Coupling of light-induced electron transfer to proton uptake in photosynthesis

Light energy is transformed into chemical energy in photosynthesis by coupling a light-induced electron transfer to proton uptake. The resulting proton gradient drives ATP synthesis. In this study, we monitored the light-induced reactions in a 100-kDa photosynthetic protein from 30 ns to 35 s by FTIR difference spectroscopy. The results provide detailed mechanistic insights into the electron and proton transfer reactions of the QA to QB transition: reduction of QA in picoseconds induces protonation of histidines, probably of His126 and His128 in the H subunit at the entrance of the proton uptake channel, and of Asp210 in the L subunit inside the channel at 12 micros and 150 micros. This seems to be a prerequisite for the reduction of QB, mainly at 150 micros. QA- is reoxidized at 1.1 ms, and a proton is transferred from Asp210 to Glu212 in the L subunit, the proton donor to QB-. Notably, our data indicate that QB is not reduced directly by QA- but presumably through an intermediary electron donor.     

Characterization of the photoreduction of the secondary quinone QB in the photosynthetic reaction center from rhodobacter capsulatus with FTIR spectroscopy

The photoreduction of the secondary quinone acceptor QB in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter (Rb.) capsulatus has been investigated by light-induced FTIR difference spectroscopy in 1H2O and 2H2O. The Q-B/QB FTIR spectra reflect reorganization of the protein upon electron transfer, changes of protonation state of carboxylic acid groups, and (semi)quinone-protein interactions. As expected from the conservation of most of the amino acids near QB in the RCs from Rb. capsulatus and Rb. sphaeroides, several protein and quinone IR bands are common to both spectra, e.g., the 1728 cm-1 band is assigned to proton uptake by a carboxylic acid residue, most probably Glu L212 as previously proposed for Rb. sphaeroides RCs. However, noticeable changes are observed at 1709 cm-1 (deprotonation of a Glu or Asp residue), 1674 and 1659 cm-1 (side chain and/or backbone), around 1540 cm-1 (amide II), and in the semiquinone absorption range. This FTIR study demonstrates that the environment of the secondary quinone in Rb. capsulatus is close but not identical to that in Rb. sphaeroides suggesting slight differences in the structural organization of side chains and/or ordered water molecules near QB.     

In bacterial reaction centers protons can diffuse to the secondary quinone by alternative pathways.

The mechanisms of proton conduction to the reduced secondary quinone in bacterial reaction centers were studied in wild-type and genetically modified reaction centers from Rhodobacter capsulatus. In the L212-213AA double mutant (L212Glu----Ala, L213Asp----Ala), reaction center function is severely altered. However, a photocompetent revertant of this strain which carries a third 'compensating' mutation, M231Arg----Leu, at about 15 A from the secondary quinone, displays the normal proton binding function of the reaction center. Furthermore, the apparent pK values of group(s) involved in the stabilization of the semiquinone anion are restored by that mutation. We conclude that L212Glu and L213Asp are not obligatory residues for proton donation to QB in Rb. capsulatus. We suggest that protons can be delivered to the QB site from the cytoplasm via a network of proton channels activated by compensatory mutations, possibly involving water molecules bound in the interior of the reaction center.     

Calculated coupling of electron and proton transfer in the photosynthetic reaction center of Rhodopseudomonas viridis.

Based on new Rhodopseudomonas (Rp.) viridis reaction center (RC) coordinates with a reliable structure of the secondary acceptor quinone (QB) site, a continuum dielectric model and finite difference technique have been used to identify clusters of electrostatically interacting ionizable residues. Twenty-three residues within a distance of 25 A from QB (QB cluster) have been shown to be strongly electrostatically coupled to QB, either directly or indirectly. An analogous cluster of 24 residues is found to interact with QA (QA cluster). Both clusters extend to the cytoplasmic surface in at least two directions. However, the QB cluster differs from the QA cluster in that it has a surplus of acidic residues, more strong electrostatic interactions, is less solvated, and experiences a strong positive electrostatic field arising from the polypeptide backbone. Consequently, upon reduction of QA or QB, it is the QB cluster, and not the QA cluster, which is responsible for substoichiometric proton uptake at neutral pH. The bulk of the changes in the QB cluster are calculated to be due to the protonation of a tightly coupled cluster of the three Glu residues (L212, H177, and M234) within the QB cluster. If the lifetime of the doubly reduced state QB2- is long enough, Asp M43 and Ser L223 are predicted to also become protonated. The calculated complex titration behavior of the strongly interacting residues of the QB cluster and the resulting electrostatic response to electron transfer may be a common feature in proton-transferring membrane protein complexes.     

