Colin A. Wraight

Electron acceptors of bacterial photosynthetic reaction centers II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex☆

The photoreduction of ubiquinone in the electron acceptor complex (QIQII) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H+ in the reduction was revealed by the pH dependence of the electron transfer events and by net H+ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash QII receives an electron via QI to form a stable ubisemiquinone anion (QII-); the second flash generates QI-. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H+, to produce QIIH2. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H+ uptake. Above pH 6 there is a progressive increase in H+ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H+ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to QII-. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H+ binding electron transfer following the second flash.

Electron acceptors of photosynthetic bacterial reaction centers. Direct observation of oscillatory behaviour suggesting two closely equivalent ubiquinones.

When reaction centers are illuminated by a series of single turnover flashes ubisemiquinone is formed and destroyed on alternate flashes. This oscillatory behaviour can be observed with both optical and electron spin resonance techniques. The oscillations are dependent upon the presence of excess ubiquinone in a manner which suggests that two molecules may act almost equivalently as metastable primary acceptors forming a two-electron gate between the one-electron primary photoact and a two-electron secondary acceptor pool.

Electron Transfer and Protein Dynamics in the Photosynthetic Reaction Center

We have measured the kinetics of electron transfer (ET) from the primary quinone (Q(A)) to the special pair (P) of the reaction center (RC) complex from Rhodobacter sphaeroides as a function of temperature (5-300 K), illumination protocol (cooled in the dark and under illumination from 110, 160, 180, and 280 K), and warming rate (1.3 and 13 mK/s). The nonexponential kinetics are interpreted with a quantum-mechanical ET model (Fermis golden rule and the spin-boson model), in which heterogeneity of the protein ensemble, relaxations, and fluctuations are cast into a single coordinate that relaxes monotonically and is sensitive to all types of relaxations caused by ET. Our analysis shows that the structural changes that occur in response to ET decrease the free energy gap between donor and acceptor states by 120 meV and decrease the electronic coupling between donor and acceptor states from 2.7 x 10(-4) cm(-1) to 1.8 x 10(-4) cm(-1). At cryogenic temperatures, conformational changes can be slowed or completely arrested, allowing us to monitor relaxations on the annealing time scale (approximately 10(3)-10(4) s) as well as the time scale of ET (approximately 100 ms). The relaxations occur within four broad tiers of conformational substates with average apparent Arrhenius activation enthalpies of 17, 50, 78, and 110 kJ/mol and preexponential factors of 10(13), 10(15), 10(21), and 10(25) s(-1), respectively. The parameterization provides a prediction of the time course of relaxations at all temperatures. At 300 K, relaxations are expected to occur from 1 ps to 1 ms, whereas at lower temperatures, even broader distributions of relaxation times are expected. The weak dependence of the ET rate on both temperature and protein conformation, together with the possibility of modeling heterogeneity and dynamics with a single conformational coordinate, make RC a useful model system for probing the dynamics of conformational changes in proteins.

Flash-induced H+ binding by bacterial photosynthetic reaction centers: Influences of the redox states of the acceptor quinones and primary donor

