P. P. Knox

Self-Regulation Phenomena Applied to Bacterial Reaction Centers 2. Nonequilibrium Adiabatic Potential: Dark and Light Conformations Revisited

Experimental and theoretical results in support of nonlinear dynamic behavior of photosynthetic reaction centers under light-activated conditions are presented. Different conditions of light adaptation allow for preparation of reaction centers in either of two different conformational states. These states were detected both by short actinic flashes and by the switching of the actinic illumination level between different stationary state values. In the second method, the equilibration kinetics of reaction centers isolated from Rhodobacter sphaeroides were shown to be inherently biphasic. The fast and slow equilibration kinetics are shown to correspond to electron transfer (charge separation) at a fixed structure and to combined electron-conformational transitions governed by the bounded diffusion along the potential surface, respectively. The primary donor recovery kinetics after an actinic flash revealed a pronounced dependence on the time interval (deltat) between cessation of a lengthy preillumination of a sample and the actinic flash. A pronounced slow relaxation component with a decay half time of more than 50 s was measured for deltat > 10 s. This component corresponds to charge recombination in reaction centers for which light-induced structural changes have not relaxed completely before the flash. The amplitude of this component depended on the conditions of the sample preparation, specifically on the type of detergent used in the preparation. The redox potential parameters as well as the structural diffusion constants were estimated for samples prepared in different ways.

The influence of structural-dynamic organization of RC from purple bacterium rhodobacter sphaeroides on picosecond stages of photoinduced reactions

Effects of the hydrogen bond network on the rate constants of energy migration (km), charge separation (ke), electron transfer to QA (kQ) and P+I- recombination in RC of Rhodobacter sphaeroides were analysed in control and modified RC preparations at different temperatures. Modification of RC were made by the addition of 40% v/v DMSO. The rate constants km, ke, kQ were evaluated from pump-and-probe measurements of the absorption difference kinetics at 665 nm corresponding to BPhL- formation and subsequent electron transfer to QA. For the investigation of P+I- recombination a primary quinone acceptor was pre-reduced in the dark by adding of 1 mg/ml of dithionite and 1 mM sodium ascorbate. Recombination kinetics were measured at 665 and 870 nm. The numerical analysis of the temperature dependence of ke and kQ was performed on the basis of the model proposed by Kakitani and Kakitani (T. Kakitani and H. Kakitani (1981), Biochim. Biophys. Acta, 635, 498-514). It was found that: (a) in control samples the molecular rate constants km, ke and kQ were about (3.4 ps)-1, (4.5 ps)-1 and (200 ps)-1, respectively; (b) under modification by DMSO these rates decrease up to (5.3 ps)-1, (10.3 ps)-1 and (500 ps)-1, respectively; (c) as the temperature drops from 300 K to 77 K the rate constant km decreases by 1.8 times in control and by 3.2 times in modified samples. In contrast to the observed km changes the increase in ke and kQ values by 2 and more times under cooling was found in control and modified RC; (d) in control preparations with QA acceptor pre-reduced in the dark the lowering of the temperature caused the increase in the time of P+I- recombination from 10 to 20 ns. After DMSO modification the kinetics of charge recombination in RC was biexponential at room temperature with tau=10 ns and tau1=0.8 ns, and at 77 K with tau=20 ns and tau1=0.6 ns, correspondingly. The results obtained reveal that in RC isolated from Rb. sphaeroides the processes of energy migration, charge separation, electron transfer to QA and ion-radical pair P+I- recombination depend on the state of hydrogen bonds of water-protein structure. Fast relaxation processes in RC structure including polarization of H-containing molecules in the surrounding of electron carriers can accept electron energy dissipated at the initial steps of energy and electron transfer. Copyright 1998 Elsevier Science B.V. All rights reserved.