The organisation of proton motive and non-proton motive redox loops in prokaryotic respiratory systems

Respiration is fundamental to the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. In principle, the free energy of a redox reaction catalysed by a membrane-bound electron transport chain is transduced via the generation of an electrochemical ion (usually proton) gradient across a coupling membrane that drives ATP synthesis. The proton motive force (pmf) can be built up by different mechanisms like proton pumping, quinone/quinol cycling or by a redox loop. The latter couples electron transport to a net proton transfer across the membrane without proton pumping. Instead, charge separation is achieved by quinone-reactive enzymes or enzyme complexes whose active sites for substrates and quinones are situated on different sides of the coupling membrane. The necessary transmembrane electron transport is usually accomplished by the presence of two haem groups that face opposite sides of the membrane. There are many different enzyme complexes that are part of redox loops and their catalysed redox reactions can be either electrogenic, electroneutral (non-proton motive) or even pmf-consuming. This article gives conceptual classification of different operational organisations of redox loops and uses this as a platform from which to explore the biodiversity of quinone/quinol-cycling redox systems.     

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

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

Calculated protein and proton motions coupled to electron transfer: electron transfer from QA- to QB in bacterial photosynthetic reaction centers.

Reaction centers from Rhodobacter sphaeroides were subjected to Monte Carlo sampling to determine the Boltzmann distribution of side-chain ionization states and positions and buried water orientation and site occupancy. Changing the oxidation states of the bacteriochlorophyll dimer electron donor (P) and primary (QA) and secondary (QB) quinone electron acceptors allows preparation of the ground (all neutral), P+QA-, P+QB-, P0QA-, and P0QB- states. The calculated proton binding going from ground to other oxidation states and the free energy of electron transfer from QA-QB to form QAQB- (DeltaGAB) compare well with experiment from pH 5 to pH 11. At pH 7 DeltaGAB is measured as -65 meV and calculated to be -80 meV. With fixed protein positions as in standard electrostatic calculations, DeltaGAB is +170 meV. At pH 7 approximately 0.2 H+/protein is bound on QA reduction. On electron transfer to QB there is little additional proton uptake, but shifts in side chain protonation and position occur throughout the protein. Waters in channels leading from QB to the surface change site occupancy and orientation. A cluster of acids (GluL212, AspL210, and L213) and SerL223 near QB play important roles. A simplified view shows this cluster with a single negative charge (on AspL213 with a hydrogen bond to SerL233) in the ground state. In the QB- state the cluster still has one negative charge, now on the more distant AspL210. AspL213 and SerL223 move so SerL223 can hydrogen bond to QB-. These rearrangements plus other changes throughout the protein make the reaction energetically favorable.     

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

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

Photoreduction of QA, QB, and cytochrome b-559 in an oxygen-evolving photosystem II preparation from the thermophilic cyanobacterium Synechococcus sp

Light-induced absorption changes in an oxygen-evolving photosystem II (PS II) preparation from the thermophilic cyanobacterium Synechococcus sp. were analyzed using continuous illumination which caused the reduction of both QA (first stable quinone electron acceptor) and QB (second quinone electron acceptor of photosystem II). In this photosystem II preparation in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) the amount of QA was estimated to be 1 per 42 chlorophylls. In the absence of DCMU, plastoquinone (1.68 per QA) was photoreduced to plastohydroquinone within a few seconds, indicating that QB is reduced and protonated during this period. An electrochromic band shift centered around 685 nm was observed with and without DCMU. The extent of this band shift caused by QB reduction per electron was about a third or half of that caused by QA reduction. A significant amount of cytochrome b-559 (0.86 per QA) was photoreduced. Only 60% of the photoreduction of cytochrome b-559 was inhibited by a DCMU concentration that inhibited electron transfer beyond QB, indicating that the site of the reduction of cytochrome b-559 is located before the QB site and possibly on the donor side of PS II.     

