N. I. Zakharova

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.

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.

Effect of replacing the primary quinone by different species on the ultrafast photosynthetic electron transfer in bacterial reaction centres.

Using picosecond absorption spectroscopy it has been shown that in Rhodobacter sphaeroides reaction centres the substitution of the primary quinone acceptor (QA), ubiquinone-10, by other quinone species (with redox potentials higher or lower than that of ubiquinone-10) has essentially no modifying effect on the reaction centre protein. The molecular relaxation processes that accompany the localization and stabilization of a photo-excited electron on the intermediate acceptor, bacteriopheophytin (I), are not affected, although the subsequent transfer of the electron from I to QA is slowed down. Consequently, this leads to a lower quantum efficiency of high rate of direct I-----QA reaction is normally due to the specificity of the primary quinone species and its binding site in the reaction centre protein which provide optimum steric and chemical conditions for an effective interaction between I and QA.

Effects of Extraction of the H-Subunit from Rhodobacter sphaeroides Reaction Centers on Relaxation Processes Associated with Charge Separation

Effects of extraction of the H-subunit from Rhodobacter sphaeroides photosynthetic reaction centers (RC) on the characteristics of the photoinduced conformational transition associated with electron transfer between photoactive bacterio-chlorophyll and primary quinone acceptor were studied. Extraction of the H-subunit (i.e., the subunit that is not directly bound to electron transfer cofactors) was found to have a significant effect on the dynamic properties of the protein–pigment complex of the RC, the effect being mediated by modification of parameters of the relaxation processes associated with charge separation.

Effects of Oxygen, Heavy Water, and Glycerol on Electron Transfer in the Acceptor Part of Rhodobacter sphaeroides Reaction Centers

The kinetics of electron transfer between primary and secondary quinone acceptors of the photosynthetic reaction center (RC) of the purple bacterium Rhodobacter sphaeroides wild type was studied at the wavelengths 400 and 450 nm. It was shown that removing of molecular oxygen from RC preparations slowed down the fast phase of the process from 4–4.5 µsec to tens of microseconds. Similar effects were observed after the incubation of RC in heavy water for 72 h or glycerol addition (90% v/v) to RC preparations. The observed effects are interpreted in terms of the influence of these agents on the hydrogen bond system of the RC. The state of this system can determine the formation of different RC conformations that are characterized by different rates of electron transfer between quinone acceptors.

Temporary stabilization of electron on quinone acceptor side of reaction centers from the bacterium Rhodobacter sphaeroides wild type and mutant SA(L223) depending on duration of light activation.

The dark reduction of photooxidized bacteriochlorophyll (P+) by photoreduced secondary quinone acceptor (QB–) in isolated reaction centers (RC) from the bacterium Rhodobacter sphaeroides wild type and mutant strain SA(L223) depending on the duration of light activation of RC was studied. The kinetics of the dark reduction of P+ decreased with increasing light duration, which is probably due to conformational changes occurring under prolonged light activation in RC from the wild type bacterium. In RC from bacteria of the mutant strain in which protonatable amino acid Ser L223 near QB is substituted by Ala, the dependence of reduction kinetics of P+ on duration of light was not observed. Such dependence, however, became observable after addition of cryoprotectors, namely glycerol and dimethylsulfoxide, to the RC samples from the mutant strain. It was concluded that substitution of Ser L223 with Ala disturbs the native mechanism of electrostatic stabilization of the electron in the RC quinone acceptor site. At the same time, an additional modification of RC hydrogen bonds by glycerol and dimethylsulfoxide probably includes various possibilities for more effective time delay of the electron on QB.

The Electrogenic Event Associated with the Reduction of the Secondary Quinone Acceptor in Rhodobacter Sphaeroides Reaction Centers

An electrometric method was used to investigate flash-induced electrogenic stages in proteoliposomes containing photosynthetic RCs of Rhodobacter sphaeroides. Besides the very fast electrogenic step associated with primary dipole formation, an additional electrogenic stage with a rise-time of 0.25 ms (pH 7.5) appeared to be induced by even numbered flashes. The maximal amplitude of this stage contributes ~0.3 to the fast phase, associated with the charge separation between P870 and QA. The similarity of the rise-time of this phase, the time of the disproportioning reaction of semiquinones QA and QB and the rate of proton uptake by RCs, its appearance only after even-numbered flashes, the sensitivity to o-phenanthroline as well as the increase of its rise-time with pH indicate that the additional electrogenic phase arises from the reaction:

Effect of D2O and cryosolvents on the redox properties of bacteriochlorophyll dimer and electron transfer processes in Rhodobacter sphaeroides reaction centers

{Q_A}^ - ({H^ + }){Q_B}^ - + {H^ + } - - \to {Q_A}{Q_B}{H_2}