Mahir D. Mamedov

Near-IR absorbance changes and electrogenic reactions in the microsecond-to-second time domain in Photosystem I.

The back-reaction kinetics in Photosystem I (PS I) were studied on the microsecond-to-s time scale in cyanobacterial preparations, which differed in the number of iron-sulfur clusters to assess the contributions of particular components to the reduction of P700+. In membrane fragments and in trimeric P700-FA/FB complexes, the major contribution to the absorbance change at 820 nm (delta A820) was the back-reaction of FA- and/or FB- with lifetimes of approximately 10 and 80 ms (approximately 10% and 40% relative amplitude). The decay of photoinduced electric potential (delta psi) across a membrane with directionally incorporated P700-FA/FB complexes had similar kinetics. HgCl2-treated PS I complexes, which contain FA but no FB, retain both of these kinetic components, indicating that neither can be assigned uniquely to a specific acceptor. These results suggest that FA- reduces P700+ directly and argue for a rapid electron equilibration between FA and FB, which would eliminate their kinetic distinction in a back-reaction. In PsaC-depleted P700-Fx cores, as well as in P700-FA/FB complexes with chemically reduced FA and FB, the major contribution to the delta A820 and the delta psi decay is a biphasic back-reaction of F-X (approximately 400 microseconds and 1.5 ms) with some contribution from A-1 (approximately 10 microseconds and 100 microseconds), the latter of which is variable depending on experimental conditions. The delta A820 decay in a P700-A1 core devoid of all iron-sulfur clusters comprises two phases with lifetimes of 10 microseconds and 130 microseconds (2.7:1 ratio). The biexponential back-reaction kinetics found for each of the electron acceptors may be related to existence of different conformational states of the PS I complex. In all preparations studied, excitation at 532 nm with flash energies exceeding 10 mJ gives rise to formation of antenna 3Chl, which also contributes to delta A820 decay on the tens-of-microsecond time scale. A distinction between delta A820 components related to back-reactions and to 3Chl decay can be made by analysis of flash saturation dependencies and by measurements of kinetics with preoxidized P700.

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (<100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A0 (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first approximately 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next approximately 40 fs the formation of a new broad band centered at approximately 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A1 (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700+A0A1) PS I reaction center (RC) from that of the open (state P700A0A1) RC reveals the pure spectrum of the P700+A0- ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An*-->k1[(PA0)*A1--><100 fs P+A0-A1]-->k2P+A0A1-, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (<100 fs) charge separation with the formation of the P700+A0-A1 state in approximately one half of the RCs, the approximately 5-ps energy transfer from antenna Chl* to P700A0A1 in the remaining RCs, and approximately 25-ps formation of the secondary radical pair P700+A0A1-.

Flash-induced electrogenic events in the photosynthetic reaction center and bc1 complexes of Rhodobacter sphaeroides chromatophores

Electrogenic events in Rb. Sphaeroides chromatophores have been studied (i) electrometrically in the chromatophore/phospholipid-impregnated collodion film system and (ii) spectrophotometrically by measuring the electrochromic spectral shift of carotenoids. Under the conditions when the bc 1 complex and ubiquinone pool were oxidized at pH 7.5, the second flashwas shown to give rise to at least two additional electrogenic phases of τ values approx. 0.2 and approx. 20 ms, which were not induced by the first flash. The fast phase was resistant to the inhibitors of the bc 1 complex, antimycin A and myxothiazol. It seems to be due to the protonation of reduced Q B in the RC complex. The slow phase was partly inhibited by antimycin A and completely by subsequent addition of myxothiazol. The antimycin-sensitive constituent of the slow phase was τ ≈ 40 ms and its rise was non-exponential. The antimycin-insensitive, myxothiazol-sensitive constituent was τ ≈ 7 ms. A comparison of (i) the kinetics of cytochrome b h redox conversion induced by the first and second flashes and (ii) the electrogenic reactions sensitive to the Q-cycle inhibitors suggests that the myxothiazol-sensitive electrogenic phase is associated with the reduction of cytochrome b h ( b -561). The antimycin-sensitive electrogenic phase apparently results from the protonation of reduced Q in the quinone-reducing center of the bct complex. Reduction of ubiquinone to ubisemiquinone by b h seems to be electrically silent, since there is no electrogenic phase to follow the kinetics of this process. Myxothiazol addedin the absence of antimycin A induced a negative electrogenic phase with an opposite polarity (τ ≈ 2.5 ms) after the second flash. This phase, completely abolished by the addition of antimycin A, is assumed to be due to the electrogenic deprotonation of the RC-reduced QH 2 which combines with center C in the bc 1 complex. The data obtained by the electrometric and spectrophotometric methods appear to be very similar, though the electrometric method is more sensitive because of the much higher signal-to-noise ratio.

