-deltaG(AB) and pH dependence of the electron transfer from P(+)Q(A)(-)Q(B) toP(+)Q(A)Q(B)(-) in Rhodobacter sphaeroides reaction centers

The electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) can occur in two phases with a well-characterized 100 micros component (tau(2)) and a faster process occurring in less than 10 micros (tau(1)). The fast reaction is clearly seen when the native ubiquinone-10 at Q(A) is replaced with naphthoquinones. The dependence of tau(1) on the free-energy difference between the P(+)Q(A)(-)Q(B) and P(+)Q(A)Q(B)(-) states (-) and on the pH was measured using naphthoquinones with different electrochemical midpoint potentials as Q(A) in Rhodobacter sphaeroides reaction centers (RCs) and in RCs where - is changed by mutation of M265 in the Q(A) site from Ile to Thr (M265IT). Q(B) was ubiquinone (UQ(B)) in all cases. Electron transfer was measured by using the absorption differences of the naphthosemiquinone at Q(A) and the ubisemiquinone at Q(B) between 390 and 500 nm. As - was changed from -90 to -250 meV tau(1) decreased from 29 to 0.2 micros. The free-energy dependence of tau(1) provides a reorganization energy of 850 +/- 100 meV for the electron transfer from Q(A)(-) to Q(B). The slower reaction at tau(2) is free-energy independent, so processes other than electron transfer determine the observed rate. The fraction of the reaction at tau(1) increases with increasing driving force and is 100% of the reaction when - is approximately 100 meV more favorable than in the native RCs with ubiquinone as Q(A). The fast phase, tau(1), is pH independent from pH 6 to 11 while tau(2) slows above pH 9. As the Q(A) isoprene tail length is increased from 2 to 10 isoprene units the fraction at tau(1) decreases. However, tau(1), tau(2), and the fraction of the reaction in each phase are independent of the tail length of UQ(B).     

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q(B) in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q(A-)Q(B)-->Q(A)Q(B-) (k(AB)(1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k(AB)(1)), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q(B) [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (-), 1601 (-) and 1467 (+) cm(-1), characteristic of Q(A) reduction. The time evolution of the spectra shows reoxidation of Q(A-) and concomitant reduction of Q(B) with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q(B-) occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm(-1) [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q(B) in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q(A-) to Q(B) and the identification of Glu-L212 as the main proton acceptor in the state Q(A)Q(B-).     

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.     

Investigation of B-branch electron transfer by femtosecond time resolved spectroscopy in a Rhodobacter sphaeroides reaction centre that lacks the Q(A) ubiquinone

The dynamics of electron transfer in a membrane-bound Rhodobacter sphaeroides reaction centre containing a combination of four mutations were investigated by transient absorption spectroscopy. The reaction centre, named WAAH, has a mutation that causes the reaction centre to assemble without a Q(A) ubiquinone (Ala M260 to Trp), a mutation that causes the replacement of the H(A) bacteriopheophytin with a bacteriochlorophyll (Leu M214 to His) and two mutations that remove acidic groups close to the Q(B) ubiquinone (Glu L212 to Ala and Asp L213 to Ala). Previous work has shown that the Q(B) ubiquinone is reduced by electron transfer along the so-called inactive cofactor branch (B-branch) in the WAAH reaction centre (M.C. Wakeham, M.G. Goodwin, C. McKibbin, M.R. Jones, Photo-accumulation of the P(+)Q(B)(-) radical pair state in purple bacterial reaction centres that lack the Q(A) ubiquinone, FEBS Letters 540 (2003) 234-240). In the present study the dynamics of electron transfer in the membrane-bound WAAH reaction centre were studied by femtosecond transient absorption spectroscopy, and the data analysed using a compartmental model. The analysis indicates that the yield of Q(B) reduction via the B-branch is approximately 8% in the WAAH reaction centre, consistent with results from millisecond time-scale kinetic spectroscopy. Possible contributions to this yield of the constituent mutations in the WAAH reaction centre and the membrane environment of the complex are discussed.     

