Orientation of the primary quinone of bacterial photosynthetic reaction centers contined in chromatophore and reconstituted membranes

The orientation of the reaction center bacteriochlorophyll dimer, (BChl)₂, and primary quinone, Q_I, has been studied by EPR in chromatophores of Rhodopseudomonas sphaeroides R26 and Chromatium vinosum and in the reconstituted membrane multilayers of the isolated Rps. sphaeroides reaction center protein. The similarity in the angular dependence of the (BChl)₂ triplet and Q_I?Fe²⁺ signals in the chromatophore and reconstituted reaction center membrane multilayers indicates that the reaction center is similarly oriented in both native and model membranes. The principle magnetic axes of the (BChl)₂ triplet are found to lie with the x direction approximately parallel to the plane of the membrane surface, and the z and y directions approx. 10–20° away from the plane of the membrane surface and membrane normal, respectively. The Q_I?Fe²⁺ signals are found to have the g 1.82 component positioned perpendicular to the plane of the membrane surface, with an orthogonal low-field transition (at g 1.68 in Rps. Sphaeroides and at g 1.62 in C. vinosum) lying parallel to the plane of the membrane surface. The orientation of Q_I was determined by the angular dependence of this signal in Fe²⁺-depleted reaction center reconstituted membrane multilayers, and it was found to be situated most likely with the plane of the quinone ring perpendicular to the plane of the membrane surface.     

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.Abbreviations Bchl bacteriochlorophyll - Bpheo bacteriopheophytin - D electron donor to P⁺ - P bacteriochlorophyll dimer - Q quinone acceptor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ₆ ubiquinone-30     

Kinetics and yields of bacteriochlorophyll fluorescence: redox and conformation changes in reaction center of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Induction of the bacteriochlorophyll fluorescence under rectangular shape of intense laser diode illumination (1 W cm(-2), 808 nm) was measured over wide time range from 10 mus to 4 s in whole cells, chromatophore and isolated reaction center protein of wild type and carotenoid-less mutant (R-26.1) of purple photosynthetic bacterium Rhodobacter sphaeroides. While the antenna-containing species showed large and positive variable fluorescence (F (v)) to initial fluorescence (F (0)) (F (v)/F (0) approximately 4.5 in whole cells), the isolated RC had negative change (F (v)/F (0) approximately -0.6) during photochemistry. In chromatophore from R-26.1, only seven times higher rate was measured than in isolated reaction center under identical experimental conditions. The enhancement effect of large antenna on the rate of photochemistry in chromatophore was partially compensated by the favorable pigment absorption properties in isolated RC. The transition from membrane bound to isolated form of the reaction center was probed by titration of zwitterionic detergent LDAO in chromatophore, and at 0.03% LDAO concentration, sharp change of the variable fluorescence was observed. The sudden drop was explained by the formation of LDAO micelles. After the photochemical phase, additional change of fluorescence yield could be observed in isolated RC considered as manifestation of long-living conformations of the trapped redox states of the protein characterized by non-exponential kinetics. Strong support was provided for use of the fluorescence induction to track structural and conformation changes at their earliest phases in chromatophores and isolated reaction centers.     

Charge separation in Rhodobacter sphaeroides mutant reaction centers with increased midpoint potential of the primary electron donor

Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll B_A at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P⁺H _A ^? state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P⁺B _A ^? H_A and P⁺B_AH _A ^? states. The data give grounds for assuming that the value of the energy difference between the states P*, P⁺B _A ^? H_A, and P⁺B_AH _A ^? at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll B_A is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P⁺B _A ^? state with respect to P*.     

Photooxidase activity of Rhodospirillum rubrum chromatophores and reaction center complexes. The role of non-cyclic electron transfer in generation of the membrane potential

The mechanism of light-induced O₂ uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O₂ uptake by the extracted chromatophores and stimulated the O₂ uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O₂. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O₂ upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl-p-phenylenediamine (Me₄Ph(NH₂)₂) or N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB^?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD⁺ or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O₂, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me₄Ph(NH₂)₂ to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me₄Ph(NH₂)₂ and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me₄Ph(NH₂)₂ and TMPD are different.     

P+HA- charge recombination reaction rate constant in Rhodobacter sphaeroides reaction centers is independent of the P/P+ midpoint potential.

