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.     

Mechanism of proton transfer inhibition by Cd(2+) binding to bacterial reaction centers: determination of the pK(A) of functionally important histidine residues

The bacterial photosynthetic reaction center (RC) uses light energy to catalyze the reduction of a bound quinone molecule Q(B) to quinol Q(B)H(2). In RCs from Rhodobacter sphaeroides the protons involved in this process come from the cytoplasm and travel through pathways that involve His-H126 and His-H128 located near the proton entry point. In this study, we measured the pH dependence from 4.5 to 8.5 of the binding of the proton transfer inhibitor Cd(2+), which ligates to these surface His in the RC and inhibits proton-coupled electron transfer. At pH <6, the negative slope of the logarithm of the dissociation constant, K(D), versus pH approaches 2, indicating that, upon binding of Cd(2+), two protons are displaced; i.e., the binding is electrostatically compensated. At pH >7, K(D) becomes essentially independent of pH. A theoretical fit to the data over the entire pH range required two protons with pK(A) values of 6.8 and 6.3 (+/-0.5). To assess the contribution of His-H126 and His-H128 to the observed pH dependence, K(D) was measured in mutant RCs that lack the imidazole group of His-H126 or His-H128 (His --> Ala). In both mutant RCs, K(D) was approximately pH independent, showing that Cd(2+) does not displace protons upon binding in the mutant RCs, in contrast to the native RC in which His-H126 and His-H128 are the predominant contributors to the observed pH dependence of K(D). Thus, Cd(2+) inhibits RC function by binding to functionally important histidines.     

Cumulant analysis of charge recombination kinetics in bacterial reaction centers reconstituted into lipid vesicles

The kinetics of charge recombination between the primary photoxidized donor (P(+)) and the secondary reduced quinone acceptor (Q(B)(-)) have been studied in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides incorporated into lecithin vesicles containing large ubiquinone pools over the temperature range 275 K = (50 +/- 15) nm). Following these premises, we describe the kinetics of P(+)Q(B)(-) recombination with a truncated cumulant expansion and relate it to P(Q) and to the free energy changes for Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer (DeltaG(AB)(o)) and for quinone binding (DeltaG(bind)(o)) at Q(B). The model accounts well for the temperature and quinone dependence of the charge recombination kinetics, yielding DeltaG(AB)(o) = -7.67 +/- 0.05 kJ mol(-1) and DeltaG(bind)(o) = -14.6 +/- 0.6 kJ mol(-1) at 298 K.     

Functional consequences of the organization of the photosynthetic apparatus in Rhodobacter sphaeroides: II. A study of PufX- membranes

In the bacterium R. sphaeroides, the polypeptide PufX is indispensable for photosynthetic growth. Its deletion is known to have important consequences on the organization of the photosynthetic apparatus. In the wild-type strain, complexes between the reaction center (RC) and the antenna (light-harvesting complex 1 (LH1)) are associated in dimers, and LH1 does not fully encircle the RC. In the absence of PufX, the complexes become monomeric, and the LH1 ring closes around the RC. We analyzed the functional consequences of PufX deletion. Some effects can be ascribed to the monomerization of the RC.LH1 complexes: the number of RCs that share a common antenna for excitation transfer or a common quinone pool become smaller. We examined the kinetic effects of the closed LH1 ring on quinone turnover: diffusion across LH1 entails a delay of approximately 1 ms, and the barrier appears to be located directly against the quinone-binding (secondary quinone acceptor (Q(B))) pocket. The diffusion of ubiquinol from the RC to the cytochrome bc1 complex is approximately 2-fold slower in the mutant, suggesting an increased distance between the two complexes. The properties of the Q(B) pocket (binding of inhibitors, stabilization of Q(B-), and rate of Q(B)-H2 formation) appear to be modified in the mutant. Another specificity of PufX- is the accumulation of closed centers in the Q(A-) (where Q(A) is the primary quinone acceptor) state as the secondary acceptor pool becomes reduced, which is probably the origin of photosynthetic incompetence. We suggest that this is related to the Q(B) pocket alterations. The malfunction of the reaction center is probably due to a faulty association with LH1 that is prevented in the PufX-containing structure.     

