Delayed fluorescence from the photosynthetic reaction center measured by electronic gating of the photomultiplier.

The decay of the delayed fluorescence (920 nm) of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides R26 in the P(+)Q(A)(-) charge-separated state (P and Q(A) are the primary donor and quinone, respectively) has been monitored in a wide (100 ns to 100 ms) time range. The photomultiplier (Hamamatsu R3310-03) was protected from the intense prompt fluorescence by application of gating potential pulses (-280 V) to the first, third, and fifth dynodes during the laser pulse. The gain of the photomultiplier dropped transiently by a factor of 1 x 10(6). The delayed fluorescence showed a smooth but nonexponential decay from 100 ns to 1 ms that was explained by the relaxation of the average free energy between P* and P(+)Q(A)(-) changing from -580 to -910 meV. This relaxation is due to the slow protein response to charge separation and can be described by a Kohlrausch relaxation function with time constant of 65 micros and a stretching exponent of alpha = 0.45.

The electric field generated by photosynthetic reaction center induces rapid reversed electron transfer in the bc1 complex.

The cytochrome bc(1) complex is the central enzyme of respiratory and photosynthetic electron-transfer chains. It couples the redox work of quinol oxidation and cytochrome reduction to the generation of a proton gradient needed for ATP synthesis. When the quinone processing Q(i)- and Q(o)-sites of the complex are inhibited by both antimycin and myxothiazol, the flash-induced kinetics of the b-heme chain, which transfers electrons between these sites, are also expected to be inhibited. However, we have observed in Rhodobacter sphaeroides chromatophores, that when a fraction of heme b(H) is reduced, flash excitation induces fast (half-time approximately 0.1 ms) oxidation of heme b(H), even in the presence of antimycin and myxothiazol. The sensitivity of this oxidation to ionophores and uncouplers, and the absence of any delay in the onset of this reaction, indicates that it is due to a reversal of electron transfer between b(L) and b(H) hemes, driven by the electrical field generated by the photosynthetic reaction center. In the presence of antimycin A, but absence of myxothiazol, the second and following flashes induce a similar ( approximately 0.1 ms) transient oxidation of approximately 10% of the cytochrome b(H) reduced on the first flash. From the observed amplitude of the field-induced oxidation of heme b(H), we estimate that the equilibrium constant for sharing one electron between hemes b(L) and b(H) is 10-15 at pH 7. The small value of this equilibrium constant modifies our understanding of the thermodynamics of the Q-cycle, especially in the context of a dimeric structure of bc(1) complex.

Site specific labeling of Rhodobacter sphaeroides reaction centers with dye probes for surface pH measurements.

Covalently bound pH sensitive dyes are an important tool for characterizing the proteolytic reactions of protein complexes that play key roles in biological energy transduction. Here we demonstrate the feasibility of this method for photosynthetic reaction centers (RCs) for the first time, by the highly selective attachment of two thiol reactive derivatives of fluorescein to the two H subunit cysteines of the photosynthetic RC from Rhodobacter sphaeroides R-26 The pK(a) shifts of the dyes upon binding to the protein and in response to high salt were measured, and interpreted based on the structure of the RC. 2-[(5-fluoresceinyl)aminocarbonyl]ethyl-methanethiosulfonate was attached to Cys H156 and fluorescein-5-maleimide to Cys H234. By following the absorption changes of bound fluorescein (500 nm), and those of the hydrophilic pH indicator 8-hydroxypyrene-1,3,6-tris-sulfonic acid (468 nm), the surface and bulk pH were monitored separately with less than 5% crosstalk. Flash-induced proton uptake and external calibrations by mixing with aliquots of acid were measured in different redox states of the RCs. The results indicate that the charge in the quinone acceptor complex after flash activation (primary quinone acceptor (Q(A))- or secondary quinone acceptor (Q(B))-) has no effect on the surface pH and potential in the vicinity of these two attachment sites, between pH 6.5 and 9. Application of the method to other surface locations is discussed.

Protein control of the redox potential of the primary quinone acceptor in reactioncCenters from Rhodobacter sphaeroides.

