Local electrostatic field induced by the carotenoid bound to the reaction center of the purple photosynthetic bacterium Rhodobacter sphaeroides

Electroabsorption (EA) spectra were recorded in the region of the reaction center (RC) Qy absorption bands of bacteriochlorophyll (Bchl) and bacteriopheophytin, to investigate the effect of carotenoid (Car) on the electrostatic environment of the RCs of the purple bacterium Rhodobacter (Rb.) sphaeroides. Two different RCs were prepared from Rb. sphaeroides strain R26.1 (R26.1-RC); R26.1 RC lacking Car and a reconstituted RC (R26.1-RC+ Car) prepared by incorporating a synthetic Car (3,4-dihydrospheroidene). Although there were no detectable differences between these two RCs in their near infrared (NIR) absorption spectra at 79 and 293 K, or in their EA spectra at 79 K, significant differences were detected in their EA spectra at 293 K. Three nonlinear optical parameters of each RC were determined in order to evaluate quantitatively these differences; transition dipole-moment polarizability and hyperpolarizability (D factor), the change in polarizability upon photoexcitation (Deltaalpha), and the change in dipole-moment upon photoexcitation (Deltamu). The value of D or Deltaalpha determined for each absorption band of the two RC samples showed similar values at 77 or 293 K. However, the Deltamu values of the special pair Bchls (P) and the monomer Bchls absorption bands showed significant differences between the two RCs at 293 K. X-ray crystallography of the two RCs has revealed that a single molecule of the solubilizing detergent LDAO occupies part of the carotenoid binding site in the absence of a carotenoid. The difference in the value of Deltamu therefore represents the differential effect of the detergent LDAO and the carotenoid on P. The change of electrostatic field around P induced by the presence of Car was determined to be 1.7 x 10(5) [V/cm], corresponding to a approximately 10% change in the electrostatic field around P.     

Orientation-resolving pulsed electron dipolar high-field EPR spectroscopy on disordered solids: I. Structure of spin-correlated radical pairs in bacterial photosynthetic reaction centers

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.     

Electron transfer kinetics in photosynthetic reaction centers embedded in polyvinyl alcohol films

The coupling between electron transfer and protein dynamics has been studied at room temperature in isolated reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in polyvinyl alcohol (PVA) films of different water/RC ratios. The kinetic analysis of charge recombination shows that dehydration of RC-containing PVA films causes reversible, inhomogeneous inhibition of electron transfer from the reduced primary quinone acceptor (Q(A)(-)) to the secondary quinone Q(B). A more extensive dehydration of solid PVA matrices accelerates electron transfer from Q(A)(-) to the primary photooxidized electron donor P(+). These effects indicate that incorporation of RCs into dehydrated PVA films hinders the conformational dynamics gating Q(A)(-) to Q(B) electron transfer at room temperature and slows down protein relaxation which stabilizes the primary charge-separated state P(+)Q(A)(-). A comparison with analogous effects observed in trehalose-coated RCs suggests that protein motions are less severely reduced in PVA films than in trehalose matrices at comparable water/RC ratios.     

Selective detection of the structural changes upon photoreactions of several redox cofactors in photosystem II by means of light-induced ATR-FTIR difference spectroscopy

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was applied for the first time to detect the structural changes upon photoreactions of redox cofactors in photosystem II (PSII). The PSII-enriched membranes from spinach were adsorbed on the surface of a silicon prism, and FTIR measurements of various redox cofactors were performed for the same sample but under different conditions by exchanging buffers in a flow cell. Light-induced FTIR difference spectra upon redox reactions of the oxygen-evolving Mn cluster, the primary quinone electron acceptor QA, the redox-active tyrosine YD, the primary electron acceptor pheophytin, and the primary electron donor chlorophyll P680 were successively recorded in buffers including different redox reagents and inhibitors. All of these cofactors remained active in the PSII membranes on the silicon surface, and the resultant spectra were basically identical to those previously recorded by the conventional transmission method. These ATR-FTIR measurements enable accurate comparison between reactions of different active sites in a single PSII sample. The present results demonstrated that the ATR-FTIR spectroscopy is a useful technique for investigation of the reaction mechanism of PSII.     