An isotope-edited FTIR investigation of the role of Ser-L223 in binding quinone (QB) and semiquinone (QB-) in the reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (Q(B)) site. Several nearby residues are important for both binding and redox chemistry involved in the light-induced conversion from Q(B) to quinol Q(B)H(2). Ser-L223 is one of the functionally important residues located near Q(B). To obtain information on the interaction between Ser-L223 and Q(B) and Q(B)(-), isotope-edited Q(B)(-)/Q(B) FTIR difference spectra were measured in a mutant RC in which Ser-L223 is replaced with Ala and compared to the native RC. The isotope-edited IR fingerprint spectra for the C=O [see text] and C=C [see text] modes of Q(B) (Q(B)(-)) in the mutant are essentially the same as those of the native RC. These findings indicate that highly equivalent interactions of Q(B) and Q(B)(-) with the protein occur in both native and mutant RCs. The simplest explanation of these results is that Ser-L223 is not hydrogen bonded to Q(B) or Q(B)(-) but presumably forms a hydrogen bond to a nearby acid group, preferentially Asp-L213. The rotation of the Ser OH proton from Asp-L213 to Q(B)(-) is expected to be an important step in the proton transfer to the reduced quinone. In addition, the reduced quinone remains firmly bound, indicating that other distinct hydrogen bonds are more important for stabilizing Q(B)(-). Implications on the design features of the Q(B) binding site are discussed.     

In vivo assembly of a truncated H subunit mutant of the <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> photosynthetic reaction centre and direct electron transfer from the Q<Subscript>A</Subscript> quinone to an electrode

We address a challenge in the engineering of proteins to redirect electron transfer pathways, using the bacterial photosynthetic reaction centre (RC) pigment–protein complex. Direct electron transfer is shown to occur from the Q_A quinone of the Rhodobacter sphaeroides RC containing a truncated H protein and bound on the quinone side to a gold electrode. In previous reports of binding to the quinone side of the RC, electron transfer has relied on the use of a soluble mediator between the RC and an electrode, in part because the probability of Q_B quinone reduction is much greater than that of direct electron transfer through the large cytoplasmic domain of the H subunit, presenting a?~?25 Å barrier. A series of C-terminal truncations of the H subunit were created to expose the quinone region of the RC L and M proteins, and all truncated RC H mutants assembled in vivo. The 45M mutant was designed to contain only the N-terminal 45 amino acid residues of the H subunit including the membrane-spanning α-helix; the mutant RC was stable when purified using the detergent N-dodecyl-β-d-maltoside, contained a near-native ratio of bacteriochlorophylls to bacteriopheophytins, and showed a charge-separated state of \({{\text{P}}^{\text{+}}}{{\text{Q}}_{\text{A}}^-}\). The 45M-M229 mutant RC had a Cys residue introduced in the vicinity of the Q_A quinone on the newly exposed protein surface for electrode attachment, decreasing the distance between the quinone and electrode to ~?12 Å. Steady-state photocurrents of up to around 200 nA/cm² were generated in the presence of 20 mM hydroquinone as the electron donor to the RC. This novel configuration yielded photocurrents orders of magnitude greater than previous reports of electron transfer from the quinone region of RCs bound in this orientation to an electrode.     

Simultaneous replacement of Asp-L210 and Asp-M17 with Asn increases proton uptake by Glu-L212 upon first electron transfer to QB in reaction centers from Rhodobacter sphaeroides.

