On the electron stabilization within the quinone acceptor part of Rhodobacter sphaeroides photosynthetic reaction centers

The time evolution of the photoinduced differential absorption spectrum of isolated Rhodobacter sphaeroides photosynthetic reaction centers was investigated. The measurements were carried out in the spectral region of 400-500 nm on the time scale of up to 200 microseconds. The spectral changes observed can be interpreted in terms of the effects of proton shift along hydrogen bonds between the primary quinone acceptor and the protein. A theoretical analysis of the spectrum time evolution was performed, which is based on the consideration of the kinetics of proton tunneling along the hydrogen bond. It was shown that the stabilization of the primary quinone electronic state occurs within the first several tens of microseconds after quinone reduction. It slows down upon the deuteration of reaction centers as well as after adding 90% of glycerol; on the other hand, it accelerates as temperature rises up to 40 degrees C.     

Revealing the involvement of extended hydrogen bond networks in the cooperative function between distant sites in bacterial reaction centers

In reaction center proteins of photosynthetic bacteria, the amplitude of proton uptake induced by the one-electron reduction of either of the two quinone electron acceptors (Q(A) and Q(B)) is an intrinsic observable of the electrostatic interactions associated with the redox function of the complex. We report here that, in Rhodobacter capsulatus, complete restoration of proton uptake (upon formation of Q(A)(-) and Q(B)(-)) to the level found in the wild type is observed in a mutant reaction center in which a tyrosine substitution in the Q(A) environment (Ala(M274) --> Tyr) is coupled with mutations of acidic residues near Q(B) (Glu(L212) --> Ala/Asp(L213) --> Ala) that initially cancel the proton uptake above pH 8. This result demonstrates that proton uptake occurs by strong cooperation between structural motifs, such as hydrogen-bonded networks, that span the 18 A distance between the two quinone acceptors.     

Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers

Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, Q_A, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320–330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution in the presence of DCBQ or DMBQ support the conclusion that inactive Photosystem II complexes have a functional water oxidation system.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - F_o initial fluorescence level using dark-adapted thylakoids - Inactive reaction centers reaction centers inactive in plastoquinone reduction - PS II Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II Department of Plant Biology, University of IllinoisDepartment of Physiology & Biophysics, University of Illinois     

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs

Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone Q_A, non-heme Fe²⁺ ion and neighboring residues_. The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the Q_A state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [Q_A-His^M219] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P⁺/Q_A⁻ recombination rate temperature dependence.     

Flash-induced proton transfer in photosynthetic bacteria

A proton electrochemical potential across the membranes of photosynthetic purple bacteria is established by a light-driven proton pump mechanism: the absorbed light in the reaction center initiates electron transfer which is coupled to the vectorial displacement of protons from the cytoplasm to the periplasm. The stoichiometry and kinetics of proton binding and release can be tracked directly by electric (glass electrodes), spectrophotometric (pH indicator dyes) and conductimetric techniques. The primary step in the formation of the transmembrane chemiosmotic potential is the uptake of two protons by the doubly reduced secondary quinone in the reaction center and the subsequent exchange of hydroquinol for quinone from the membrane quinone-pool. However, the proton binding associated with singly reduced promary and/or secondary quinones of the reaction center is substoichiometric, pH-dependent and its rate is electrostatically enhanced but not diffusion limited. Molecular details of protonation are discussed based on the crystallographic structure of the reaction center of purple bacteriaRb. sphaeroides andRps. viridis, structure-based molecular (electrostatic) calculations and mutagenesis directed at protonatable amino acids supposed to be involved in proton conduction pathways.     

Anomalous acceleration of the photocycle in photosynthetic reaction centers inhibited on the acceptor side

The rate of the photocycle (quinone reduction cycle) was measured under continuous light excitation in an isolated reaction center protein of the photosynthetic bacterium Rhodobacter sphaeroides. The rate is determined by the slowest step of the photocycle, which could be the photochemistry (charge separation), the quinone/quinol and cytochrome c(2+)/c(3+) exchanges, or proton delivery to the secondary quinone. The photocycle was driven by high light intensity of a laser diode (5 W/cm(2) at 808 nm) to avoid light limitation of the observed rate. The fast turnover of the reaction center (up to 10(3) s(-1)) was slowed down by inhibition of the proton delivery to the secondary quinone by transition metal ions (Cd(2+) and Ni(2+)), by mutation of a key protonatable group (L213Asp --> Asn), or by use of low-affinity ubiquinone (UQ(0)) to the secondary quinone binding site. Although in all of these cases the rate of turnover was 2-3 orders of magnitude less than that of the primary photochemistry, marked light intensity dependence was observed. The rate of the photocycle increased from 7 s(-1) (Ni(2+), low light intensity) to 27 s(-1) (high light intensity) at pH 8.4. The anomalous reacceleration is due to alternative events on the acceptor side induced by continuous excitation. We argue that the continuous excitation of the protein trapped in the reduced acceptor (Q(A)(-)Q(B)(-)) state produces short-lived reduced bacteriopheophytin (I(-)) that delivers activation energy to anomalous changes on the acceptor side as second interquinone electron transfer before proton uptake or increase of the quinone dissociation constant.     