The kinetics of the P+HA- (oxidized donor, reduced bacteriopheophytin acceptor) recombination reaction was measured in a series of reaction center mutants of Rhodobacter sphaeroides with altered P/P+ midpoint potentials between 410 and 765 mV. The time constant for P+HA- recombination was found to range between 14 and 26 ns and was essentially independent of P/P+ midpoint potential. Previous work has shown that the time constant for initial electron transfer in these mutants at room temperature is also only weakly dependent on the P/P+ midpoint potential, ranging from about 2.5 ps to about 50 ps. These results, taken together, imply that heterogeneity in the P/P+ midpoint potential within the reaction center population is not likely the dominant cause of the substantial kinetic complexity observed in the decay of the excited singlet state of P on the picosecond to nanosecond time scale. In addition, the pathway of P+HA- decay appears to be direct or via P+BA- rather than proceeding back through P, even in the highest-potential mutant, as is evident from the fact that the rate of P+HA- recombination is unaltered by pushing P+HA- much closer to P in energy. Finally, the midpoint potential independence of the P+HA- recombination rate constant suggests that the slow rate of P+HA- recombination arises from an inherent limitation in the maximum rate of this process rather than because it occurs in the inverted region of a classical Marcus rate vs free energy curve.     

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 (Q₁Q₁₁) 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 Q₁₁ receives an electron via Q₁ to form a stable ubisemiquinone anion (Q?^?₁₁); the second flash generates Q?^?₁. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H⁺, to produce Q₁₁H₂. 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 Q?^?₁₁. 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.     

Mössbauer spectroscopy studies of photosynthetic reaction centers from Rhodopseudomonas sphaeroides R-26

⁵⁷Fe Mössbauer spectroscopy measurements on reaction centers differing in ubiquinone content, detergent, oxidation state, or the presence of o-phenanthroline all show a single quadrupole doublet of similar splitting (ΔE_Q), center shift (δ) and temperature dependence. The results are indicative of high-spin Fe²⁺ with an approximately invariant first coordination sphere. A crystal field model with strong electron delocalization can account for the temperature dependence of ΔE_Q, but further data are needed to achieve a unique parameterization.     

Characterization of a semi-stable,charge-separated state in reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides, subjected to continuous illumination in the presence of an inhibitor of the Q_A to Q_B electron transfer, the oxidation of P870 consisted of several kinetic phases with a fast initial reaction followed by very slow accumulation of P870⁺ with a halftime of several minutes. When the light was turned off, a phase of fast charge recombination was followed by an equally slow reduction of P870⁺. In reaction centers depleted of Q_B, where forward electron transfer from Q_A is also prevented, the slow reactions were also observed but with different kinetic properties. The kinetic traces of accumulation and decay of P870⁺ could be fitted to a simple three-state model where the initial, fast charge separation is followed by a slow reversible conversion to a long-lived, charge-stabilized state. Spectroscopic examination of the charge-separated, semi-stable state, using optical absorbance and EPR spectroscopy, suggests that the unpaired electron on the acceptor side is located in an environment significantly different from normal. The activation parameters and enthalpy and entropy changes, determined from the temperature dependence of the slow conversion reaction, suggest that this might be coupled to changes in the protein structure of the reaction centers, supporting the spectroscopic results. One model that is consistent with the present observations is that reaction centers, after the primary charge separation, undergo a slow, light-induced change in conformation affecting the acceptor side. This revised version was published online in June 2006 with corrections to the Cover Date.     

Turnover of ubiquinone-0 at the acceptor side of photosynthetic reaction center

The steady-state operation of photosynthetic reaction center from Rhodobacter sphaeroides was investigated by measuring the rate of cytochrome photo-oxidation under intensive continuous illumination (808 nm, 5 W cm(-2)). The native quinone UQ(10) in Q(B) binding site of the reaction center was substituted by tailless UQ(0) and the binding parameters and the turnover rate of the UQ(0) was studied to test the recently discovered light-intensity dependent acceptor side effect (Gerencsér and Maróti 2006). The binding parameters of UQ(0) (k (on) = 2.1 x 10(5) M(-1) s(-1) and k (off) = 100 s(-1)) were characteristic to the RC exposed to high light-intensity. The dissociation constant (K (D) = 480 muM) determined under high light intensity is 2-3 times larger than that determined from flash-experiments. The light-intensity dependent acceleration of cytochrome turnover measured on reaction center of inhibited proton binding was independent of the type of the quinone and was sensitive only to the size ("pressure") of the quinone pool. The dissociation constants of different types of semiquinones show similarly high (several orders of magnitude) increase in the modified conformation of the Q(B) binding pocket due to high intensity of illumination. This result indicates the exclusive role of the quinone headgroup in the binding of semiquinone to different conformations of the protein.     

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).     