Femtosecond spectroscopy of the primary charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation in the QY and Soret bands

The primary charge separation and electron-transfer processes of photosynthesis occur in the reaction center (RC). Isolated RCs of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus were studied at room temperature by using femtosecond transient absorption spectroscopy with selective excitation. Upon excitation in the Q(Y) absorbance band of the bacteriochlorophyll (BChl) dimer (P) at 865 nm, a 7.0 +/- 0.5 ps kinetic component was observed in the 538 nm region (Q(X) band of the bacteriopheophytin (BPheo)), 750 nm region (Q(Y) band of the BPheo), and 920 nm region (stimulated emission of the excited-state of P), indicating that this lifetime represents electron transfer from P to BPheo. The same time constant was also observed upon 740 nm or 800 nm excitation. A longer lifetime (300 +/- 30 ps), which was assigned to the time of reduction of the primary quinone, Q(A), was also observed. The transient absorption spectra and kinetics all indicate that only one electron-transfer branch is involved in primary charge separation under these excitation conditions. However, the transient absorption changes upon excitation in the Soret band at 390 nm reveal a more complex set of energy and electron-transfer processes. By comparison to studies on the RCs of the purple bacterium Rhodobacter sphaeroides, we discuss the possible mechanism of electron-transfer pathway dependence on excitation energy and propose a model of the Cf. aurantiacus RC that better explains the observed results.     

Structure of the charge separated state P865(+)Q(A)- in the photosynthetic reaction centers of Rhodobacter sphaeroides by quantum beat oscillations and high-field electron paramagnetic resonance: evidence for light-induced Q(A)- reorientation

The structure of the secondary radical pair, P865(+)Q(A)-, in fully deuterated and Zn-substituted reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides R-26 has been determined by high-time resolution and high-field electron paramagnetic resonance (EPR). A computer analysis of quantum beat oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the orientation of the various magnetic tensors of P865(+)Q(A)- with respect to a magnetic reference frame. The orientation of the g-tensor of P865(+) in an external reference system is adapted from a single-crystal W-band (95 GHz) EPR study [Klette, R.; T?rring, J. T.; Plato, M.; M?bius, K.; B?nigk, B.; Lubitz, W. J. Phys. Chem. 1993, 97, 2015-2020]. Thus, we obtain the three-dimensional structure of the charge separated state P865(+)Q(A)- on a nanosecond time scale after light-induced charge separation. Comparison with crystallographic data reveals that the position of the quinone is essentially the same as that in the X-ray structure. However, the head group of Q(A)- has undergone a 60 degrees rotation in the ring plane relative to its orientation in the crystal structure. Analysis suggests that the two different QA conformations are functionally relevant states which control the electron-transfer kinetics from Q(A)- to the secondary quinone acceptor QB. It appears that the rate-limiting step of this reaction is a reorientation of Q(A)- in its binding pocket upon light-induced reduction. The new kinetic model accounts for striking observations by Kleinfeld et al. who reported that electron transfer from Q(A)- to QB proceeds in RCs cooled to cryogenic temperature under illumination but does not proceed in RCs cooled in the dark [Kleinfeld, D.; Okamura, M. Y.; Feher, G. Biochemistry 1984, 23, 5780-5786].     

Transmembrane charge transfer in photosynthetic reaction centers: some similarities and distinctions

This mini review presents a general comparison of structural and functional peculiarities of three types of photosynthetic reaction centers (RCs)--photosystem (PS) II, RC from purple bacteria (bRC) and PS I. The nature and mechanisms of the primary electron transfer reactions, as well as specific features of the charge transfer reactions at the donor and acceptor sides of RCs are considered. Comparison of photosynthetic RCs shows general similarity between the core central parts of all three types, between the acceptor sides of bRC and PS II, and between the donor sides of bRC and PS I. In the latter case, the similarity covers thermodynamic, kinetic and dielectric properties, which determine the resemblance of mechanisms of electrogenic reduction of the photooxidized primary donors. Significant distinctions between the donor and acceptor sides of PS I and PS II are also discussed. The results recently obtained in our laboratory indicate in favor of the following sequence of the primary and secondary electron transfer reactions: in PS II (bRC): Р(680)(Р(870)) → Chl(D1)(В(А)) → Phe(bPhe) → Q(A); and in PS I: Р(700) → А(0А)/A(0B) → Q(A)/Q(B).     