Modification of protein hydrogen bonds influences the efficiency of picosecond electron transfer in bacterial photosynthetic reaction centers

Picosecond absorption spectroscopy was used to monitor laser‐induced oxidation‐reductions of reaction center (RC) bacteriochlorophyll (P) and bacteriopheophytin (I) in Rhodopseudomonas sphaeroides RC preparations on exposure to different chemicals. The D2O isotope substitution of H2O or partial substitution of water by organic solvents (ethylene glycol, glycerol, propylene glycol, dimethyl sulfoxide) causes the appearance of a fast, nanosecond component of P+ reduction, the result of an increased probability of recombination of the primary ion‐radical products P+I− → PI. The effect is accompanied by a noticeable slowing down of electron transfer from photoreduced bacteriopheophytin to the primary quinone acceptor QA. The effect of the organic solvents, known as cryoprotectors, is correlated with their degree of hydrophobicity, i.e. the ability to penetrate the RC protein and interact with bound water and protein hydrogen bonds. The conclusion drawn from the data is that the dielectric relaxation processes through which the intermediate energy levels of the carriers in the PIQA system are lowered to levels necessary for the stabilization of the photochemically separated charges proceed with the involvement of protons of the nearest water‐protein surrounding of the RC pigments and electron transport cofactors.

Effects of mutual influence of photoinduced electron transitions and slow structural rearrangements in bacterial photosynthetic reaction centers

We describe the phenomenon of light-induced structural transformations in the reaction centers (RC) of photosynthetic bacteria which makes self-regulation of the RC charge separation efficiency possible. The nature of the effect is that the light-driven electron transfer (ET) between the RC redox-cofactors causes structural changes in the protein-cofactors system and this in turn affects the ET kinetics. If the electron-conformation interaction is strong enough, then such self-regulation gives birth to a new RC conformational state of enhanced charge separation efficiency. We show experimental results of stationary and kinetic absorbance change characteristics under different photoexcitation conditions, indicating structural rearrangements on a rather long (minutes) time scale, mainly within the secondary acceptor binding pocket. To simplify the description, in constructing a theory of structure-function reorganization in the RC we employ the adiabatic approach. Final expressions enable us to make qualitative comparison with experimentally observed kinetics of the fast and slow stages of ‘free’ and ‘structurally controlled’ electron relaxation, respectively.

Relaxation mechanism of molecular systems containing hydrogen bonds and free energy temperature dependence of reaction of charges recombination within Rhodobacter sphaeroides RC

The mechanism of protonic relaxation is shown to take place in molecular systems containing hydrogen bonds. The mechanism arises from the proton redistribution between two stable states on hydrogen bond lines. This redistribution occurs due to changes of hydrogen bond double well potential, brought about by changes of the electronic state of a molecular system. A characteristic of the relaxation process is that it takes place due to the proton tunneling along hydrogen bonds. The charge shift causes electrostatic potential variation in the electron localization area, which leads to the shift of molecular system energy levels and changes its redox potential. The characteristic time of the protonic relaxation is shown to depend essentially on hydrogen bond bending strain, which increases with the temperature rise and decreases abruptly the efficiency of proton redistribution. Hence, the rate of this process decreases with temperature, in contrast to the activation process. This relaxation process is shown to be responsible for energetic characteristics of recombination reaction P+QA--->PQA (free energy difference DeltaG and/or reorganization energy lambda), temperature dependence in Rhodobacter sphaeroides RC.

Energetics and mechanisms of high efficiency of charge separation and electron transfer processes in Rhodobacter sphaeroides reaction centers