P680 (PD1PD2) and ChlD1 as alternative electron donors in photosystem II core complexes and isolated reaction centers

Low temperature (77-90 K) measurements of absorption spectral changes induced by red light illumination in isolated photosystem II (PSII) reaction centers (RCs, D1/D2/Cyt b559 complex) with different external acceptors and in PSII core complexes have shown that two different electron donors can alternatively function in PSII: chlorophyll (Chl) dimer P(680) absorbing at 684 nm and Chl monomer Chl(D1) absorbing at 674 nm. Under physiological conditions (278 K) transient absorption difference spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach PSII core complexes excited at 710 nm. It was shown that the initial electron transfer reaction takes place with a time constant of ~0.9 ps. This kinetics was ascribed to charge separation between P(680)* and Chl(D1) absorbing at 670 nm accompanied by the formation of the primary charge-separated state P(680)(+)Chl(DI)(-), as indicated by 0.9-ps transient bleaching at 670 nm. The subsequent electron transfer from Chl(D1)(-) occurred within 13-14 ps and was accompanied by relaxation of the 670-nm band, bleaching of the Pheo(D1) Q(x) absorption band at 545 nm, and development of the anion-radical band of Pheo(D1)(-) at 450-460 nm, the latter two attributable to formation of the secondary radical pair P(680)(+)Pheo(D1)(-). The 14-ps relaxation of the 670-nm band was previously assigned to the Chl(D1) absorption in isolated PSII RCs [Shelaev, Gostev, Nadtochenko, Shkuropatov, Zabelin, Mamedov, Semenov, Sarkisov and Shuvalov, Photosynth. Res. 98 (2008) 95-103]. We suggest that the longer wavelength position of P(680) (near 680 nm) as a primary electron donor and the shorter wavelength position of Chl(D1) (near 670 nm) as a primary acceptor within the Q(y) transitions in RC allow an effective competition with an energy transfer and stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as the primary electron donor and Pheo(D1) as the primary acceptor cannot be ruled out, the 20-fs excitation at the far-red tail of the PSII core complex absorption spectrum at 710 nm appears to induce a transition to a low-energy state P(680)* with charge-transfer character (probably P(D1)(δ+)P(D2)(δ-)) which results in an effective electron transfer from P(680)* (the primary electron donor) to Chl(D1) as the intermediary acceptor.

Electrogenesis associated with proton transfer in the reaction center protein of the purple bacterium Rhodobacter sphaeroides

Electrogenic events in the photosynthetic reaction center complex (RC), accompanying single‐ and two‐electron reduction of the secondar quinone acceptor Qb, were investigated. In the presence of inhibitors of electron transfer via the bc1‐complex, the kinetics of formation of the transmembrane electric potential difference induced by two successive light flashes exhibit a few phases. Besides the fast phase A which is due to the charge separation between the bacteriochlorophyll dimer P and primary quinone acceptor qa, two slower atrazine‐sensitive phases, BI and BII, were observed. Phase BI is suggested to be due to proton transfer between the amino acid residues of the reaction center protein, and phase BII due to proton uptake during the second flash‐induced formation of ubiquinol. A possible model of electrogenesis in the acceptor moiety of the RC is discussed.

O2 reduction by photosystem I involves phylloquinone under steady-state illumination.

O2 reduction was investigated in photosystem I (PS I) complexes isolated from cyanobacteria Synechocystis sp. PCC 6803 wild type (WT) and menB mutant strain, which is unable to synthesize phylloquinone and contains plastoquinone at the quinone‐binding site A1. PS I complexes from WT and menB mutant exhibited different dependencies of O2 reduction on light intensity, namely, the values of O2 reduction rate in WT did not reach saturation at high intensities, in contrast to the values in menB mutant. The obtained results suggest the immediate phylloquinone involvement in the light‐induced O2 reduction by PS I.