Reversible binding of metal ions onto bacterial layers revealed by protonation-induced ATR-FTIR difference spectroscopy

The ability of microorganisms to adhere to abiotic surfaces and the potentialities of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy have been exploited to study protonation and heavy metal binding events onto bacterial surfaces. This work represents the first attempt to apply on bacteria the recently developed method known as perfusion-induced ATR-FTIR difference spectroscopy. Such a technique allows measurement of even slight changes in the infrared spectrum of the sample, deposited as a thin layer on an ATR crystal, while an aqueous solution is perfused over its surface. Solutions at different pH have been used for inducing protonation/deprotonation of functional groups lying on the surface of Rhodobacter sphaeroides cells, chosen as a model system. The interaction of Ni(2+) with surface protonable groups of this microorganism has been investigated with a double-difference approach exploiting competition between nickel cations and protons. Protonation-induced difference spectra of simple model compounds have been acquired to guide band assignment in bacterial spectra, thus allowing identification of major components involved in proton uptake and metal binding. The data collected reveal that carboxylate moieties on the bacterial surface of R. sphaeroides play a role in extracellular biosorption of Ni(2+), establishing with this ion relatively weak coordinative bonds.     

Light- and redox-dependent thermal stability of the reaction center of the photosynthetic Bacterium rhodobacter sphaeroides

Irreversible loss of the photochemical activity and damage of the pigments (bacteriochlorophyll [Bchl] monomer, Bchl dimer [P] and bacteriopheophytin) by combined treatment with intense and continuous visible light and elevated temperature have been studied in a deoxygenated solution of reaction center (RC) protein from the nonsulfur purple photosynthetic bacterium Rhodobacter sphaeroides. Both the fraction of RC in the charge-separated redox state (P+Q-, where Q is a quinone electron acceptor) and the degradation of the pigments showed saturation as a function of increasing light intensity up to 400 mW cm(-2) (488/515 nm) or 1100 microE m(-2) s(-1) (white light). The thermal denaturation curves of the RC in the P+Q- redox state demonstrated broadening and 10-20 degrees C shift to lower temperature (after 30-90 min heat treatment) compared with those in the PQ redox state. Similar but less striking behavior was seen for RC of other redox states (P+Q and PQ-) generated either by light or by electrochemical treatment in the dark. These experiments suggest that it is not the intense light per se but the changes in the redox state of the protein that are responsible for the increased sensitivity to photo- and heat damage. The RC with a charge pair (P+Q-) is more vulnerable to elevated temperature than the RC with (P+Q or PQ-) or without (PQ) a single charge. To reveal both the thermodynamic and kinetic aspects of the denaturation, a simple three-state model of coupled reversible thermal and irreversible kinetic transitions is presented. These effects may have relevance to the heat stability of other redox proteins in bioenergetics.     

Multichannel flash spectroscopy of the reaction centers of wild-type and mutant Rhodobacter sphaeroides: bacteriochlorophyllB-mediated interaction between the carotenoid triplet and the special pair

Multichannel flash spectroscopy (with microsecond time resolution) has been applied to carotenoid (Car)-containing and Car-less reaction centers (RC) of Rhodobacter sphaeroides with a view to investigate the interaction between the Car and its neighboring pigments at room temperature. Under neutral redox potential conditions, where the primary quinone acceptor (QA) is oxidized, the light-induced spectral changes in the 350-1000 nm region are attributed to the photochemical oxidation of the special pair (denoted here as P870), the generation of P870(+)QA(-), and the attendant electrochromism of adjacent chromophores. A bathochromic shift of <1 nm in the visible absorption region of Car reveals the sensitivity of Car to the P870 photooxidation. Under low redox potential conditions, where QA is reduced, P870 triplets (P870(+)) are formed. The time-resolved triplet-minus-singlet (TmS) spectrum of Car-less RC shows a deep bleaching at 870 nm, which belongs to P870(+), and additional (but smaller) bleaching at 800 nm; the entire spectrum decays at the same rate (with a lifetime of about 50 micros). The bleaching at 800 nm arises from the pigment interaction between P870(+) and the accessory bacteriochlorophylls on A and B branches (BA,B). In Car-containing RC, the TmS spectra of Car are accompanied by two smaller, negative signals--a sharp peak at 809 +/- 2 nm and a broad band at 870 nm--which decay at the same rate as the TmS spectrum of Car (ca 10 micros). The former is ascribed to the perturbation, by Car(+), of the absorption spectrum of BB; the latter, to the TmS spectrum of P870(+), a species that appears to be in approximate thermal equilibrium with Car(+). These assignments are consistent with the absorption-detected magnetic resonance spectra obtained by other workers at low temperatures.     