Low-temperature interquinone electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis: characterization of Q(B)- states by high-frequency electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR)

High-frequency electron paramagnetic resonance (HF EPR) techniques have been employed to look for localized light-induced conformational changes in the protein environments around the reduced secondary quinone acceptor (Q(B)(-)) in Rhodobacter sphaeroides and Blastochloris viridis RCs. The Q(A)(-) and Q(B)(-) radical species in Fe-removed/Zn-replaced protonated RCs substituted with deuterated quinones are distinguishable with pulsed D-band (130 GHz) EPR and provide native probes of both the low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer event and the structure of trapped conformational substates. We report here the first spectroscopic evidence that cryogenically trapped, light-induced changes enable low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer in the B. viridis RC and the first observation of an inactive, trapped P(+)Q(B)(-) state in both R. sphaeroides and B. viridis RCs that does not recombine at 20 K. The high resolution and orientational selectivity of HF electron-nuclear double resonance (ENDOR) allows us to directly probe protein environments around Q(B)(-) for distinct P(+)Q(B)(-) kinetic RC states by spectrally selecting specific nuclei in isotopically labeled samples. No structural differences in the protein structure near Q(B)(-) or reorientation (within 5 degrees ) of Q(B)(-) was observed with HF ENDOR spectra of two states of P(+)Q(B)(-): "active" and "inactive" states with regards to low-temperature electron transfer. These results reveal a remarkably enforced local protein environment for Q(B) in its reduced semiquinone state and suggest that the conformational change that controls reactivity resides beyond the Q(B) local environment.     

Electrogenic proton transfer in Rhodobacter sphaeroides reaction centers: effect of coenzyme Q(10) substitution by decylubiquinone in the Q(B) binding site.

An electrometric technique was used to investigate the effect of coenzyme Q(10) (UQ), substitution by decylubiquinone (dQ) at the Q(B) binding site of reaction centers (UQ-RC and dQ-RC, respectively) on the electrogenic proton transfer kinetics upon Q(B) reduction in Rhodobacter sphaeroides chromatophores. Unlike dQ-RC, the kinetics of the second flash-induced proton uptake in UQ-RC clearly deviated from the mono-exponential one. The activation energy (about 30 kJ/mol) and the pH profile of the kinetics in dQ-RC were similar to those in UQ-RC, with the power law approximation used in the latter case. The interpretation of the data presumed the quinone translocation between the two binding positions within the Q(B) site. It is proposed that the native isoprenyl side chain (in contrast to decyl chain) favors the equilibrium binding of neutral quinone at the redox-active 'proximal' position, but causes a higher barrier for the hydroquinone movement from 'proximal' to 'distal' position.     

The L(M196)H mutation in Rhodobacter sphaeroides reaction center results in new electrostatic interactions

Photosynthesis Research - New histidine residue was introduced in M196 position in the reaction center of Rhodobacter sphaeroides in order to alter polarity of the BChl dimer’s protein...     

Mutation of the Chlamydomonas reinhardtii analogue of residue M210 of the Rhodobacter sphaeroides reaction center slows primary electron transfer in Photosystem II

Primary charge separation within Photosystem II (PS II) is much slower (time constant 21 ps) than the equivalent step in the related reaction center (RC) found in purple bacteria ( 3 ps). In the case of the bacterial RC, replacement of a specific tyrosine residue within the M subunit (at position 210 in Rhodobacter sphaeroides), by a leucine residue slows down charge separation to 20 ps. Significantly the analogous residue in PS II, within the D2 polypeptide, is a leucine not a tyrosine (at position D2-205, Chlamydomonas reinhardtii numbering). Consequently, it has been postulated [Hastings et al. (1992) Biochemistry 31: 7638–7647] that the rate of electron transfer could be increased in PS II by replacing this leucine residue with tyrosine. We have tested this hypothesis by constructing the D2-Leu205Tyr mutant in the green alga, Chlamydomonas reinhardtii, through transformation of the chloroplast genome. Primary charge separation was examined in isolated PS II RCs by time-resolved optical spectroscopy and was found to occur with a time constant of 40 ps. We conclude that mutation of D2-Leu205 to Tyr does not increase the rate of charge separation in PS II. The slower kinetics of primary charge separation in wild type PS II are probably not due to a specific difference in primary structure compared with the bacterial RC but rather a consequence of the P680 singlet excited state being a shallower trap for excitation energy within the reaction center.     