Temporary Stabilization of Electron on Quinone Acceptor Side of Reaction Centers from the Bacterium Rhodobacter sphaeroides Wild Type and Mutant SA(L223) Depending on Duration of Light Activation

The dark reduction of photooxidized bacteriochlorophyll (P+) by photoreduced secondary quinone acceptor (QB-) in isolated reaction centers (RC) from the bacterium Rhodobacter sphaeroides wild type and mutant strain SA(L223) depending on the duration of light activation of RC was studied. The kinetics of the dark reduction of P+ decreased with increasing light duration, which is probably due to conformational changes occurring under prolonged light activation in RC from the wild type bacterium. In RC from bacteria of the mutant strain in which protonatable amino acid Ser L223 near QB is substituted by Ala, the dependence of reduction kinetics of P+ on duration of light was not observed. Such dependence, however, became observable after addition of cryoprotectors, namely glycerol and dimethylsulfoxide, to the RC samples from the mutant strain. It was concluded that substitution of Ser L223 with Ala disturbs the native mechanism of electrostatic stabilization of the electron in the RC quinone acceptor site. At the same time, an additional modification of RC hydrogen bonds by glycerol and dimethylsulfoxide probably includes various possibilities for more effective time delay of the electron on QB.     

Mutations in the D1 subunit of photosystem II distinguish between quinone and herbicide binding sites.

The structure-activity relationships of the plastoquinone QB binding domain in the D1 subunit of photosystem II (PSII) were investigated by characterization of mutations introduced in the D1 protein. Eight novel point mutations in the gene psbA, which encodes D1, were generated in the cyanobacterium Synechocystis PCC6803 by site-specific mutagenesis in vitro. The effects of the resulting modifications in D1 on electron transfer in PSII and on herbicide binding were analyzed. The results extend the structural analogies between the secondary quinone binding site in D1 and in subunit L of the photosynthetic reaction center in purple bacteria. The involvement of Phe255, Ser264, and Leu271 of D1 in plastoquinone binding and electron transfer in PSII was established. An indirect effect of Tyr254 on the binding of QB was demonstrated. Changes in binding of herbicides and QB to D1 as a result of the mutations revealed specific interactions between amino acid residues in D1 and the plastoquinone and distinguished between the binding sites of QB and herbicides.     

Absence of a bicarbonate-depletion effect in electron transfer between quinones in chromatophores and reaction centers of Rhodobacter sphaeroides

Higher plants, algae, and cyanobacteria are known to require bicarbonate ions for electron flow from the first stable electron acceptor quinone QA to the second electron acceptor quinone QB, and to the intersystem quinone pool. It has been suggested that in Photosystem II of oxygenic photosynthesis, bicarbonate ion functions to maintain the reaction center in a proper conformation and, perhaps, to provide the protons needed to stabilize the semiquinone (QB-). In this paper, we show that bicarbonate ions do not influence the electron flow, from the quinone QA to QB and beyond, in the photosynthetic bacterium Rhodobacter sphaeroides. No measurable effect of bicarbonate depletion, obtained by competition with formate, was observed on cytochrome b-561 reduction in chromatophores; on the flash-dependent oscillation of semiquinone formation in reaction centers; on electron transfer from QA- to QB; or on either the fast or slow recovery of the oxidized primary donor (P+) which reflects the P+QA- ----PQA or the P+QB- ----PQB reaction. The lack of an observed effect in Rhodobacter sphaeroides in contrast to the effect seen in Photosystem II is suggested to be due to the amino-acid sequence differences between the reaction centers of the two systems.     

The interaction of quinone and detergent with reaction centers of purple bacteria. I. Slow quinone exchange between reaction center micelles and pure detergent micelles.