LIGHT-INDUCED ELECTRON TRANSPORT ACROSS SEMICONDUCTOR ELECTRODE/REACTION-CENTER FILM/ELECTROLYTE INTERFACES

Abstract— Reaction center (RC) complexes isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26 were dried as a film onto platinum and semiconductor (SnO₂) electrodes. The light-induced primary charge separation which occurs across the biological complex couples electrically with the SnO₂ but not with the metal electrode on the time scale of observation. As the working electrode in a two-electrode photoelectrochemical cell, RC-coated SnO₂ generated photovoltages as high as 80 mV and photocurrents as high as 0.5µA·cm² when exposed to light of λ >600nm. The number of quinone molecules per RC strongly influences the photovoltage and photocurrent observed. Photo-effects generated by RC electrodes persist after several days of storage; however, the kinetics and polarity of the effects are subject to change. The potential use of RC electrodes lies more as a new probe of photosynthetic electron transport rather than as a solar energy conversion device because modification to the RCs and their environment affect the electrical properties of the cell. An energy-level model is proposed to explain how the photoelectrochemical cell functions.     

Fast reversible electron transfer for photosynthetic reaction center from wild type Rhodobacter sphaeroides re-constituted in polycation sandwiched monolayer film

Direct reversible electron transfer for photosynthetic reaction center from wild type Rhodobacter sphaeroides re-constituted in polycation sandwiched monolayer film was observed in this work. The redox potential E0' = 0.46 V vs. NHE for first primary donor redox couple P/P+ was accurately measured from reversible CV or SWV peaks, which were quite close to those obtained from optic redox titration method. Reaction center (RC) in film was found re-constituted in such an ordered way that the orientation of RC favored the electron transfer in film. Thus, the protein electroactivity seems to be turned on in this artificial biomimic thin film. Furthermore, RC in the film features a photo-induced redox-peak fluctuation, suggesting an intact and functional state for RC in such film. Redox peaks were also found dependent of pH, implying a proton-coupled electron transfer occurring in film. Charge recombination was observed accompanied with change of electrochemical driving force. Electrochemical model assuming several classes of electroactive sites in the films on the electrode with a dispersion of standard potentials successfully fits SWV experimental data at different pulse height and frequency.     

Inter- and intraspecific variation in excited-state triplet energy transfer rates in reaction centers of photosynthetic bacteria

In protein-cofactor reaction center (RC) complexes of purple photosynthetic bacteria, the major role of the bound carotenoid (C) is to quench the triplet state formed on the primary electron donor (P) before its sensitization of the excited singlet state of molecular oxygen from its ground triplet state. This triplet energy is transferred from P to C via the bacteriochlorophyll monomer B(B). Using time-resolved electron paramagnetic resonance (TREPR), we have examined the temperature dependence of the rates of this triplet energy transfer reaction in the RC of three wild-type species of purple nonsulfur bacteria. Species-specific differences in the rate of transfer were observed. Wild-type Rhodobacter capsulatus RCs were less efficient at the triplet transfer reaction than Rhodobacter sphaeroides RCs, but were more efficient than Rhodospirillum rubrum RCs. In addition, RCs from three mutant strains of R. capsulatus carrying substitutions of amino acids near P and B(B) were examined. Two of the mutant RCs showed decreased triplet transfer rates compared with wild-type RCs, whereas one of the mutant RCs demonstrated a slight increase in triplet transfer rate at low temperatures. The results show that site-specific changes within the RC of R. capsulatus can mimic interspecies differences in the rates of triplet energy transfer. This application of TREPR was instrumental in defining critical energetic and coupling factors that dictate the efficiency of this photoprotective process.     