The flash-induced proton-binding behavior of reaction centers from Rhodobacter sphaeroides was examined, over a wide range of pH, as a function of the one-electron redox states of the acceptor quinones (QAQ−A and QBQ−B) and the primary donor (P+P). Below about pH 9, the P+Q− states (P+Q−A and P+Q−B), generated in the absence of exogenous electron donor to P+, fail to take up protons stoichiometrically, as established in the previous paper (Maroti, P. and Wraight, C.A. (1988) Biochim. Biophys. Acta 934, 314–328). When P+ is rereduced by a donor, to yield the state PQ−, proton binding is enhanced in this lower pH range. In the case of QB-reconstituted reaction centers, the net proton binding stoichiometry (H+P+) for PQ−B is about 0.85, between pH 7 and pH 9, approaching the stoichiometric value expected for a pH-dependent redox midpoint potential of QBQ−B. The shortfall from H+P+ = 1.0 can be accounted for by the involvement of four protonatable groups with different pK values depending on the redox state of QB. The pK shifts vary from 0.3 to 1.5 pH units, with the lower pK groups exhibiting the smaller pK shifts. The enhancement of proton binding, associated with the rereduction of P+, is interpreted as a response of the same groups to the redox state of P+P, with the lower pK groups exhibiting the largest pK shifts - up to 1.0 pH unit. A similar general behavior is seen for reaction centers lacking QB, or in the presence of terbutryn, a competitive inhibitor of QB-binding. Quantitatively, the rereduction of P+ does not restore such high levels of H+ binding for PQ−A or PQ−A+ terbutryn (H+P+ ⩽ 0.5 at all pH values), but the behavior can be similarly accounted for by four protonatable groups that are somewhat less responsive to the redox states of QA and P. The pK values for the three different acceptor configurations (QB, QA and QA+ terbutryn) are similar but not identical, and depend on the redox states of the primary donor and the acceptor quinones, and on the occupancy of the QB-binding site. The pK values are discussed in terms of possible structural determinants of the quinone binding sites. The data define protonation networks for the reaction center states PQ, P+Q− and PQ−, and allow one to deduce the properties of a fourth state: P+Q. The derived pK values predict the occurrence of H+ release from the state P+Q, at low pH, and this was confirmed by using ferricyanide to reoxidize Q− following a flash to generate P+Q. The proposed protonation scheme allows the calculation of the pH dependence of the one-electron transfer equilibrium between P+Q−AQB and P+QAQ−B. This agrees well with the measured value, derived from the kinetics of charge recombination. However, the pK changes, derived from the enhanced proton binding that accompanies rereduction of P+, give rise to a substantial discrepancy between the calculated and measured values for the PQ−AQB ↔ PQAQ−B equilibrium. The pH dependences of the redox midpoint potentials (Em) of QAQ−A, QBQ−B and P+P are also calculated. Good agreement between calculated and measured values is obtained for P+P, and between the calculated value for QBQ−B and that expected from studies on chromatophores. However, the calculated pH dependence of Em(QAQ−A) is at variance with that measured in isolated reaction centers or chromatophores. These discrepancies are discussed but not resolved.

Photoelectrochemical complexes for solar energy conversion that chemically and autonomously regenerate

Naturally occurring photosynthetic systems use elaborate pathways of self-repair to limit the impact of photo-damage. Herein, we demonstrate a complex that mimics this process consisting of two recombinant proteins, phospholipids and a carbon nanotube. The components self-assemble into a configuration in which an array of lipid bilayers aggregate on the surface of the carbon nanotube, creating a platform for the attachment of light-converting proteins. The system can disassemble upon the addition of a surfactant and reassemble on its removal over an indefinite number of cycles. The assembly is thermodynamically meta-stable and can only transition reversibly if the rate of surfactant removal exceeds about 10−5 sec−1. Only in the assembled state do the complexes exhibit photoelectrochemical activity. We demonstrate a regeneration cycle that uses surfactant to switch between assembled and disassembled states, resulting in increased photo-conversion efficiency of more than 300% over 168 hours and an indefinite extension of the systems lifetime.

A crucial role for AspL213 in the proton transfer pathway to the secondary quinone of reaction centers from Rhodobacter sphaeroides

The aspartic acid at position 213 of the L-subunit (AspL213) of reaction centers from Rb. sphaeroides is one of two acidic residues in the binding site of the secondary quinone (QB). Alteration of AspL213 to asparagine by site-directed mutagenesis drastically affected the light-induced proton and electron transfer functions leading to formation of quinol (QBH2). The first electron transfer was slowed to a half-time in the millisecond time range, but the equilibrium (Q−AQB ↔ QAQ−B) was substantially increased in favor of QB reduction and the pH dependence of the equilibrium was altered. The stabilization of Q−B is suggested to result from the uncharged nature of the substitution, with the implication that AspL213 is normally ionized and presents an electrostatic restriction to the first electron transfer. The second electron transfer (Q−AQ−B + 2H+ ↔ QAQBH2) was even more severely inhibited and was at least 104-times slower than the wild type at pH > 6.5, and after only two flashes the RCs were blocked in the Q−AQ−B state. At lower pH some transfer activity was restored, although with a rate still 103-times slower than the wild type. The kinetics of the second electron transfer at low pH corresponded exactly to the kinetics of proton uptake. These data are interpreted as implying an essential role for AspL213 in the proton transfer pathway leading to the formation of QH2 after the second flash.

THE ROLE OF QUINONES IN BACTERIAL PHOTOSYNTHESIS

The position and role of quinones in electron transport systems have been areas of intermittent research activity since their discovery over 25 yr ago. Mitchell’s recent suggestions of an explicit role for the semiquinone forms of these redox agents have focused attention on the essentially mechanistic problem of interposing these generally two-electron redox couples in a chain of one-electron carriers [40,41,42]. The ripeness of this field for a new perspective is evident from the explosion of experimental and theoretical studies that has followed. In the area of bacterial photosynthesis, alone, there has been a dramatic increase in quinone-related studies and, consequently, in quantitative data. In this review, I have placed some emphasis on the role of specialized quinones in mediating electron transfer between the reaction center and the electron transport chain because the quantitative nature of the information available is beginning to reveal complex, dyanmic interactions between components which will, I believe, have a far-reaching influence on our view of electron transport, energy coupling and membrane phenomena in general.