The kinetics of light-induced electron transfer in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides were studied in the presence of the detergent lauryldimethylamine-N-oxide (LDAO). After the light-induced electron transfer from the primary donor (P) to the acceptor quinone complex, the dark re-reduction of P+ reflects recombination from the reduced acceptor quinones, QA- or QB-. The secondary quinone, QB, which is loosely bound to the RC, determines the rate of this process. Electron transfer to QB slows down the return of the electron to P+, giving rise to a slow phase of the recovery kinetics with time tau P approximately 1 s, whereas charge recombination in RCs lacking QB generates a fast phase with time tau AP approximately 0.1 s. The amount of quinone bound to RC micelles can be reduced by increasing the detergent concentration. The characteristic time of the slow component of P+ dark relaxation, observed at low quinone content per RC micelle (at high detergent concentration), is about 1.2-1.5 s, in sharp contrast to expectations from previous models, according to which the time of the slow component should approach the time of the fast component (about 0.1 s) when the quinone concentration approaches zero. To account for this large discrepancy, a new quantitative approach has been developed to analyze the kinetics of electron transfer in isolated RCs with the following key features: 1) The exchange of quinone between different micelles (RC and detergent micelles) occurs more slowly than electron transfer from QB- to P+; 2) The exchange of quinone between the detergent "phase" and the QB binding site within the same RC micelle is much faster than electron transfer between QA- and P+; 3) The time of the slow component of P+ dark relaxation is determined by (n) > or = 1, the average number of quinones in RC micelles, calculated only for those RC micelles that have at least one quinone per RC (in excess of QA). An analytical function is derived that relates the time of the slow component of P+ relaxation, tau P, and the relative amplitude of the slow phase. This provides a useful means of determining the true equilibrium constant of electron transfer between QA and QB (LAB), and the association equilibrium constant of quinone binding at the QB site (KQ+). We found that LAB = 22 +/- 3 and KQ = 0.6 +/- 0.2 at pH 7.5. The analysis shows that saturation of the QB binding site in detergent-solubilized RCs is difficult to achieve with hydrophobic quinones. This has important implications for the interpretation of apparent dependencies of QB function on environmental parameters (e.g. pH) and on mutational alterations. The model accounts for the effects of detergent and quinone concentration on electron transfer in the acceptor quinone complex, and the conclusions are of general significance for the study of quinone-binding membrane proteins in detergent solutions.     

Evidence for delocalized anticooperative flash induced proton binding as revealed by mutants at the M266His iron ligand in bacterial reaction centers

Bacterial reaction centers (RCs) convert light energy into chemical free energy via the double reduction and protonation of the secondary quinone electron acceptor, QB, to the dihydroquinone QBH2. Two RC mutants (M266His --> Leu and M266His --> Ala) with a modified ligand of the non-heme iron have been studied by flash-induced absorbance change spectroscopy. No important changes were observed for the rate constants of the first and second electron transfers between the first quinone electron acceptor, QA, and QB. However, in the M266HL mutant a destabilization of approximately 40 meV of the free energy level of QA- was observed, at variance with the M266HA mutant. The superposition of the three-dimensional X-ray structures of the three proteins in the QA region provides no obvious explanation for the energy modification in the M266HL mutant. The shift of the midpoint redox potential of QA/QA- in M266HL caused accelerated recombination of the charges in the P+ QA- state of the RCs where the native QA was replaced by a low potential anthraquinone (AQA). As previously reported for the native RCs, in the M266HL we observed a biphasicity of the P+ AQA- --> P AQA charge recombination. Interestingly, both phases present a similar acceleration in the M266HL mutant with respect to the wild type. The pH dependencies of the proton uptake upon QA- and QB- formations are superimposable in both mutants but very different from those of native RCs. The data measured in mutants are similar to those that we previously obtained on strains modified at various sites of the cytoplasmic region. The similarity of the response to these different mutations is puzzling, and we propose that it arises from a collective behavior of multiple acidic residues resulting in strongly anticooperative proton binding. The unspecific disappearance of the high pH band of proton uptake observed in all these mutants appears as the natural consequence of removing any member of an interactive proton cluster. This long range interaction also accounts for the similar responses to mutations of the proton uptake pattern induced by either QA- or QB-. We surmise that the presence of an extended protonated water H-bond network providing protons to QB is responsible for these effects.     

Amino acid residues involved in the coordination and assembly of the manganese cluster of photosystem II. Proton-coupled electron transport of the redox-active tyrosines and its relationship to water oxidation

The combination of site-directed mutagenesis, isotopic labeling, new magnetic resonance techniques and optical spectroscopic methods have provided new insights into cofactor coordination and into the mechanism of electron transport and proton-coupled electron transport in photosystem II. Site-directed mutations in the D1 polypeptide of this photosystem have implicated a number of histidine and carboxylate residues in the coordination and assembly of the manganese cluster, responsible for photosynthetic water oxidation. Many of these are located in the carboxy-terminal region of this polypeptide close to the processing site involved in its maturation. This maturation is a required precondition for cluster assembly. Recent proposals for the mechanism of water oxidation have directly implicated redox-active tyrosine Y(Z) in this mechanism and have emphasized the importance of the coupling of proton and electron transfer in the reduction of Y(Z)(radical) by the Mn cluster. The interaction of both homologous redox-active tyrosines Y(Z) and Y(D) with their respective homologous proton acceptors is discussed in an effort to better understand the significance of such coupling.     

A serine-to-threonine substitution in the triazine herbicide-binding protein in potato cells results in atrazine resistance without impairing productivity.