Influence of cardiolipin on the functionality of the Q(a) site of the photosynthetic bacterial reaction center

The effect of cardiolipin on the functionality of the Q(A) site of a photosynthetic reaction center (RC) was studied in RCs from the purple non-sulfur bacterium Rhodobacter sphaeroides by means of time-resolved absorbance measurements. The binding of the ubiquinone-10 to the Q(A) site of the RC embedded in cardiolipin or lecithin liposomes has been followed at different temperatures and phospholipid loading. A global fit of the experimental data allowed us to get quite reliable values of the thermodynamic parameters joined to the binding process. The presence of cardiolipin does not affect the affinity of the Q(A) site for ubiquinone but has a marked influence on the rate of P+QA(-) --> PQA electron transfer. The P+QA(-) charge recombination kinetics has been examined in liposomes made of cardiolipin/lecithin mixtures and in detergent (DDAO) micelles doped with cardiolipin. The electron-transfer rate constant increases upon cardiolipin loading. It appears that the main effect of cardiolipin on the electron transfer can be ascribed to a destabilization of the charge-separated state. Results obtained in micelles and vesicles follow the same titration curve when cardiolipin concentration evaluated with respect to the apolar phase is used as a relevant variable. The dependence of the P+QA(-) recombination rate on cardiolipin loading suggests two classes of binding sites. In addition to a high-affinity site (compatible with previous crystallographic studies), a cooperative binding, involving about four cardiolipin molecules, takes place at high cardiolipin loading.     

Type 1 reaction center of photosynthetic heliobacteria

The reaction center (RC) of heliobacteria contains iron-sulfur centers as terminal electron acceptors, analogous to those of green sulfur bacteria as well as photosystem I in cyanobacteria and higher plants. Therefore, they all belong to the so-called type 1 RCs, in contrast to the type 2 RCs of purple bacteria and photosystem II containing quinone molecules. Although the architecture of the heliobacterial RC as a protein complex is still unknown, it forms a homodimer made up of two identical PshA core proteins, where two symmetrical electron transfer pathways along the C2 axis are assumed to be equally functional. Electrons are considered to be transferred from membrane-bound cytochrome c (PetJ) to a special pair P800, a chlorophyll a-like molecule A0, (a quinone molecule A1) and a [4Fe-4S] center Fx and, finally, to 2[4Fe-4S] centers FA/FB. No definite evidence has been obtained for the presence of functional quinone acceptor A1. An additional interesting point is that the electron transfer reaction from cytochrome c to P800 proceeds in a collisional mode. It is highly dependent on the temperature, ion strength and/or viscosity in a reaction medium, suggesting that a heme-binding moiety fluctuates in an aqueous phase with its amino-terminus anchored to membranes.     

Variation of Ser-L223 hydrogen bonding with the QB redox state in reaction centers from Rhodobacter sphaeroides

Ser-L223 is close to ubiquinone (Q(B)) in the B-branch of the bacterial photosynthetic reaction center (bRC) from Rhodobacter (Rb) sphaeroides. Therefore, the presence of a hydrogen bond (H bond) between the two was naturally proposed from the crystal structure. The hydrogen bonding pattern of Q(B) from the light-exposed structure was studied by generating hydrogen atom coordinates based on the CHARMM force field. In the Q(B) neutral charge state (Q(B)(0)), no H bond was found between the oxygen of the OH group from Ser-L223 and the carbonyl oxygen of Q(B) that is distal to the non-heme iron. In the reduced state (Q(B)(-)), however, Ser-L213 was found to form an H bond with Q(B) only when Asp-L213 is protonated by more than 0.75 H(+). This indicates the significance of the protonation of Asp-L213 in forming an H bond between Ser-L223 and Q(B). We found that the driving force to form the H bond between Ser-L223 and Q(B) is enhanced by the positively charged Arg-L217. The calculated Q(B) redox potentials with or without this H bond discriminated two ET rates, which are close to the faster and slower time phases observed in UV-Vis and FTIR studies. Together with the calculated redox potential of the quinones, this H-bond formation could play a key role in conformational gating for the ET process from Q(A) to Q(B).     