The role of the protein environment in determining the redox midpoint potential (E(m)) of Q(A), the primary quinone of bacterial reaction centers, was investigated by mutation of isoleucine at position 265 of the M subunit in Rhodobacter sphaeroides. Isoleucine was changed to threonine, serine, and valine, yielding mutants M265IT, M265IS, and M265IV, respectively. All three mutants, with smaller residues replacing isoleucine, exhibited decreased binding affinities of the Q(A) site for various quinone analogues, consistent with an enlargement or loosening of the headgroup binding domain and a decrease in the van der Waals contact for small quinones. In all other respects, M265IV was like the wild type, but the polar mutants, M265IT and M265IS, had unexpectedly dramatic decreases in the redox midpoint potential of Q(A), resulting in faster rates of P(+)Q(A)(-) charge recombination. For both anthraquinone and native ubiquinone, the in situ E(m) of Q(A) was estimated to be approximately 100 and 85 mV lower in M265IT and M265IS, respectively. The effect on E(m)(Q(A)) indicates destabilization of the semiquinone or stabilization of the quinone. This is suggested to arise from either (i) electrostatic interaction between the partial charges or dipole of the residue hydroxyl group and the charge distribution of quinone and semiquinone states with particular influence near the C4 carbonyl group or (ii) from hydrogen bonding interactions between the hydroxyl oxygen and the N(delta)H of histidine M219, causing a weakening of the hydrogen bond to the C4 carbonyl. The rate of the first electron transfer (k(AB)(()(1)())) in the polar mutants was the same as in the wild type at low pH but decelerated at higher pH with altered pH dependence. The rate of the second electron transfer (k(AB)(()(2)())) was 3-4-fold greater than in the wild type over the whole pH range from 4 to 11, consistent with a larger driving force for electron transfer derived from the lower E(m) of Q(A).

Preferential binding of equine ferricytochrome c to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides.

Redox titration of horse heart cytochrome c (cyt c), in the presence of varying concentrations of detergent-solubilized photosynthetic reaction center (RC) from Rhodobacter sphaeroides, revealed an RC concentration-dependent decrease in the measured cyt c midpoint potential that is indicative of a 3.6 +/- 0.2-fold stronger binding affinity of oxidized cytochrome to a single binding site. This effect was correlated with preferential binding in the functional complex by redox titration of the fraction of RCs exhibiting microsecond, first-order, special pair reduction by cytochrome. A binding affinity ratio of 3.1 +/- 0.4 was determined by this second technique, confirming the result. Redox titration of flash-induced intracomplex electron transfer also showed the association in the electron transfer-active complex to be strong, with a dissociation constant of 0.17 +/- 0.03 microM. The tight binding is associated with a slow off-rate which, in the case of the oxidized form, can influence the kinetics of P(+) reduction. The pitfalls of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions are discussed with relation to the first electron transfer from primary to secondary RC quinone acceptors. The results shed some light on the diversity of kinetic behavior reported for the cytochrome to RC electron-transfer reaction.

DCCD inhibits the reactions of the iron-sulfur protein in Rhodobacter sphaeroides chromatophores.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1).

Aspartate-187 of cytochrome b is not needed for DCCD inhibition of ubiquinol: cytochrome c oxidoreductase in Rhodobacter sphaeroides chromatophores.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-state proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. In chromatophores of the purple bacterium Rhodobacter sphaeroides, this has been associated with the specific labeling of a surface-exposed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang et al. (1998) Arch. Biochem. Biophys. 352, 193-198]. To explore the possible role of this amino acid residue in the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and the electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores from wild type (WT) and mutant cells, in which aspartate-187 of cytochrome b (Asp(B187)) has been changed to asparagine (mutant B187 DN). The kinetics and amplitude of phase III of the electrochromic shift of carotenoids, reflecting electrogenic reactions in the bc(1) complex, and of the redox changes of cytochromes and reaction center, were similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chromatophores. The dependence of the kinetics and amplitude of phase III of the electrochromic shift on DCCD concentration was identical in WT and B187DN chromatophores, indicating that covalent modification of Asp(B187) is not specifically responsible for the effect of DCCD-induced effects of cytochrome bc(1) complex. Furthermore, no evidence for differential inhibition of electrogenesis and electron transport was found in either strain. We conclude that Asp(B187) plays no crucial role in the protonogenic reactions of bc(1) complex, since its replacement by asparagine does not lead to any significant effects on either the electrogenic reactions of bc(1) complex, as revealed by phase III of the electrochromic shift of carotenoids, or sensitivity of turnover to DCCD.