Electron transfer in deuterated reaction centers of Rhodobacter sphaeroides at 90 K according to femtosecond spectroscopy data

The primary act of charge separation was studied in P⁺B_A ^– and P⁺H_A ^– states (P, primary electron donor; B_A and H_A, primary and secondary electron acceptor) of native reaction centers (RCs) of Rhodobacter sphaeroides R-26 using femtosecond absorption spectroscopy at low (90 K) and room temperature. Coherent oscillations were studied in the kinetics of the stimulated emission band of P* (935 nm), of absorption band of B_A ^– (1020 nm) and of absorption band of H_A (760 nm). It was found that in native RCs kept in heavy water (D₂O) buffer the isotopic decreasing of basic oscillation frequency 32 cm ^–1 and its overtones takes place by the same factor 1.3 in the 935, 1020, and 760 nm bands in comparison with the samples in ordinary water H₂O. This suggests that the femtosecond oscillations in RC kinetics with 32 cm ^–1 frequency may be caused by rotation of hydrogen-containing groups, in particular the water molecule which may be placed between primary electron donor P_B and primary electron acceptor B_A. This rotation may appear also as high harmonics up to sixth in the stimulated emission of P*. The rotation of the water molecule may modulate electron transfer from P* to B_A. The results allow for tracing of the possible pathway of electron transfer from P* to B_A along a chain consisting of polar atoms according to the Brookhaven Protein Data Bank (1PRC): Mg(P_B)-N-C-N(His M200)-HOH-O = B_A. We assume that the role of 32-cm ^–1 modulation in electron transfer along this chain consists of a fixation of electron density at B_A ^– during a reversible electron transfer, when populations of P* and P⁺B_A ^– states are approximately equal.     

Effects of oxygen on the dark recombination between photoreduced secondary quinone and oxidized bacteriochlorophyll in Rhodobacter sphaeroides reaction centers

The influence of duration of exposure to actinic light (from 1 sec to 10 min) and temperature (from 3 to 35^°C) on the temporary stabilization of the photomobilized electron in the secondary quinone acceptor (Q_B) locus of Rhodobacter sphaeroides reaction centers (RC) was studied under aerobic or anaerobic conditions. Optical spectrophotometry and ESR methods were used. The stabilization time increased significantly upon increasing the exposure duration under aerobic conditions. The stabilization time decreased under anaerobic conditions, its dependence on light exposure duration being significantly less pronounced. Generation of superoxide radical in photoactivated aerobic samples was revealed by the ESR method. Possible interpretation of the effects is suggested in terms of interaction between the semiquinone Q_B with oxygen, the interaction efficiency being determined by the conformational transitions in the structure of RC triggered by actinic light on and off.     

Kinetics and thermodynamics of the P870+Q−A → P870+Q−B reaction in isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides

The temperature dependences of the P870⁺Q^?_A → P870Q_A and P870⁺Q^?_B → P870Q_B recombination reactions were measured in reaction centers from Rhodopseudomonas sphaeroides. The data indicate that the P870⁺Q^?_B state decays by thermal repopulation of the P870⁺Q^?_A state, followed by recombination. ΔG° for the P870⁺Q^?_A → P870⁺Q^?_B reaction is ?6.89 kJ · mol^?1, while ΔH° = ?14.45 kJ · mol^?1 and ?TΔS° = + 7.53 kJ · mol^?1. The activation ethalpy, $\text{H}{}^3$, for the P870⁺Q^?_A Δ P870⁺Q^?_B reaction is +56.9 kJ · mol^?1, while the activation entropy is near zero. The results permit an estimate of the shape of the potential energy curve for the P870⁺Q^?_A → P870⁺Q^?_B electron transfer reaction.     

Kinetic study of PF and CarT states in the LM subunit purified from the wild-type Rhodobacter sphaeroides reaction centers

The kinetics of absorbance changes related to the charge-separated state, P^F, and to the formation and decay of the carotenoid triplet state (Car^T) were studied in the LM reaction center subunit isolated from a wild-type strain of the purple bacterium Rhodobacter sphaeroides (strain Y). The P^F lifetime is lengthened (20±1.5 ns) in the LM complex as compared to the intact reaction centers (11±1 ns). The yield of the carotenoid triplet formation is higher (0.28±0.01) in the LM complex than in native reaction centers. We interpret our results in terms of perturbations of a first-order reaction connecting the singlet and the triplet state of the radical-pair state. Our results, together with those of a recent work (Agalidis, I., Nuijs, A.M. and Reiss-Husson, F. (1987) Biochim. Biophys. Acta (in press)) are consistent with a high I to Q_A electron transfer rate in this LM subunit, which is metal-depleted.The LM complex is considerably more sensitive than the reaction centers to photooxidative damage in the presence of oxygen. This is not readily accounted for simply by the higher carotenoid triplet yield, and may suggest a greater accessibility of the internal structures in the absence of the H-subunit.The lifetime of the carotenoid triplet decay (6.4±0.3 s) in the LM subunit is unchanged compared to the native reaction centers.Abbreviations BChl bacteriochlorophyll - Bph bacteriopheophytin - Car carotenoid - Chl chlorophyll - cyt cytochrome - L, M and H subunits light, medium and heavy subunits of the reaction center complex - P^R triplet electronic state of the primary electron donor - P; Q_A the first stable electron acceptor, a bound quinone - RC reaction center - LDAO lauryldimethylamine N-oxide - SDS sodium dodecyl sulfate - UQ ubiquinone This paper is published in our new format. All future authors are requested to follow our new instructions (see Photosynthesis Research 10:519–526, 1986)—Editor.     