Enthalpy/entropy driven activation of the first interquinone electron transfer in bacterial photosynthetic reaction centers embedded in vesicles of physiologically important phospholipids

The thermodynamics and kinetics of light-induced electron transfer in bacterial photosynthetic RCs are sensitive to physiologically important lipids (phosphatidylcholine, cardiolipin and phosphatidylglycerol) in the environment. The analysis of the temperature-dependence of the rate of the P(+)Q(A)(-)Q(B)-->P(+)Q(A)Q(B)(-) interquinone electron transfer revealed high enthalpy change of activation in zwitterionic or neutral micelles and vesicles and low enthalpy change of activation in vesicles constituted of negatively charged phospholipids. The entropy change of activation was compensated by the changes of enthalpy, thus the free energy change of activation ( approximately 500 meV) did not show large variation in vesicles of different lipids.     

Heterodimeric versus homodimeric structure of the primary electron donor in Rhodobacter sphaeroides reaction centers genetically modified at position M202

Using light-induced Fourier-transform infrared (FTIR) difference spectroscopy of the photo-oxidation of the primary donor (P) in chromatophores from Rhodobacter sphaeroides, we examined a series of site-directed mutants with His M202 changed to Gly, Ser, Cys, Asn or Glu in order to assess the ability of these side chains to ligate the Mg atom of one of the two bacteriochlorophylls (BChl) constituting P. In the P+QA-/PQA FTIR difference spectra of the mutants HG(M202), HS(M202), HC(M202) and HN(M202), the presence of a specific electronic transition at approximately 2650-2750 cm-1 as well as of associated vibrational (phase-phonon) bands at approximately 1560, 1480 and 1290 cm-1 demonstrate that these mutants contain a BChl/BChl homodimer like that in native reaction centers with the charge on P+ shared between the two coupled BChl. In contrast, the absence of all of these bands in HE(M202) shows that this mutant contains a BChl/bacteriopheophytin heterodimer with the charge localized on the single BChl, as previously determined for the mutant HL(M202). Furthermore, the spectra of the heterodimers HE(M202) and HL(M202) are very similar in the 4000-1200 cm-1 IR range. Perturbations of the 10a-ester and 9-keto carbonyl modes for both the P and P+ states are observed in the homodimer mutants reflecting slight variations in the conformation and/or in position of P. These perturbations are likely to be due to a repositioning of the dimer in the new protein cavity generated by the mutation.     

Conformational control of the Q(A) to Q(B) electron transfer in bacterial reaction centers: evidence for a frozen conformational landscape below -25 degrees C

The competition between the P(+)Q(A)(-) --> PQ(A) charge recombination (P, bacteriochlorophyll pair acting as primary photochemical electron donor) and the electron transfer to the secondary quinone acceptor Q(A)(-)Q(B) --> Q(A)Q(B)(-) (Q(A) and Q(B), primary and secondary electron accepting quinones) was investigated in chromatophores of Rb. capsulatus, varying the temperature down to -65 degrees C. The analysis of the flash-induced pattern for the formation of P(+)Q(A)Q(B)(-) shows that the diminished yield, when lowering the temperature, is not due to a homogeneous slowing of the rate constant k(AB) of the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer but to a distribution of conformations that modulate the electron transfer rate over more than 3 orders of magnitude. This distribution appears "frozen", as no dynamic redistribution was observed over time ranges > 10 s (below -25 degrees C). The kinetic pattern was analyzed to estimate the shape of the distribution of k(AB), showing a bell-shaped band on the high rate side and a fraction of "blocked" reaction centers (RCs) with very slow k(AB). When the temperature is lowered, the high rate band moves to slower rate regions and the fraction of blocked RCs increases at the expense of the high rate band. The RCs that recombine from the P(+)Q(A)Q(B)(-) state appear temporarily converted to a state with rapid k(AB), indicating that the stabilized state described by Kleinfeld et al. (Biochemistry 1984, 23, 5780-5786) is still accessible at -60 degrees C.