The structure of chromatophores from purple photosynthetic bacteria fused with lipid-impregnated collodion films determined by near-field scanning optical microscopy.

Lipid-impregnated collodion (nitrocellulose) films have been frequently used as a fusion substrate in the measurement and analysis of electrogenic activity in biological membranes and proteoliposomes. While the method of fusion of biological membranes or proteoliposomes with such films has found a wide application, little is known about the structures formed after the fusion. Yet, knowledge of this structure is important for the interpretation of the measured electric potential. To characterize structures formed after fusion of membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides with lipid-impregnated collodion films, we used near-field scanning optical microscopy. It is shown here that structures formed from chromatophores on the collodion film can be distinguished from the lipid-impregnated background by measuring the fluorescence originating either from endogenous fluorophores of the chromatophores or from fluorescent dyes trapped inside the chromatophores. The structures formed after fusion of chromatophores to the collodion film look like isolated (or sometimes aggregated, depending on the conditions) blisters, with diameters ranging from 0.3 to 10 microm (average approximately 1 microm) and heights from 0.01 to 1 microm (average approximately 0.03 microm). These large sizes indicate that the blisters are formed by the fusion of many chromatophores. Results with dyes trapped inside chromatophores reveal that chromatophores fused with lipid-impregnated films retain a distinct internal water phase.

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 (Fermi's 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.

Kinetics of H+ ion binding by the P+QA-state of bacterial photosynthetic reaction centers: rate limitation within the protein.

The kinetics of flash-induced H+ ion binding by isolated reaction centers (RCs) of Rhodobacter sphaeroides, strain R-26, were measured, using pH indicators and conductimetry, in the presence of terbutryn to block electron transfer between the primary and secondary quinones (QA and QB), and in the absence of exogenous electron donors to the oxidized primary donor, P+, i.e., the P+QA-state. Under these conditions, proton binding by RCs is to the protein rather than to any of the cofactors. After light activation to form P+QA-, the kinetics of proton binding were monoexponential at all pH values studied. At neutral pH, the apparent bimolecular rate constant was close to the diffusional limit for proton transfer in aqueous solution (approximately 10(11) M-1 s-1), but increased significantly in the alkaline pH range (e.g., 2 x 10(13) M-1 s-1 at pH 10). The average slope of the pH dependence was -0.4 instead of -1.0, as might be expected for a H+ diffusion-controlled process. High activation energy (0.54 eV at pH 8.0) and weak viscosity dependence showed that H+ ion uptake by RCs is not limited by diffusion. The salt dependence of the H+ ion binding rate and the pK values of the protonatable amino acid residues of the reaction center implicated surface charge influences, and Gouy-Chapman theory provided a workable description of the ionic effects as arising from modulation of the pH at the surface of the RC. Incubation in D2O caused small increases in the pKs of the protonatable groups and a small, pH (pD)-dependent slowing of the binding rate. The salt, pH, temperature, viscosity, and D2O dependences of the proton uptake by RCs in the P+QA- state were accounted for by three considerations: 1) parallel pathways of H+ delivery to the RC, contributing to the observed (net) H+ disappearance; 2) rate limitation of the protonation of target groups within the protein by conformational dynamics; and 3) electrostatic influences of charged groups in the protein, via the surface pH.

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.

Reactions of isocytochrome c2 in the photosynthetic electron transfer chain of Rhodobacter sphaeroides.