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the first electron transfer to the secondary quinone acceptor Q(B) is coupled to the protonation of Glu-L212, located approximately 5 A from the center of Q(B). Upon the second electron transfer to Q(B), Glu-L212 is involved in fast proton delivery to the reduced Q(B). Since Asp-L210 and Asp-M17 play an important role in the proton transfer to the Q(B) site [Paddock, M. L., Adelroth, P., Chang, C., Abresch, E. C., Feher, G., and Okamura, M. Y. (2001) Biochemistry 40, 6893-6902], we investigated the effects of replacing one or both Asp residues with Asn on proton uptake by Glu-L212 using FTIR difference spectroscopy. Upon the first electron transfer to Q(B), the amplitude of the proton uptake by Glu-L212 at pH 8 is increased in the single and double mutant RCs, as is evident from the larger intensity (by 35-55%) of the carboxylic acid band at 1727 cm(-1) in the Q(B)(-)/Q(B) difference spectra of mutant RCs, compared to that at 1728 cm(-1) in native RCs. This implies that the extent of ionization of Glu-L212 in the Q(B) ground state is greater in the mutants than in native RCs and that Asp-M17 and Asp-L210 are at least partially ionized near neutral pH in native RCs. In addition, no changes in the protonation state or the environment of these two residues are detected upon Q(B) reduction. The absence of the 1727 cm(-1) signal in all of the RCs lacking Glu-L212, confirms that the positive band at 1728-1727 cm(-1) probes the protonation of Glu-L212 in native and mutant RCs.     

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.     

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].     

Quinone reduction via secondary B-branch electron transfer in mutant bacterial reaction centers

Symmetry-related branches of electron-transfer cofactors-initiating with a primary electron donor (P) and terminating in quinone acceptors (Q)-are common features of photosynthetic reaction centers (RC). Experimental observations show activity of only one of them-the A branch-in wild-type bacterial RCs. In a mutant RC, we now demonstrate that electron transfer can occur along the entire, normally inactive B-branch pathway to reduce the terminal acceptor Q(B) on the time scale of nanoseconds. The transmembrane charge-separated state P(+)Q(B)(-) is created in this manner in a Rhodobacter capsulatus RC containing the F(L181)Y-Y(M208)F-L(M212)H-W(M250)V mutations (YFHV). The W(M250)V mutation quantitatively blocks binding of Q(A), thereby eliminating Q(B) reduction via the normal A-branch pathway. Full occupancy of the Q(B) site by the native UQ(10) is ensured (without the necessity of reconstitution by exogenous quinone) by purification of RCs with the mild detergent, Deriphat 160-C. The lifetime of P(+)Q(B)(-) in the YFHV mutant RC is >6 s (at pH 8.0, 298 K). This charge-separated state is not formed upon addition of competitive inhibitors of Q(B) binding (terbutryn or stigmatellin). Furthermore, this lifetime is much longer than the value of approximately 1-1.5 s found when P(+)Q(B)(-) is produced in the wild-type RC by A-side activity alone. Collectively, these results demonstrate that P(+)Q(B)(-) is formed solely by activity of the B-branch carriers in the YFHV RC. In comparison, P(+)Q(B)(-) can form by either the A or B branches in the YFH RC, as indicated by the biexponential lifetimes of approximately 1 and approximately 6-10 s. These findings suggest that P(+)Q(B)(-) states formed via the two branches are distinct and that P(+)Q(B)(-) formed by the B side does not decay via the normal (indirect) pathway that utilizes the A-side cofactors when present. These differences may report on structural and energetic factors that further distinguish the functional asymmetry of the two cofactor branches.     

Identification of the proton pathway in bacterial reaction centers: cooperation between Asp-M17 and Asp-L210 facilitates proton transfer to the secondary quinone (QB)