Effects of environmental changes due to D(2)O/H(2)O substitution and cryosolvent addition on the energetics of the special pair and the rate constants of forward and back electron transfer reactions in the picosecond-nanosecond time domain have been studied in isolated reaction centers (RC) of the anaxogenic purple bacterium Rhodobacter sphaeroides. The following results were obtained: (i). replacement of H(2)O by D(2)O or addition of either 70% (v/v) glycerol or 35% (v/v) DMSO do not influence the absorption spectra; (ii). in marked contrast to this invariance of absorption, the maxima of fluorescence spectra are red shifted relative to control by 3.5, 6.8 and 14.5 nm for RCs suspended in glycerol, D(2)O or DMSO, respectively; (iii). D(2)O/H(2)O substitution or DMSO addition give rise to an increase of the time constants of charge separation (tau(e)), and Q(A)(-) formation (tau(Q)) by a factors of 2.5-3.1 and 1.7-2.5, respectively; (iv). addition of 70% glycerol is virtually without effect on the values of tau(e) and tau(Q); (v). the midpoint potential E(m) of P/P(+) is shifted by about 30 and 45 mV towards higher values by addition of 70% glycerol and 35% DMSO, respectively, but remains invariant to D(2)O/H(2)O exchange; and (vi). an additional fast component with tau(1)=0.5-0.8 ns in the kinetics of charge recombination P(+)H(A)(-)-->P*(P)H(A) emerges in RC suspensions modified either by D(2)O/H(2)O substitution or by DMSO treatment. The results have been analysed with special emphasis on the role of deformations of hydrogen bonds for the solvation mechanism of nonequilibrium states of cofactors. Reorientation of hydrogen bonds provides the major contribution of the very fast environmental response to excitation of the special pair P. The Gibbs standard free energy gap between 1P* and P(+)B(A)(-) due to solvation is estimated to be approximately 70, 59 and 48 meV for control, D(2)O- and DMSO-treated RC samples, respectively.

Influence of chemical chaperones on the properties of lysozyme and the reaction center protein from Rhodobacter sphaeroides

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. The chosen chemical chaperones differ strongly in their structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described in the framework of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic core) moieties; this provides for additional regulation of the functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in the denaturation temperature and an increase in the amplitude of equilibrium fluctuations, allowing it to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in this case. Therefore, methylresorcinol does not affect the functional properties of the RC protein. It is concluded that different chaperones at the same concentration as well as one and the same chaperone at different concentrations produce protein 3D structures differing in dynamic and functional characteristics.

Photo-induced electron transport and water state in Rhodospirillum rubrum chromatophores.

It is shown that in bacterial chromatophores the pronounced changes in the free water content with a proton spin-spin relaxation time (T2) of 10(-3)--10(-2) s does not influence the efficiency of electron transfer from the photosynthetic reaction centre to the membrane pool of secondary acceptors. An abrupt inhibition of this process occurs only after the loss of the water with faster proton spin-spin relaxation time (T2 of 10(-4) s). The process is reversible. The water fraction in question is obviously bound to the chromatophore proteins and forms the primary hydration layer.

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 (QB) 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 QB with oxygen, the interaction efficiency being determined by the conformational transitions in the structure of RC triggered by actinic light on and off.

Dipyridamole and its derivatives modify the kinetics of the electron transport in reaction centers from Rhodobacter sphaeroides

A well known vasodilator dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d]pyrim idine, and its derivatives have recently been shown as potential co-activators (modulators) in the phenomenon of multidrug resistance (MDR) in cancer therapy. They inhibit the specific function of a transmembrane P-glycoprotein responsible for the ex-flux of anti-cancer drugs from tumor cells. To clarify molecular mechanisms of the anti-MDR activity of DIP and its two derivatives, RA25 and RA47, we have studied their effects on electron transport in reaction centers (RC) from purple photosynthetic bacteria Rb. sphaeroides, using RC as a model system. Increasing concentrations of DIP and RA47 progressively accelerate the back electron transfer from the primary quinone acceptor QA to the bacteriochlorophyll dimer Bchl2 (Bchl2+ -QA- recombination). In the absence of o-phenantroline, when both quinone acceptors QA and QB are involved in the electron transport, RA47 is more effective than DIP. DIP stabilizes the electron on the secondary quinone acceptor QB, the effect manifested as the retardation of Bchl2+ -QB- recombination. Effects of RA25 are negligible in all cases. The drugs are proposed to change the electron transport affecting the RC structural dynamics and the stabilization of the electron on quinone acceptors through modification of H-bonds in the system.