Rhodobacter sphaeroides strains lacking cytochrome c2 (cyt c2), the normal electron donor to P870+ in light-oxidized reaction center (RC) complexes, are unable to grow photosynthetically. However, spd mutations that suppress the photosynthetic deficiency of cyt c2 mutants elevate levels of the cyt c2 isoform, isocyt c2. We monitored photosynthetic electron transfer in whole cells, in chromatophores, and with purified components to ascertain if and how isocyt c2 reduced light-oxidized RC complexes. These studies revealed that several fundamental aspects of photosynthetic electron transfer were similar in strains that use isocyt c2 and wild-type cells. For example, P870+ reduction accompanied cytochrome c oxidation. In addition, photosynthetic electron transfer was blocked by the well-known cyt bc1 complex inhibitors antimycin and myxothiazol. However, even at the increased isocyt c2 levels present in these strains (approximately 40% that of cyt c2 in wild-type cells), there was little, if any, of the rapid (< 5 microns) electron transfer to P870+ that is characteristic of cytochromes bound to RC complexes at the time of the light flash. Thus, it appears that isocyt c2 function limits the in vivo rate of P870+ reduction. Indeed, at low ionic strength in vitro, the apparent affinity of isocyt c2 for RC complexes (KD approximately 40 microM) is significantly lower than that of cyt c2 (KD approximately 1.0 microM). This reduced affinity does not appear to result from an altered mode of RC binding by isocyt c2 since electrostatic interactions make similar overall contributions to the binding of both cyt c2 and isocyt c2 to this membrane-bound redox partner. Thus, sequence, structural, or local conformational differences between cyt c2 and isocyt c2 significantly alter their apparent affinities for this physiologically relevant redox partner.

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

Oxygen evolution in photosynthesis: from unicycle to bicycle.

Flash-induced oxygen evolution in the thylakoids of plants and algae exhibits damped oscillations with period four. These are well described by the S-state model of Kok et al. [Kok, B., Forbush, B. & McGloin, M. (1970) Photochem. Photobiol. 11, 457-475], with damping provided by empirical misses and double hits in the reaction center of photosystem II. Here we apply a mechanistic interpretation of misses as mainly determined by reaction centers that are inactive at the time of the flash due to the presence of either P+ or QA, according to the electron transfer equilibria on the donor and acceptor sides of the reaction center. Calculation of misses on this basis, using known or estimated values of the equilibrium constants for electron transfer between the S states and tyrosine Yz, between Yz and P680, as well as between the acceptor plastoquinones, allows a natural description of the flash number dependence of oxygen evolution. The calculated misses are different for each flash-induced reaction center transition. Identification of this mechanism underlying the miss factor for each transition leads to the recognition of two different reaction sequence cycles of photosystem II, with different transition probabilities, producing an intrinsic heterogeneity of photosystem II activity.

Kinetic factors in the bicycle model of oxygen evolution by Photosystem II.

Flash-induced oxygen evolution and many related processes in thylakoids of oxygenic organisms are modulated with period four by the S-state transitions associated with the oxygen evolving system of Photosystem II (PS II). To analyze these phenomena, we have interpreted the S-state model on the basis of the charge accumulating activities on both sides of PS II-4 charges on the donor side and 2 charges on the acceptor side. This results in the recognition of two parallel reaction center cycles V and W of PS II function (V.P. Shinkarev and C.A. Wraight (1993) Proc Natl Acad Sci USA 90: 1834-1838). The description of damping of the period four oscillations is here extended to include kinetic sources of misses in both cycles. Such misses arise in reaction centers (RCs) in which back reaction between P(+) and QA (-) occurs before the electron transfer equilibria on the donor and acceptor sides of the RC are reached. These are in addition to misses which are determined by reaction centers (RCs) that are inactive at the time of the flash due to the presence of either P(+) or QA (-) according to the electron transfer equilibria on the donor and acceptor sides of the RC. Using known or estimated values of the equilibrium and rate constants for donor and acceptor side reactions of the RC, this provides a natural and quantitatively reasonable description of the flash number dependence of oxygen evolution and other period four modulated processes of PS II. The estimated miss factors are different for both cycles V and W and are dependent on flash number and pH. Estimates based on existing data show that miss factors of the first type (kinetic) are dominant at low pH, while those of the second type (equilibrium) are dominant at high pH.

Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g., Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+Q-A or P+Q-B, respectively; (b) the kinetics of electron transfer from Q-A to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (tau = 3.5 ps), followed by reduction of the bacteriopheophytin (tau = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (tau = 410 +/- 30 and 47 +/- 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type).(ABSTRACT TRUNCATED AT 400 WORDS)

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)

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.

Investigations on the influence of headgroup substitution and isoprene side-chain length in the function of primary and secondary quinones of bacterial reaction centers.

The contributions of headgroup and side-chain in the binding and function of the primary (QA) and secondary (QB) quinones of isolated reaction centers (RCs) from Rhodobacter sphaeroides were investigated. Various ubiquinones and structurally similar quinones were reconstituted into RCs depleted of one (1Q-RCs) or both (0Q-RCs) quinones. The influence of partition coefficients on the apparent binding affinities was minimized by expressing dissociation constants in terms of the mole fraction of quinone partitioned into the detergent. It was then apparent that the size of the isoprenyl side-chain was of little consequence in determining the binding affinity or the functional competence of either QA or QB, although an alkyl chain of equivalent size was a poor substitute. The degree of substitution of the headgroup, however, was a sensitive determinant of binding. For both quinone sites, the trisubstituted plastoquinones bond more weakly than the fully substituted ubiquinones. Similarly, for binding to the QA site, duroquinone (tetramethylbenzoquinone) bound much more strongly than trimethylbenzoquinone. The affinity of the QA site for ubiquinones was about 20-times stronger than the QB site, but the QB site is probably not more specific than the QA site. However, QB function depends on a suitable redox free-energy drop from QA as well as binding, and of all the quinones tested only the ubiquinones simultaneously supported full QA and QB activity. Even plastoquinone-A, which fills both roles in Photosystem II, was unable to do so in bacterial RCs, although it did bind. The unique ability of ubiquinones to both bind and provide the appropriate redox span is discussed. The temperature dependence of binding of the isoprenyl ubiquinones at the QA site changed markedly with chain length. For Q-10-Q-7, the binding enthalpy was positive and net binding was entirely driven by entropic factors. For the shorter-chain ubiquinones, Q-6-Q-1, both entropy and enthalpy of binding were favorable. This strong entropy-enthalpy compensation is suggested to arise from antagonistic interactions (anticooperativity) between headgroup and tail binding. For QB function by hydrophobic quinones, the temperature dependence of the micelle properties prevented easy access to thermodynamic parameters. However, for water-soluble Q-0, binding to the QB site was determined to be enthalpically driven.(ABSTRACT TRUNCATED AT 400 WORDS)

Sulfhydryl modifying reagents inhibit Q A (-) oxidation in reaction centers from Rhodobacter sphaeroides and Capsulatus, but not Rhodopseudomonas viridis.

The effects of various sulfhydryl-modifying reagents on reaction centers (RCs) from purple photosynthetic bacteria have been examined, with particular emphasis on the activity of the acceptor quinones, QA and QB, comprising the two electron gate. Mercurial reagents, especially p-chloromercuribenzenesulfonate (pCMBS), were effective in inhibiting QB function in RCs from Rhodobacter sphaeroides and Rb. capsulatus, but not in Rhodopseudomonas viridis. The inhibition was fully reversible by dialysis against dithiothreitol (DTT). The effect on QB function was not an apparent one mediated by an alteration in the redox potential of QA. N-ethylmaleimide (NEM) had no effect on any of the quinone functions, even at very high concentrations. Comparison of the X-ray structures of the RCs from Rb. sphaeroides and Rp. viridis and the known amino acid sequences for all three bacterial RCs suggest that a cysteine residue at position 108 in the L subunit of the Rhodobacter species is the most likely candidate for the site of action of the mercurial reagents. This was strongly supported by the absence of any effect of pCMBS on a site specific mutation of Rb. sphaeroides (L108CS) with residue L108 changed from cysteine to serine. These results imply a long distance (>20 Å) effect on the functioning of QB, perhaps involving a relatively gross structural alteration.