The reaction center (RC) from Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, Q(B) (the secondary quinone electron acceptor), to form quinol, Q(B)H2. Asp-L210 and Asp-M17 have been proposed to be components of the pathway for proton transfer [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. To test the importance of these residues for efficient proton transfer, the rates of the proton-coupled electron-transfer reaction k(AB)(2) (Q(A-*)Q(B-*) + H+ <==>Q(A-*)Q(B)H* --> Q(A)Q(B)H-) and its associated proton uptake were measured in native and mutant RCs, lacking one or both Asp residues. In the double mutant RCs, the k(AB)(2) reaction and its associated proton uptake were approximately 300-fold slower than in native RCs (pH 8). In contrast, single mutant RCs displayed reaction rates that were < or =3-fold slower than native (pH 8). In addition, the rate-limiting step of k(AB)(2) was changed from electron transfer (native and single mutants) to proton transfer (double mutant) as shown from the lack of a dependence of the observed rate on the driving force for electron transfer in the double mutant RCs compared to the native or single mutants. This implies that the rate of the proton-transfer step was reduced (> or =10(3)-fold) upon replacement of both Asp-L210 and Asp-M17 with Asn. Similar, but less drastic, differences were observed for k(AB)(1), which at pH > or =8 is coupled to the protonation of Glu-L212 [(Q(A-*)Q(B))-Glu- + H+ --> (Q(A)Q(B-*)-GluH]. These results show that the pathway for proton transfer from solution to reduced Q(B) involves both Asp-L210 and Asp-M17, which provide parallel branches to the proton-transfer pathway and through their electrostatic interaction have a cooperative effect on the proton-transfer rate. A possible mechanism for the cooperativity is discussed.     

Cu2+ site in photosynthetic bacterial reaction centers from Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis

The interaction of metal ions with isolated photosynthetic reaction centers (RCs) from the purple bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis has been investigated with transient optical and magnetic resonance techniques. In RCs from all species, the electrochromic response of the bacteriopheophytin cofactors associated with Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer is slowed in the presence of Cu(2+). This slowing is similar to the metal ion effect observed for RCs from Rb. sphaeroides where Zn(2+) was bound to a specific site on the surface of the RC [Utschig et al. (1998) Biochemistry 37, 8278]. The coordination environments of the Cu(2+) sites were probed with electron paramagnetic resonance (EPR) spectroscopy, providing the first direct spectroscopic evidence for the existence of a second metal site in RCs from Rb. capsulatus and Rps. viridis. In the dark, RCs with Cu(2+) bound to the surface exhibit axially symmetric EPR spectra. Electron spin echo envelope modulation (ESEEM) spectral results indicate multiple weakly hyperfine coupled (14)N nuclei in close proximity to Cu(2+). These ESEEM spectra resemble those observed for Cu(2+) RCs from Rb. sphaeroides [Utschig et al. (2000) Biochemistry 39, 2961] and indicate that two or more histidines ligate the Cu(2+) at the surface site in each RC. Thus, RCs from Rb. sphaeroides, Rb. capsulatus, and Rps. viridis each have a structurally analogous Cu(2+) binding site that is involved in modulating the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer process. Inspection of the Rps. viridis crystal structure reveals four potential histidine ligands from three different subunits (M16, H178, H72, and L211) located beneath the Q(B) binding pocket. The location of these histidines is surprisingly similar to the grouping of four histidine residues (H68, H126, H128, and L211) observed in the Rb. sphaeroides RC crystal structure. Further elucidation of these Cu(2+) sites will provide a means to investigate localized proton entry into the RCs of Rb. capsulatus and Rps. viridis as well as locate a site of protein motions coupled with electron transfer.     

Effect of dipyridamole on the recombination kinetics between photooxidized bacteriochlorophyll and photoreduced primary quinone in reaction centres of purple bacteria

The action of dipyridamole (DIP) on dark recombination between the photooxidized special pair bacteriochlorophyll BChl2+ and reduced primary quinone acceptor Q(A)- in the reaction centres (RCs) of the bacteria Rhodobacter sphaeroides was studied in the presence of different detergents (LDAO, Triton X-100, sodium cholate, sodium dodecyl sulfate). DIP accelerated this reaction approximately 4-5-fold. In RCs with the extracted H-subunit, the effect of DIP was observed at lower concentrations. The possibility of modification of the RC structure-dynamic state by DIP (including changes in RC hydrogen bonds) is proposed. The modification obviously disturbs the processes of the long-life electrostatic stabilization of Q(A)-.     