A mutation of the psbA gene was identified in photoautotrophic potato (Solanum tuberosum L. cv Superior x U.S. Department of Agriculture line 66-142) cells selected for resistance to 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine (atrazine). Photoaffinity labeling with 6-azido-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine detected a thylakoid membrane protein with a M(r) of 32,000 in susceptible, but not in resistant, cells. This protein was identified as the secondary quinone acceptor of photosystem II (QB) protein. Atrazine resistance in selected cells was attributable to a mutation from AGT (serine) to ACT (threonine) in codon 264 of the psbA gene that encodes the QB protein. Although the mutant cells exhibited extreme levels of resistance to atrazine, no concomitant reductions in photosynthetic electron transport or cell growth rates compared to the unselected cells were detected. This is in contrast with the losses in productivity observed in atrazine-resistant mutants that contain a glycine-264 alteration.     

Small weak acids stimulate proton transfer events in site-directed mutants of the two ionizable residues, GluL212 and AspL213, in the QB-binding site of Rhodobacter sphaeroides reaction center.

Mutations of the two ionizable residues, GluL212 and AspL213, in the secondary quinone (QB) binding site of reaction centers (RCs) from Rhodobacter sphaeroides cause major dysfunctions in the proton transfer processes leading to the formation of quinol. Mutant RCs with AspL213----Asn are especially severely blocked, and the rate of the proton-limited transfer of the second electron is at least 10(4) times slower than in the wild-type. Small, weak acids, such as azide/hydrazoic acid (N3-/HN3;pK approximately 4.7) accelerated the electron transfer rate in mutant RCs in a pH and concentration-dependent manner, consistent with their functioning as protein-penetrating protonophores, delivering protons to the QB site in a non-specific, diffusive process. Other small weak acids acted similarly with efficacies dependent on their size and pK values. In terms of the concentration of protonated species, the relative effectiveness was: nitrite greater than cyanate approximately formate greater than azide much greater than acetate. The behavior of bacterial RCs containing the AspL213----Asn mutation resembles that of bicarbonate-depleted photosystem II, and the mutational block is partially alleviated by bicarbonate. The possibility is discussed that bicarbonate acts in PS II as an analogue to the carboxylic acid residues of the bacterial proton conduction pathway.     

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone-10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash-induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+QA-) formation. Since the C. aurantiacus RC lacks H-subunit, this subunit was not an obligatory component for electrogenic QB protonation.     

pH modulates the quinone position in the photosynthetic reaction center from Rhodobacter sphaeroides in the neutral and charge separated states

The structure of the photosynthetic reaction-center from Rhodobacter sphaeroides has been determined at four different pH values (6.5, 8.0, 9.0, 10.0) in the neutral and in charge separated states. At pH 8.0, in the neutral state, we obtain a resolution of 1.87 A, which is the best ever reported for the bacterial reaction center protein. Our crystallographic data confirm the existence of two different binding positions of the secondary quinone (QB). We observe a new orientation of QB in its distal position, which shows no ring-flip compared to the orientation in the proximal position. Datasets collected for the different pH values show a pH-dependence of the population of the proximal position. The new orientation of QB in the distal position and the pH-dependence could be confirmed by continuum electrostatics calculations. Our calculations are in agreement with the experimentally observed proton uptake upon charge separation. The high resolution of our crystallographic data allows us to identify new water molecules and external residues being involved in two previously described hydrogen bond proton channels. These extended proton-transfer pathways, ending at either of the two oxo-groups of QB in its proximal position, provide additional evidence that ring-flipping is not required for complete protonation of QB upon reduction.     

A modeling and simulation perspective on the mechanism and function of respiratory complex I

Respiratory complex I is a giant redox-driven proton pump, and central to energy production in mitochondria and bacteria. It catalyses the reduction of quinone to quinol, and converts the free energy released into the endergonic proton translocation across the membrane. The proton pumping sets up the proton electrochemical gradient, which propels the synthesis of ATP. Despite the availability of extensive biochemical, biophysical and structural data on complex I, the mechanism of coupling between the electron and proton transfer reactions remain uncertain. In this work, we discuss current state-of-the-art in the field with particular emphasis on the molecular mechanism of respiratory complex I, as deduced from computational modeling and simulation approaches, but in strong alliance with the experimental data. This leads to novel synthesis of mechanistic ideas on a highly complex enzyme of the electron transport chain that has been associated with a number of mitochondrial and neurodegenerative disorders.