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs

Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone Q(A), non-heme Fe(2+) ion and neighboring residues(.) The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the Q(A) state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [Q(A)-His(M219)] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P(+)/Q(A)(-) recombination rate temperature dependence.     

Oxidation-reduction thermodynamics of the acceptor quinone complex in whole-membrane fragments from Chloroflexus aurantiacus

Oxidation-reduction thermodynamic equilibria involving the quinone-acceptor complex have been examined in whole-membrane fragments from Chloroflexus aurantiacus. The primary quinone acceptor was titrated by monitoring the amount of cytochrome c554 photooxidized by a flash of light as a function of the redox potential. In contrast to previous data obtained in purified plasma membranes, in which the primary quinone acceptor exhibited a midpoint potential equal to -50 mV at pH 8.2, in whole-membrane fragments it titrated at -210 mV (pH 8.0), with a pH dependence of -60 mV/pH up to a pK value of 9.3. o-Phenanthroline, an inhibitor of electron transfer from the primary to the secondary quinone acceptor, shifted the Em/pH curve of the primary acceptor to higher redox potentials. The midpoint potential of the secondary quinone acceptor and its dependence on pH has been determined by comparing the kinetics of the charge recombination processes within the reaction center complex in the presence and in the absence of o-phenanthroline. It is concluded that both the primary and the secondary quinone acceptors interact with a proton, with pK values of 9.3 and of approximately 10.2 respectively. At physiological pH the electron appears to be stabilized on the secondary with respect to the primary quinone acceptor by approximately 60 meV.     

Some experiments on the primary electron acceptor in reaction centres from Rhodopseudomonas sphaeroides

The bacterial reaction center absorbance change at 450 nm (A-450) assigned to an anionic semiquinone, has been suggested as a candidate for the reduced form of the primary electron acceptor in bacterial photosynthesis. In reaction centers of Rhodopseudomonas sphaeroides we have found kinetic discrepancies between the decay of A-450 and the recovery of photochemical competence. In addition, no proton uptake is measurable on the first turnover, although subsequent ones elicit one proton bound per electron. These results are taken to indicate that the acceptor reaction after a long dark period may be different for the first turnover than for subsequent ones. It is suggested that A-450 is still a likely candidate for the acceptor function but that in reaction centers, additional quinone may act as an adventitious primary acceptor when the “true” primary acceptor is reduced. Alternatively, the primary acceptor may act in a “ping-pong” fashion with respect to subsequent photoelectrons.     

Some experiments on the primary electron acceptor in reaction centres from Rhodopseudomonas sphaeroides.

The bacterial reaction center absorbance change at 450 nm (A-450) assigned to an anionic semiquinone, has been suggested as a candidate for the reduced form of the primary electron acceptor in bacterial photosynthesis. In reaction centers of Rhodopseudomonas sphaeroides we have found kinetic discrepancies between the decay of A-450 and the recovery of photochemical competence. In addition, no proton uptake is measurable on the first turnover, although subsequent ones elicit one proton bound per electron. These results are taken to indicate that the acceptor reaction after a long dark period may be different for the first turnover than for subsequent ones. It is suggested that A-450 is still a likely candidate for the acceptor function but that in reaction centers, additional quinone may act as an adventitious primary acceptor when the "true" primary acceptor is reduced. Alternatively, the primary acceptor may act in a "ping-pong" fashion with respect to subsequent photoelectrons.     