Exploring the energy profile of the Q(A)(-) to Q(B) electron transfer reaction in bacterial photosynthetic reaction centers: pH dependence of the conformational gating step

Both large- and small-scale conformational changes are needed as proteins carry out reactions. However, little is known about the identity, energy of, and barriers between functional substates on protein reaction coordinates. In isolated bacterial photosynthetic reaction centers, the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), is rate limited by conformational changes at low pH and by proton binding at high pH. The kinetics and thermodynamics of this reaction were determined between 200 and 300 K from pH 6 to pH 10.5. A model with two substates of the reactant, P(+)Q(A)(-)Q(B), one protonated (state A) and one unprotonated (alpha), and one state of the product, P(+)Q(A)Q(B)(-) (B), was able to simulate the dependence of the rate on temperature and pH fairly well. The equilibrium between the three states were measured in situ at each temperature. Proton binding (alpha to A transition) has a favorable DeltaH and unfavorable DeltaS as does the conformational changes required for electron transfer at low pH (A to B). The pK for the A to alpha transition is 9.7 at room temperature, consistent with previous measurements, and equivalent to 13.5 at 200 K. The activation barriers were determined for each transition. Both the alpha to A and the A to B transitions are limited primarily by the activation enthalpy with modest DeltaS.     

The site-directed mutation I(L177)H in Rhodobacter sphaeroides reaction center affects coordination of P(A) and B(B) bacteriochlorophylls

To explore the influence of the I(L177)H single mutation on the properties of the nearest bacteriochlorophylls (BChls), three reaction centers (RCs) bearing double mutations were constructed in the photosynthetic purple bacterium Rhodobacter sphaeroides, and their properties and pigment content were compared with those of the correspondent single mutant RCs. Each pair of the mutations comprised the amino acid substitution I(L177)H and another mutation altering histidine ligand of BChl P(A) or BChl B(B). Contrary to expectations, the double mutation I(L177)H+H(L173)L does not bring about a heterodimer RC but causes a 46nm blue shift of the long-wavelength P absorbance band. The histidine L177 or a water molecule were suggested as putative ligands for P(A) in the RC I(L177)H+H(L173)L although this would imply a reorientation of the His backbone and additional rearrangements in the primary donor environment or even a repositioning of the BChl dimer. The crystal structure of the mutant I(L177)H reaction center determined to a resolution of 2.9? shows changes at the interface region between the BChl P(A) and the monomeric BChl B(B). Spectral and pigment analysis provided evidence for β-coordination of the BChl B(B) in the double mutant RC I(L177)H+H(M182)L and for its hexacoordination in the mutant reaction center I(L177)H. Computer modeling suggests involvement of two water molecules in the β-coordination of the BChl B(B). Possible structural consequences of the L177 mutation affecting the coordination of the two BChls P(A) and B(B) are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. II. Free energy and kinetic relations between the acceptor states Q(-)A Q(-)B and QAQ(2-)B

Thermodynamic equilibria and electron transfer kinetics involving the quinone acceptor complex in reaction centers from Rhodopseudomonas sphaeroides were investigated. We focussed on reactions involving the two-electron states QA Qn and QAQ~-, described by the scheme DQAQa~-D +X,~~A- , ~~a- ~k~ .~D+ "r~~ AK~'La2- - k~2~ k~lk O~ (2)D+~D The equilibrium partitioning between QA Q n and QAQ 2n- was determined spectroscopically from either the concentration of oxidized cytochrome c or the concentration of semiquinone after successive flashes of light.At pH < 9.5, QAQ2n - is stabilized relative to QAQn, while for pH > 9.5, QAQB is energetically favored.The reduction of QA, to form QAQ~, is not associated with a protonation step (pK< 8). However, the reduction of Q~, to form the final state QAQ~-, is accompanied by an uptake of a proton (pK >/10.7). The preferential interaction of a proton with QAQ2n - provides the driving force for the forward electron transfer.The shift toward the photochemically inactive state QAQa with increasing pH may serve as a feedback mechanism in photosynthetic organisms to limit the rise in intracellular pH. The electron-transfer rate constants were determined from the observed kinetics and the equilibria between the states QAQ2n - and QA Q n. The forward rate constant z-.A~2n~ was approximately proportional to the proton concentration, whereas kta2A~ depended only weakly on pH. The recombination kinetics of D +QAQ2n- was biphasic. The slow rate agreed with the predicted charge recombination via the intermediate state D +QAQff; the fast rate may be due to the recombination from a separate (conformational) state. The results of this work were combined with those of a previous study on reactions involving the one-electron precursor states QAQa and QAQn(Kleinfeld, D., Okamura, M.Y., and Feher, G. (1984) Biochim. Biophys. Acta 766, 126-140). The overall sequence for the protonation of the reaction center in response to successive reductions of the accept or complex involves the uptake of one proton for each electron transferred to QB- This sequential uptake initiates the formation of a proton gradient across the cell membrane.     

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.     

Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides reconstituted with anthraquinone as primary quinone Q(A)

In the photosynthetic reaction centre (RC) from the purple bacterium Rhodobacter sphaeroides, the primary quinone, a ubiquinone-10 (Q(A)), has been substituted by anthraquinone. Three-dimensional crystals have been grown from the modified RC and its structure has been determined by X-ray crystallography to 2.4 A resolution. The bindings of the head-group from ubiquinone-10 and of the anthraquinone ring are very similar. In particular, both rings are parallel to each other and the hydrogen bonds connecting the native ubiquinone-10 molecule to AlaM260 and HisM219 are conserved in the anthraquinone containing RC. The space of the phytyl tail missing in the anthraquinone exchanged RC is occupied by the alkyl chain of a detergent molecule. Other structural changes of the Q(A)-binding site are within the limit of resolution. Our structural data bring strong credit to the very large amount of spectroscopic data previously achieved in anthraquinone-replaced RCs and which have participated in the determination of the energetics of the quinone system in bacterial RCs.     

Identification of a hydrogen bond in the phe M197-->Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X-ray crystallography and FTIR spectroscopy

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875-12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.     

Identification of a novel protonation pattern for carboxylic acids upon Q(B) photoreduction in Rhodobacter sphaeroides reaction center mutants at Asp-L213 and Glu-L212 sites

In the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides, light energy is rapidly converted to chemical energy through coupled electron-proton transfer to a buried quinone molecule Q(B). Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations that are observable using light-induced Fourier transform infrared (FTIR) difference spectroscopy. Upon formation, Q(B)(-) induces protonation of Glu-L212, located within 5 A of Q(B), resulting in a IR signal at 1728 cm(-1). However, no other IR signal is observed within the classic absorption range of protonated carboxylic acids (1770-1700 cm(-1)). In particular, no signal for Asp-L213 is found despite its juxtaposition to Q(B) and importance for proton uptake on the second electron-transfer step. In an attempt to uncover the reason behind this lack of signal, the microscopic electrostatic environment in the vicinity of Q(B) was modified by interchanging Asp and Glu at the L213 and L212 positions. The Q(B)(-)/Q(B) FTIR spectrum of the Asp-L212/Glu-L213 swap mutant in the 1770-1700 cm(-1) range shows several distinct new signals, which are sensitive to (1)H/(2)H isotopic exchange, indicating that the reduction of Q(B) results in the change of the protonation state of several carboxylic acids. The new bands at 1752 and 1747 cm(-1) were assigned to an increase of protonation in response to Q(B) reduction of Glu-L213 and Asp-L212, respectively, based on the effect of replacing them with their amine analogues. Since other carboxylic acid signals were observed, it is concluded that the swap mutations at L212 and L213 affect a cluster of carboxylic acids larger than the L212/L213 acid pair. Implications for the native reaction center are discussed.     

Exploring the energy landscape for Q(A)(-) to Q(B) electron transfer in bacterial photosynthetic reaction centers: effect of substrate position and tail length on the conformational gating step

The ability to initiate reactions with a flash of light and to monitor reactions over a wide temperature range allows detailed analysis of reaction mechanisms in photosynthetic reaction centers (RCs) of purple bacteria. In this protein, the electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) is rate-limited by conformational changes rather than electron tunneling. Q(B) movement from a distal to a proximal site has been proposed to be the rate-limiting change. The importance of quinone motion was examined by shortening the Q(B) tail from 50 to 5 carbons. No change in rate was found from 100 to 300 K. The temperature dependence of the rate was also measured in three L209 proline mutants. Under conditions where Q(B) is in the distal site in wild-type RCs, it is trapped in the proximal site in the Tyr L209 mutant [Kuglstatter, A., et al. (2001) Biochemistry 40, 4253-4260]. The electron transfer slows at low temperature for all three mutants as it does in wild-type protein, indicating that conformational changes still limit the reaction rate. Thus, Q(B) movement is unlikely to be the sole, rate-limiting conformational gating step. The temperature dependence of the reaction in the L209 mutants differs somewhat from wild-type RCs. Entropy-enthalpy compensation reduces the difference in rates and free energy changes at room temperature.     

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.     

Exploring the primary electron acceptor (QA)-site of the bacterial reaction center from Rhodobacter sphaeroides. Binding mode of vitamin K derivatives.

