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.     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selective perturbation of the second electron transfer step in mutant bacterial reaction centers

In order to specifically perturb the primary electron acceptor B(A) -- a monomeric bacteriochlorophyll (BChl) a -- involved in bacterial photosynthetic charge separation (CS), the protein environment of B(A) in the reaction center (RC) of Rhodobacter sphaeroides was modified by site-directed mutagenesis. Isolated RCs were characterized by redox titrations, low temperature optical spectroscopy, ENDOR/TRIPLE resonance spectroscopy and femtosecond time-resolved spectroscopy. Two mutations were studied: In the GS(M203) mutant a serine is introduced near the ring E keto group of B(A), while in FY(L146) a phenylalanine near the ring A acetyl group of B(A) is replaced by tyrosine. In all mutations the oxidation potential of the primary electron donor P as well as the electronic structure of both the P(*+) radical cation and the radical anion of the secondary electron acceptor, H(A)(*-), are not significantly altered compared to the wild type (WT), while changes of the optical absorption spectra at 77 K in the BChl Q(X) and Q(Y) regions are observed. The GS(M203) mutation only leads to a minor retardation of the CS reactions at room temperature, whereas for FY(L146) significant deviations from the native electron transfer (ET) rates could be detected: In addition to a faster first (2.9 ps) and a slower second (1 ps) ET step, a new 8-ps time constant was found in the FY(L146) mutant, which can be ascribed to a fraction of RCs with slowed down secondary ET. The results allow us to address the functional role of the acetyl group of B(A) and question the role of the free energy changes as the main determining factor of ET rates in RCs. It is concluded that structural rearrangements alter the electronic coupling between the pigments and thereby influence the rate of fast CS.     

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.     

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.     

High yield of secondary B-side electron transfer in mutant Rhodobacter capsulatus reaction centers

From the crystal structures of reaction centers (RCs) from purple photosynthetic bacteria, two pathways for electron transfer (ET) are apparent but only one pathway (the A side) operates in the native protein-cofactor complex. Partial activation of the B-side pathway has unveiled the true inefficiencies of ET processes on that side in comparison to analogous reactions on the A side. Of significance are the relative rate constants for forward ET and the competing charge recombination reactions. On the B side, these rate constants are nearly equal for the secondary charge-separation step (ET from bacteriopheophytin to quinone), relegating the yield of this process to < 50%. Herein we report efforts to optimize this step. In surveying all possible residues at position 131 in the M subunit, we discovered that when glutamic acid replaces the native valine the efficiency of the secondary ET is nearly two-fold higher than in the wild-type RC. The positive effect of M131 Glu is likely due to formation of a hydrogen bond with the ring V keto group of the B-side bacteriopheophytin leading to stabilization of the charge-separated state involving this cofactor. This change slows charge recombination by roughly a factor of two and affords the improved yield of the desired forward ET to the B-side quinone terminal acceptor.     

Vibrational coherence in bacterial reaction centers with genetically modified B-branch pigment composition

Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as PhiB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (PhiB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA- absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than approximately 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA-) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+ BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P*-->P+ BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the PhiB molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and PhiB, the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+ PhiB- charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of PhiB in the B-branch within about 40 fs, which is earlier by approximately 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.     

Temperature dependence of the primary electron transfer reaction in pigment-modified bacterial reaction centers

The initial electron transfer steps in pigment modified reaction centers, where bacteriopheophytin is replaced by plant pheophytin (R26.Phe-a RCs) have been investigated over a wide temperature range by femtosecond time-resolved spectroscopy. The experimental data obtained in the maximum of the bacteriochlorophyll anion band at 1020 nm show the existence of a high and long-lived population of the primary acceptor P⁺B_A ^– even at 10 K. The data suggest a stepwise electron transfer mechanism with B_A as primary acceptor also in the low temperature domain. A detailed data analysis suggests that the pigment modification leads to a situation with almost isoenergetic primary and secondary acceptor levels, approximately 450 cm^–1 below P^*. A Gaussian distribution (with = 400 cm ^–1) of the G values has to be assumed to account for the strong dispersive character of the kinetics in this sample. Based on these assumptions, a model is presented that reproduces the observed kinetics, heterogeneity and temperature dependence.     