Plasticity of the Quinone-binding Site of the Complex II Homolog Quinol:Fumarate Reductase

Respiratory processes often use quinone oxidoreduction to generate a transmembrane proton gradient, making the 2H⁺/2e⁻ quinone chemistry important for ATP synthesis. There are a variety of quinones used as electron carriers between bioenergetic proteins, and some respiratory proteins can functionally interact with more than one quinone type. In the case of complex II homologs, which couple quinone chemistry to the interconversion of succinate and fumarate, the redox potentials of the biologically available ubiquinone and menaquinone aid in driving the chemical reaction in one direction. In the complex II homolog quinol:fumarate reductase, it has been demonstrated that menaquinol oxidation requires at least one proton shuttle, but many of the remaining mechanistic details of menaquinol oxidation are not fully understood, and little is known about ubiquinone reduction. In the current study, structural and computational studies suggest that the sequential removal of the two menaquinol protons may be accompanied by a rotation of the naphthoquinone ring to optimize the interaction with a second proton shuttling pathway. However, kinetic measurements of site-specific mutations of quinol:fumarate reductase variants show that ubiquinone reduction does not use the same pathway. Computational docking of ubiquinone followed by mutagenesis instead suggested redundant proton shuttles lining the ubiquinone-binding site or from direct transfer from solvent. These data show that the quinone-binding site provides an environment that allows multiple amino acid residues to participate in quinone oxidoreduction. This suggests that the quinone-binding site in complex II is inherently plastic and can robustly interact with different types of quinones.     

Initial stage of coupling electron and proton transport in the vicinity of the secondary quinone in photosynthesis of bacteria and plants

The coupling of electron and proton transport in the vicinity of the secondary quinone QB in the reaction center of bacteria and photosystem II of higher plants was investigated. The energy levels and wave functions of the proton in the system QB--histidine L 190 were calculated. It was shown that the proton of histidine forms a hydrogen bond with the doubly reduced quinone QB2-. A new scheme of proton transport through histidine L 190 and its coupling with electron transport was proposed.     

Uncoupling of electron and proton transfers in the photocycle of bacterial reaction centers under high light intensity

Photosynthetic reaction centers produce and export oxidizing and reducing equivalents in expense of absorbed light energy. The formation of fully reduced quinone (quinol) requires a strict (1:1) stoichiometric ratio between the electrons and H(+) ions entering the protein. The steady-state rates of both transports were measured separately under continuous illumination in the reaction center from the photosynthetic bacterium Rhodobacter sphaeroides. The uptake of the first proton was retarded by different methods and made the rate-limiting reaction in the photocycle. As expected, the rate constant of the observed proton binding remained constant (7 s(-)(1)), but that of the cytochrome photooxidation did show a remarkably large increase from 14 to 136 s(-)(1) upon increase of the exciting light intensity up to 5 W/cm(2) (808 nm) at pH 8.4 in the presence of NiCl(2). This corresponds to about 20:1 (e(-):H(+)) stoichiometric ratio. The observed enhancement is linearly proportional to the light intensity and the rate constant of the proton uptake by the acceptor complex and shows saturation character with quinone availability. For interpretation of the acceleration of cytochrome turnover, an extended model of the photocycle is proposed. A fraction of photochemically trapped RC can undergo fast (>10(3) s(-)(1)) conformational change where the semiquinone loses its high binding affinity (the dissociation constant increases by more than 5 orders of magnitude) and dissociates from the Q(B) binding site of the protein with a high rate of 4000 s(-)(1). Concomitantly, superoxide is being produced. No H(+) ion is taken up, and no quinol is created by the photocycle which is operating in about 25% of the reaction centers at the highest light intensity (5500 s(-)(1)) and slowest proton uptake (3.5 s(-)(1)) used in our experiments. The possible physical background of the light-induced conformational change and the relationship between the energies of dissociation and redox changes of the quinone in the Q(B) binding sites are discussed.     

Effect of hydrogen bonds on the energetics of electron transfer

For a model system consisting of a special pair of bacteriochlorophyll molecules (P) and a primary quinone with the nearest environment (QA) (which are acceptor and donor in the recombination reaction in Rhodobacter sphaeroides reaction center, respectively), energies of P+QA(-) and PQA states were calculated. Calculations were performed using several stable QA conformations differing by the positions of hydrogen bond protons. Essential influence of proton positions on the energy of vertical transition P+QA(-) --> PQA was shown.     