Microsecond photooxidation kinetics of cytochrome c2 from Rhodopseudomonas sphaeroides: in vivo and solution studies

In the photosyn~eti~ bacteria, stabilization of the primary charge separation between the bacteriochlorophyll primary donor and the quinone acceptor proceeds almost universally by the rapid photooxidation of a c-type cytochrome [ 11. In some species, the cytochrome is tightly bound to the reaction center and oxidation is roughly monophasic and extremely rapid, with halftimes of 0.1-I @s [I]. Ln Rhodopseudomonas sphaeroides, a species otherwise better characterized than any other, the kinetics of cytochrome c2 photooxidation are biphasic and are currently not well understood. As was first shown [I ,2] the in vivo kinetics can be well reproduced in form using isolated reaction centers incorporated into phospholipid vesicles. We have found the kinetics to be very similar for isolated reaction centers either in detergent solubilized form or incorporated into phospholipid vesicles (131, unpubli~ed obse~ations). The correspondence between the reconstituted kinetics and those in vivo has been uncertain, however, because the fast oxidation phase has never been sufficiently resolved, although a halftime of 1 O-30 ps has been estimated [4,5]. We report here on the resolution of this rapid process which proves to be considerably faster than has been supposed for this species [4,5]. We also report some observations on the slow phase which suggest a kinetic scheme for the photooxidation of cytochrome both in vivo and in vitro.

Flash-induced H+ binding by bacterial photosynthetic reaction centers: Comparison of spectrophotometric and conductimetric methods

H+ binding following flash excitation of isolated reaction centers from Rhodobacter sphaeroides was measured using a conductimetric method. A procedure is described for calibrating the H+ change and the method is compared to the more widely used spectrophotometric assay of pH-indicator dyes. The two methods agree extremely well and confirm a severe paradox in the pH dependence of the H+ binding stoichiometry - that reaction centers in the light-activated, charge-separation state, P+(QAQB)−, fail to bind net protons at pH values near neutral or lower, although proton binding is seen at higher pH. This is in contrast to expectations arising from the equilibrium redox behavior of the acceptor quinones, QA and QB, and with the pH dependence of the one-electron equilibrium between Q−AQB and QAQ−B. The observed proton binding is discussed in terms of multiple protonation equilibria linked to the redox states of the acceptor quinones and of the primary donor.

The acceptor quinone complex of Rhodopseudomonas viridis reaction centers

The acceptor complex of isolated reaction centers from Rhodopseudomonas viridis contains both menaquinone and ubiquinone. In a series of flashes the ubiquinone was observed to undergo binary oscillations in the formation and disappearance of a semiquinone, indicative of secondary acceptor (QB) activity. The oscillating signal, Q-B, was typical of a ubisemiquinone anion with a peak at 450 nm (delta epsilon = 6 mM-1 X cm-1) and a shoulder at 430 nm. Weak electrochromic bandshifts in the infrared were also evident. The spectrum of the reduced primary acceptor (Q-A) exhibited a major peak at 412 nm (delta epsilon = 10 mM-1 X cm-1) consistent with the assignment of menaquinone as QA. The Q-A spectrum also had minor peaks at 385 and 455 nm in the blue region. The same spectrum was recorded after quantitative removal of the secondary acceptor, when only menaquinone was present in the reaction centers. Spectral features in the near-infrared due to Q-A were attributed to electrochromic effects on bacteriochlorophyll (BChl) b and bacteriopheophytin (BPh) b pigments resulting in a distinctive split peak at 810 and 830 nm (delta epsilon = 8 mM-1 X cm-1). The menaquinone was identified as 2-methyl-3-nonylisoprenyl-1,4-naphthoquinone (menaquinone-9). The native QA activity was uniquely provided by this menaquinone and ubiquinone was not involved. QB activity, on the other hand, displayed at least a 40-fold preference for ubiquinone (Q-10) as compared to menaquinone. Thus, both quinone-binding sites display remarkable specificity for their respective quinones. In the absence of donors to P+, charge recombination of the P+Q-A and P+Q-B pairs had half-times of 1.1 +/- 0.2 and 110 +/- 20 ms, respectively, at pH 9.0, indicating an electron-transfer equilibrium constant (Kapp2) of at least 100 for Q-AQB in equilibrium QAQ-B. Also observed was a slow recombination of the cytochrome c-558+ Q-A pair, with t 1/2 = 2 +/- 0.5 s at pH 6.