Incorporation of a high potential quinone reveals that electron transfer in Photosystem I becomes highly asymmetric at low temperature

Photosystem I (PS I) has two nearly identical branches of electron-transfer co-factors. Based on point mutation studies, there is general agreement that both branches are active at ambient temperature but that the majority of electron-transfer events occur in the A-branch. At low temperature, reversible electron transfer between P(700) and A(1A) occurs in the A-branch. However, it has been postulated that irreversible electron transfer from P(700) through A(1B) to the terminal iron-sulfur clusters F(A) and F(B) occurs via the B-branch. Thus, to study the directionality of electron transfer at low temperature, electron transfer to the iron-sulfur clusters must be blocked. Because the geometries of the donor-acceptor radical pairs formed by electron transfer in the A- and B-branch differ, they have different spin-polarized EPR spectra and echo-modulation decay curves. Hence, time-resolved, multiple-frequency EPR spectroscopy, both in the direct-detection and pulse mode, can be used to probe the use of the two branches if electron transfer to the iron-sulfur clusters is blocked. Here, we use the PS I variant from the menB deletion mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize phylloquinone, to incorporate 2,3-dichloro-1,4-naphthoquinone (Cl(2)NQ) into the A(1A) and A(1B) binding sites. The reduction midpoint potential of Cl(2)NQ is approximately 400 mV more positive than that of phylloquinone and is unable to transfer electrons to the iron-sulfur clusters. In contrast to previous studies, in which the iron-sulfur clusters were chemically reduced and/or point mutations were used to prevent electron transfer past the quinones, we find no evidence for radical-pair formation in the B-branch. The implications of this result for the directionality of electron transfer in PS I are discussed.

EPR study of light-induced regulation of photosynthetic electron transport in Synechocystis sp. strain PCC 6803

The kinetics of the light‐induced redox changes of the photosystem 1 (PS 1) primary donor P700 in whole cells of the cyanobacteria Synechocystis sp. PCC 6803 were studied by the electron paramagnetic resonance method. It was shown that the linear photosynthetic electron transport in cyanobacteria was controlled by two main mechanisms: (i) oxygen‐dependent acceleration of electron transfer from PS 1 to NADP+ due to activation of the Calvin cycle reactions and (ii) retardation of electron flow between two photosystems governed by a transmembrane proton gradient. In addition to the linear photosynthetic electron transport, cyanobacteria were capable of maintaining alternative pathways involving cyclic electron transfer around PS 1 and respiratory chains.

Electrogenicity at the donor/acceptor sides of cyanobacterial photosystem I

To study electrogenesis the photosystem I particles fromSynechococcus elongatus were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference generation has been measured by a direct electrometric method in proteoliposomes adsorbed on a phospholipid-impregnated collodion film. The photoelectric response has been found to involve three electrogenic stages associated with (i) iron-sulfur center Fx reduction by the primary electron donor P700, (ii) electron transfer between iron-sulfur centers Fx and FA/FB, and (iii) reduction of photo-oxidized P700+ by reduced cytochromec553. The relative magnitudes of phases (ii) and (iii) comprised about 20% of phase (i).

Electrogenic reduction of the primary electron donor P700+ in photosystem I by redox dyes.

The kinetics of reduction of the photo‐oxidized primary electron donor P700+ by redox dyes N,N,N′,N′‐tetramethyl‐p‐phenylendiamine, 2,6‐dichlorophenol‐indophenol and phenazine methosulfate was studied in proteoliposomes containing Photosystem I complexes from cyanobacteria Synechocystis sp. PCC 6803 using direct electrometrical technique. In the presence of high concentrations of redox dyes, the fast generation of a membrane potential related to electron transfer between P700 and the terminal iron‐sulfur clusters FA/FB was followed by a new electrogenic phase in the millisecond time domain, which contributes approximately 20% to the overall photoelectric response. This phase is ascribed to the vectorial transfer of an electron from the redox dye to the protein‐embedded chlorophyll of P700+. Since the contribution of this electrogenic phase in the presence of artificial redox dyes is approximately equal to that of the phase observed earlier in the presence of cytochrome c 6, it is likely that electrogenic reduction of P700+ in vivo occurs due to vectorial electron transfer within RC molecule rather than within the cytochrome c 6‐P700 complex.