Mimicking the photosynthetic system with strong hydrogen bonds to promote proton electron concerted reactions

A Copper(2+) complex with a Cu^II–C bond containing sp³ configuration was used to investigate the role of strong hydrogen bonds in proton coupled electron transfer (PCET) reactions. The only example of a Cu^II–C system realized so far is that using tris-(pyridylthio)methyl (tptm) as a tetradentate tripodal ligand. Using this ligand, [CuF(tptm)] and [Cu(tptm)(OH)] have been prepared. The former complex forms supra-molecular arrays of layers of the complex between which hydroquinone is intercalated in the crystalline phase. This hydroquinone intercalation crystal was prepared via the photochemical conversion of quinone during the crystallization process. This conversion reaction probably involves a proton coupled electron transfer process. The nuclear magnetic resonance spectroscopic analysis of the reaction mixture shows the presence of Cu(III) during the conversion reaction. These results strongly suggest the presence of the molecular aggregate of the [CuF(tptm)] complex, water and quinone in the solution phase during the quinone to hydroquinone conversion. The presence of this type of aggregate requires a strong hydrogen bond between the [CuF(tptm)] complex and water. The presence of this particular hydrogen bond is a unique character of such a complex that has the Cu^II–C bond. This complex is used as a model for photosynthetic water splitting since the photoconversion of quinone to hydroquinone also involves the production of oxygen from water.     

X-Ray structure determination of three mutants of the bacterial photosynthetic reaction centers from Rb. sphaeroides; altered proton transfer pathways

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the reduction of a bound quinone molecule Q(B) is coupled with proton uptake. When Asp-L213 is replaced by Asn, proton transfer is inhibited. Proton transfer was restored by two second-site revertant mutations, Arg-M233-->Cys and Arg-H177-->His. Kinetic effects of Cd(2+) on proton transfer showed that the entry point in revertant RCs to be the same as in the native RC. The structures of the parental and two revertant RCs were determined at resolutions of 2.10, 1.80, and 2.75 A. From the structures, we were able to delineate alternate proton transfer pathways in the revertants. The main changes occur near Glu-H173, which allow it to substitute for the missing Asp-L213. The electrostatic changes near Glu-H173 cause it to be a good proton donor and acceptor, and the structural changes create a cavity which accommodates water molecules that connect Glu-H173 to other proton transfer components.     

Kinetics of the quinone binding reaction at the QB site of reaction centers from the purple bacteria Rhodobacter sphaeroides reconstituted in liposomes.

Transmembrane proton translocation in the photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides is driven by light and performed by two transmembrane complexes; the photosynthetic reaction center and the ubiquinol-cytochrome c oxidoreductase complex, coupled by two mobile electron carriers; the cytochrome and the quinone. This paper focuses on the kinetics and thermodynamics of the interaction between the lipophylic electron carrier ubiquinone-10 and the photosynthetic enzyme reconstituted in liposomes. The collected data were simulated with an existing recognized kinetic scheme and the kinetic constants of the uptake (7.2 x 107 M(-1) x s(-1)) and release (40 s(-1)) processes of the ligand were inferred. The results obtained for the quinone release kinetic constant are comparable to the rate of the charge recombination reaction from the state D(+)QA(-). Values for the kinetic constants are discussed as part of the overall photocycle, suggesting that its bottleneck may not be the quinone uptake reaction in agreement with a previous report.     

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.     

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.     

Atomic level insight into the oxidative half-reaction of aromatic amine dehydrogenase

The quinoprotein aromatic amine dehydrogenase (AADH) uses a covalently bound tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. Recent crystal structures have provided insight into the reductive half-reaction. In contrast, no atomic details are available for the oxidative half-reaction. The TTQ O7 hydroxyl group is protonated during reduction, but it is unclear how this proton can be removed during the oxidative half-reaction. Furthermore, compared with the electron transfer from the N-quinol form, electron transfer from the non-physiological O-quinol form to azurin is significantly slower. Here we report crystal structures of the O-quinol, N-quinol, and N-semiquinone forms of AADH. A comparison of oxidized and substrate reduced AADH species reveals changes in the TTQ-containing subunit, extending from residues in the immediate vicinity of the N-quinol to the putative azurin docking site, suggesting a mechanism whereby TTQ redox state influences interprotein electron transfer. In contrast, chemical reduction of the TTQ center has no significant effect on protein conformation. Furthermore, structural reorganization upon substrate reduction places a water molecule near TTQ O7 where it can act as proton acceptor. The structure of the N-semiquinone, however, is essentially similar to oxidized AADH. Surprisingly, in the presence of substrate a covalent N-semiquinone substrate adduct is observed. To our knowledge this is the first detailed insight into a complex, branching mechanism of quinone oxidation where significant structural reorganization upon reduction of the quinone center directly influences formation of the electron transfer complex and nature of the electron transfer process.