Iron as a bound secondary electron donor in modified bacterial reaction centers

The binding and oxidation of ferrous iron were studied in wild-type reaction centers and in mutants that have been modified to be both highly oxidizing and able to bind manganese [Thielges et al. (2005) Biochemistry 44, 7389-7394]. After illumination of wild-type reaction centers, steady-state optical spectroscopy showed that the oxidized bacteriochlorophyll dimer, P+, could oxidize iron but only as a second-order reaction at iron concentrations above 100 microM. In the modified reaction centers, P+ was reduced by iron in the presence of sodium bicarbonate with dissociation constants of approximately 1 microM for two mutants with different metal-binding sites. Transient optical spectroscopy showed that P+ was rapidly reduced with first-order rates of 170 and 275 s-1 for the two mutants. The dependence of the amplitude of this rate on the iron concentration yielded a dissociation constant of approximately 1 microM for both mutants, in agreement with the steady-state determination. The oxidation of bound iron by P+ was confirmed by the observation of a light-induced EPR signal centered at g values of 2.2 and 4.3 and attributed to high-spin Fe3+. Bicarbonate was required at pH 7 for low dissociation constants for both iron and manganese binding. The similarity between iron and manganese binding in these mutants provides insight into general properties of metal-binding sites in proteins.     

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.     

Resolution of electron and proton transfer events in the electrochromism associated with quinone reduction in bacterial reaction centers

We have measured the electrochromic response of the bacteriopheophytin, BPh, and bacteriochlorophyll, BChl, cofactors during the Q_A ^–Q_B Q_AQ_B ^– electron transfer in chromatophores of Rhodobacter (Rb.) capsulatus and Rb. sphaeroides. The electrochromic response rises faster in chromatophores and is more clearly biexponential than it is in isolated reaction centers. The chromatophore spectra can be interpreted in terms of a clear kinetic separation between fast electron transfer and slower non-electron transfer events such as proton transfer or protein relaxation. The electrochromic response to electron transfer exhibits rise times of about 4 µs (70%) and 40 µs (30%) in Rb. capsulatus and 4 µs (60%) and 80 µs (40%) in Rb. sphaeroides. The BPh absorption band is shifted to nearly equivalent positions in the Q_A ^– and nascent Q_B ^– states, indicating that the electrochromic perturbation of BPh absorption from the newly formed Q_B ^– state is comparable to that of Q_A ^– . Subsequently, partial attenuation of the Q_B ^– electrochromism occurs with a time constant on the order of 200 µs. This can be attributed to partial charge compensation by H⁺ (or other counter ion) movement into the Q_B pocket. Electron transfer events were found to be slower in detergent isolated RCs than in chromatophores, more nearly monoexponential, and overlap H⁺ transfer, suggesting that a change in rate-limiting step has occurred upon detergent solubilization.     

Blue light drives B-side electron transfer in bacterial photosynthetic reaction centers.

The core of the photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer. Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near-infrared light. Here, it is shown that excitation with blue light into the Soret band of the reaction center gives rise to electron transfer along the alternate or B-side pathway, resulting in a charge-separated state involving the anion of the B-side bacteriopheophytin. This electron transfer is much faster than normal A-side transfer, apparently occurring within a few hundred femtoseconds. At low temperatures, the B-side charge-separated state is stable for at least 1 ns, but at room temperature, the B-side bacteriopheophytin anion is short-lived, decaying within approximately 15 ps. One possible physiological role for B-side electron transfer is photoprotection, rapidly quenching higher excited states of the reaction center.     

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.     

An assessment of the mechanism of initial electron transfer in bacterial reaction centers

Subpicosecond time-resolved photodichroism measurements on Rhodobacter sphaeroides R26 reaction centers are reported in the key region between 620 and 740 nm, where the anions of both bacteriopheophytin and bacteriochlorophyll (BChl) have their most diagnostic absorption bands. These measurements fail to resolve clearly the formation of a reduced BChl species. The implications of this for elucidating the role of the accessory BChl in the initial stage of charge separation are discussed.     

Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane.     

Effects of freezing out protein vibrational modes on electron transfer kinetics in bacterial reaction centers

We propose a model that some vibrational modes of the protein in bacterial photosynthetic reaction centers may be frozen at low temperatures. The freezing of the protein-environmental motion can affect the electron transfer rate through changes in the reorganization energy and the free energy gap. We offer a qualitative explanation of the different kinetics of the ET processes in reaction centers which are cooled in the dark and cooled under illumination.     

High throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.     

Kinetics of electron transfer between the primary and the secondary electron acceptor in reaction centers from Rhodopseudomonas sphaeroides

Photoreduction of the two ubiquinone molecules, UQ₁ and UQ₂, bound to purified reaction center from Rhodopseudomonas sphaeroides induces different absorption band shifts of bacteriochlorophyll and bacteriopheophytin molecules depending on which ubiquinone is photoreduced. This allows us to study electron transfer between UQ₁ and UQ₂ directly by absorption spectrometry. The results support a model in which electrons are transferred one by one from UQ₁ to UQ₂ with a half-time of 200 μs, and two by two from fully reduced UQ₂ to the secondary acceptor pool.     

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.