The redox midpoint potential of the primary quinone of reaction centers in chromatophores of Rhodobacter sphaeroides is pH independent

The redox midpoint potential (E (m)) of the primary quinone of bacterial reaction centers, Q(A), in native membranes (chromatophores) measured by redox potentiometry is reported to be pH dependent (-60 mV/pH) up to a highly distinctive pK ( a ) (9.8 in Rba. sphaeroides) for the reduced state. In contrast, the E (m) of Q(A) in isolated RCs of Rba. sphaeroides, although more variable, has been found to be essentially pH-independent by both redox potentiometry and by delayed fluorescence, which determines the free energy (DeltaG (P*A)) of the P(+)Q (A) (-) state relative to P*. Delayed fluorescence was used here to determine the free energy of P(+)Q (A) (-) in chromatophores. The emission intensity in chromatophores is two orders of magnitude greater than from isolated RCs largely due to the entropic effect of antenna pigments "drawing out" the excitation from the RC. The pH dependence of DeltaG (P*A) was almost identical to that of isolated RCs, in stark contrast with potentiometric redox titrations of Q(A). We considered that Q(A) might be reduced by disproportionation with QH(2) through the Q(B) site, so the titration actually reflects the quinone pool, giving the -60 mV/pH unit dependence expected for the Q/QH(2) couple. However, the parameters necessary to achieve a strong pH-dependence are not in good agreement with expected properties of Q(A) and Q(B). We also consider the possibility that the time scale of potentiometric titrations allows the reduced state (Q (A) (-) ) to relax to a different conformation that is accompanied by stoichiometric H(+) binding. Finally, we discuss the choice of parameters necessary for determining the free energy level of P(+)Q (A) (-) from delayed fluorescence emission from chromatophores of Rba. sphaeroides.     

Protein/lipid interaction in the bacterial photosynthetic reaction center: phosphatidylcholine and phosphatidylglycerol modify the free energy levels of the quinones

The role of characteristic phospholipids of native membranes, phosphatidylcholine (PC), phosphatidylglycerol (PG), and cardiolipin (CL), was studied in the energetics of the acceptor quinone side in photosynthetic reaction centers of Rhodobacter sphaeroides. The rates of the first, k(AB)(1), and the second, k(AB)(2), electron transfer and that of the charge recombination, k(BP), the free energy levels of Q(A)(-)Q(B) and Q(A)Q(B)(-) states, and the changes of charge compensating protein relaxation were determined in RCs incorporated into artificial lipid bilayer membranes. In RCs embedded in the PC vesicle, k(AB)(1) and k(AB)(2) increased (from 3100 to 4100 s(-1) and from 740 to 3300 s(-1), respectively) and k(BP) decreased (from 0.77 to 0.39 s(-1)) compared to those measured in detergent at pH 7. In PG, k(AB)(1) and k(BP) decreased (to values of 710 and 0.26 s(-1), respectively), while k(AB)(2) increased to 1506 s(-1) at pH 7. The free energy between the Q(A)(-)Q(B) and Q(A)Q(B)(-) states decreased in PC and PG (DeltaG degrees (Q)A-(Q)B(-->)(Q)A(Q)B- = -76.9 and -88.5 meV, respectively) compared to that measured in detergent (-61.8 meV). The changes of the Q(A)/Q(A)(-) redox potential measured by delayed luminescence showed (1) a differential effect of lipids whether RC incorporated in micelles or vesicles, (2) an altered binding interaction between anionic lipids and RC, (3) a direct influence of PC and PG on the free energy levels of the primary and secondary quinones probably through the intraprotein hydrogen-bonding network, and (4) a larger increase of the Q(A)/Q(A)(-) free energy in PG than in PC both in detergent micelles and in single-component vesicles. On the basis of recent structural data, implications of the binding properties of phospholipids to RC and possible interactions between lipids and electron transfer components will be discussed.     

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

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

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.     

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.