Chlorophyll fluorescence decay kinetics was measured in sulfur deprived cells of green alga Chlamydomonas reinhardtii with a home made picosecond fluorescence laser spectrometer. The measurements were carried out on samples either shortly adapted to the dark ('Fo conditions') or treated to reduce Qa ('Fm conditions'). Bi-exponential fitting of decay kinetics was applied to distinguish two components one of them related to energy trapping (fast component) and the other to charge stabilization and recombination in PS 2 reaction centers (slow component). It was found that the slow component yield increased by 2.0 and 1.2 times when measured under 'Fo' and 'Fm conditions', respectively, in sulfur deprived cells as compared to control ones. An additional rapid rise of the slow component yield was observed when incubation was carried out in a sealed bioreactor and cell culture turned to anaerobic conditions. The obtained results strongly indicate the existence of the redox control of PS 2 activity during multiphase adaptation of C. reinhardtii to sulfur deficiency stress. Probable mechanisms responsible for the observed increased recombinant fluorescence yield in starved cells are discussed.
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.
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 DrO isotope substitution of H20 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+II + 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, kncwn 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.
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