The functional replacement of the primary ubiquinone (QA) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides with synthetic vitamin K derivatives has provided a powerful tool to investigate the electron transfer mechanism. To investigate the binding mode of these quinones to the QA binding site we have determined the binding free energy and charge recombination rate from QA(-) to D+ (kAD) of 29 different 1,4-naphthoquinone derivatives with systematically altered structures. The most striking result was that none of the eight tested compounds carrying methyl groups in both positions 5 and 8 of the aromatic ring exhibited functional binding. To understand the binding properties of these quinones on a molecular level, the structures of the reaction center-naphthoquinone complexes were predicted with ligand docking calculations. All protein--ligand structures show hydrogen bonds between the carbonyl oxygens of the quinone and AlaM260 and HisM219 as found for the native ubiquinone-10 in the X-ray structure. The center-to-center distance between the naphthoquinones at QA and the native ubiquinone-10 at QB (the secondary electron acceptor) is essentially the same, compared to the native structure. A detailed analysis of the docking calculations reveals that 5,8-disubstitution prohibits binding due to steric clashes of the 5-methyl group with the backbone atoms of AlaM260 and AlaM249. The experimentally determined binding free energies were reproduced with an rmsd of approximately 4 kJ x mol(-1) in most cases providing a valuable tool for the design of new artificial electron acceptors and inhibitors.     

Monitoring the pH dependence of IR carboxylic acid signals upon Q(B)- formation in the Glu-L212 --> Asp/Asp-L213 --> Glu swap mutant reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (QB) site. Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations in the 1770-1700 cm-1 spectral range that are sensitive to 1H/2H isotopic exchange. With respect to the native RC, a novel protonation pattern for carboxylic acids upon QB photoreduction has been identified in the Glu-L212 --> Asp/Asp-L213 --> Glu mutant RC using light-induced FTIR difference spectroscopy (Nabedryk, E., Breton, J., Okamura, M. Y., and Paddock, M. L. (2004) Biochemistry 43, 7236-7243). These carboxylic acids are structurally close and have been implicated in proton transfer to reduced QB. In this work, we extend previous studies by measuring the pH dependence of the QB-/QB FTIR difference spectra of the mutant in 1H2O and 2H2O. Large pH dependent changes were observed in the 1770-1700 cm-1 spectral range between pH 8 and pH 4. The IR fingerprints of the protonating carboxylic acids upon QB- formation were obtained from the calculated double-difference spectra 1H2O minus 2H2O. These IR fingerprints are specific for each pH, indicative of the contribution of different titrating groups. In particular, the 1752 cm-1 signal indicates that Glu-L213 protonates upon QB- formation at pH >or= 5, whereas the 1746 cm-1 signal indicates protonation of Asp-L212 even at pH 4. An unidentified carboxylic acid absorbing at approximately 1765 cm-1 could be the proton donor between pH 8 and 5. The observation that in the swap mutant there are several uniquely behaving carboxylic acids shows that electrostatic interactions occurring between them are sufficiently modified from the native RC to reveal their IR signatures.     

Phosphatidylglycerol requirement for the function of electron acceptor plastoquinone Q(B) in the photosystem II reaction center

Phosphatidylglycerol (PG), a ubiquitous constituent of thylakoid membranes of chloroplasts and cyanobacteria, is demonstrated to be essential for the functionality of plastoquinone electron acceptor Q(B) in the photosystem II reaction center of oxygenic photosynthesis. Growth of the pgsA mutant cells of Synechocystis sp. PCC6803 that are defective in phosphatidylglycerolphosphate synthase and are incapable of synthesizing PG, in a medium without PG, resulted in a 90% decrease in PG content and a 50% loss of photosynthetic oxygen-evolving activity as reported [Hagio, M., Gombos, Z., Várkonyi, Z., Masamoto, K., Sato, N., Tsuzuki, M., and Wada, H. (2000) Plant Physiol. 124, 795-804]. We have studied each step of the electron transport in photosystem II of the pgsA mutant to clarify the functional site of PG. Accumulation of Q(A)(-) was indicated by the fast rise of chlorophyll fluorescence yield under continuous and flash illumination. Oxidation of Q(A)(-) by Q(B) plastoquinone was shown to become slow, and Q(A)(-) reoxidation required a few seconds when measured by double flash fluorescence measurements. Thermoluminescence measurements further indicated the accumulation of the S(2)Q(A)(-) state but not of the S(2)Q(B)(-) state following the PG deprivation. These results suggest that the function of Q(B) plastoquinone was inactivated by the PG deprivation. We assume that PG is an indispensable component of the photosystem II reaction center complex to maintain the structural integrity of the Q(B)-binding site. These findings provide the first clear identification of a specific functional site of PG in the photosynthetic reaction center.