Different temperature dependencies of the charge recombination reaction in photoreaction centers isolated from different bacterial species

We compared the temperature dependency of the rate of the charge recombination reaction in photoreaction centers isolated from Ectothiorhodospira sp. and from Rhodospirillum rubrum G9. We also examined the temperature dependency of the bandwidth and peak wavelength of their far-red absorption band. In both preparations, the peak wavelength and the bandwidth vary monotonically with temperature between 80 and 300 K. However, the rate of the charge recombination reaction has a quite different temperature dependency. In the preparation from R. rubrum, the reaction is accelerated 5-fold in a typical sigmoidal fashion as the temperature is lowered from 300 to 80 K. In the preparation from Ectothiorhodospira sp., the reaction is accelerated monotonically only about 1.5-fold in the same temperature range. At temperatures below 100 K, the rates are similar in the two preparations. We interpret the temperature dependency of the charge recombination reaction in terms of an activationless electron-transfer model formulated by Jortner (Jortner, J. (1980) Biochim. Biophys. Acta 394, 193–230). The minimal model provides a good fit for the temperature dependency of charge recombination in the preparation from Ectothiorhodospira sp. However, to fit the temperature dependency of the R. rubrum preparation with the same model, we must further postulate that the electronic coupling factor varies with temperature in this preparation. We find that, in both preparations, the temperature dependency of the far-red absorption bandwidth is consistent with the assumption that similar vibrational modes are involved in electron transfer and in electronic excitation.     

Magnetophotoselection of the triplet state of reaction centers from Rhodopseudomonas sphaeroides R-26

Reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, give rise to large triplet state EPR signals upon illumination at low temperature (11 K). Utilizing monochromatic polarized light to generate the EPR spectra (magnetophotoselection) we have shown that the intensities of the observed triplet signals are strongly dependent upon the wavelength and polarization direction of the excitation. These data can be used to calculate the orientations of the excited transition moments with respect to each other and with respect to the triplet state principal magnetic axes system. Our quantitative approach is to follow the procedure outlined in a previous publication (Frank, H.A., Friesner, R., Nairn, J.A., Dismukes, G.C. and Sauer, K. (1979) Biochim. Biophys. Acta 547, 484–501) where computer simulations of the observed triplet state spectra were employed.The results presented in the present work indicate that the transition moment at 870 nm which is associated with the bacteriochlorophyll ‘special pair’ lies almost entirely along one of the principal magnetic axes of the triplet state. Also, the 870 nm transition moment makes an angle of approx. 60° with the 546 nm transition moment which is associated with a bacteriopheophytin. This latter result is in agreement with previous photoselection studies on the same bacterial species (Vermeglio, A., Breton, J., Paillotin, G. and Cogdell, R. (1978) Biochim. Biophys. Acta 501, 514–530).     

The interaction of the reaction center secondary quinone with the ubiquinone-cytochrome c2 oxidoreductase in Rhodopseudomonas sphaeroides chromatophores

(1) Two populations of reaction centers in the chromatophore membrane can be distinguished under some conditions of initial redox poise (300 mV < E_h < 400 mV): those which transfer a reducing equivalent after the first flash from the secondary quinone (Q_II) of the reaction center to cytochrome b of the ubiquinone-cytochrome c₂ oxidoreductase; and those which retain the reducing equivalent on Q^?_II until a second flash is given. These two populations do not exchange on a time scale of tens of seconds. (2) At redox potentials higher than 400 mV, Q^?_II generated after the first flash is no longer able to reduce cytochrome b-560 even in those reaction centers associated with an oxidoreductase. Under these conditions, doubly reduced Q_II generated by a second flash is required for cytochrome b reduction, so that the Q_II effectively functions as a two-electron gate into the oxidoreductase at these high potentials. (3) At redox potentials below 300 mV, although the two populations of Q_II are no longer distinguishable, cytochrome b reduction is still dependent on only part of the reaction center population. (4) Proton binding does not oscillate under any condition tested.     

Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280–320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P⁺(Q_AQ_B)^– state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (Q_B ^–) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the Q_B binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the Q_AQ_B quinone acceptor complex by decreasing the binding affinity of Q_B and shifting the electron equilibration from Q_AQ_B ^– to Q_A ^–Q_B. The inhibitory effect is likely to be caused by modification of the protein environment around the Q_B binding pocket and mediated by the semiquinone form of Q_B. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the Q_AQ_B acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.Abbreviations Bchl bacteriochlorophyll - P Bchl dimer - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - RC reaction center - UV-B ultraviolet-B