 7 for RCs containing Glu (native), Gln (EQ H173), or Asp (ED H173) at the H173 site. No new bands were observed in the carboxylic acid region (1770-1700 cm-1) in any of the mutant RCs compared to native RCs. In addition, the positive band at 1728 cm-1, previously assigned to Glu L212 [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., Mäntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732], remained present in all of the mutant RCs. This result shows that Glu H173 is not a major contributor to proton uptake upon QB- formation and further strengthens the assignment of the 1728 cm-1 band to Glu L212. An increase in the 1728 cm-1 band was observed in the EQ H173 RCs compared to that of either the ED H173 or native RCs. These changes are consistent with Glu and Asp at H173 remaining ionized in the QB and QB- states. Changes in the absorption regions of the semiquinone and amide or side chain groups in the spectra of the mutant RCs suggest slight changes in the protein structure compared to those of native RCs, which could contribute to the altered kinetics observed in the mutant RCs.