Alexey Yu. Semenov

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-.

Spectral, redox and kinetic characteristics of high-potential cytochrome c hemes in Rhodopseudomonas viridis reaction center

Redox, optical and kinetic characteristics of the four‐heme cytochrome c tightly bound to the reaction center complexes of Rhodopseudomonas viridis have been studied. The two high‐potential hemes, previously thought to be identical, are shown to differ in midpoint potentials, absorption spectra and kinetics of photooxidation. One heme is characterized by E m = 380 ± 10 mV, and a split α‐band (a peak at 559 nm and a shoulder at 553 nm) whereas the other has an E m = 310±10 mV and a symmetrical α‐band at 556 nm. Kinetics of laser flash oxidation of the c‐ heme by the photogenerated P‐960+ (τ ~ 0.3 μs) matches closely that of the bacteriochlorophyll reduction and precedes oxidation of the c‐556 heme, the latter occurring with τ ~ 2.5 μs concurrently with heme c‐ re‐reduction. The data point to heme c‐ being an immediate electron donor to P‐960+. Accordingly, this heme is tentatively identified with the iron‐porphyrin group proximal to the bacteriochlorophyll special pair in the three‐dimensional model of Rps. viridis reaction centers complexes [(1985) Nature 318, 618‐624]. Thus, the following reaction sequence is assumed: c‐556 → c‐559 → P‐960+.

Lipid-impregnated filters as a tool for studying the electric current-generating proteins.

Abstract Porous filters and collodion film impregnated with decane solution of phospholipids, were used for measurements of electric potential differences generated by bacteriorhodopsin, chromatophore redox chain, H+-ATPase, pyrophosphatase, and mitochondrial respiratory chain. It was shown that reconstituted proteoliposomes, containing e.g., bacteriorhodopsin or natural coupling membrane vesicles, such as Rhodospirillum rubrum chromatophores, can be attached to a filter surface by means of Ca2+ or Mg2+ ions. Addition of the respective energy source was found to result in electric potential difference being generated across the filter. This effect was measured directly by Ag AgCl electrodes immersed into electrolyte solutions on both sides of the filter. Using a phospholipid-impregnated collodion film one can measure electric responses as fast as 300 nsec. The phospholipid-impregnated filters turned out to be sensitive and reliable electrodes for measuring the concentration of synthetic penetrating ions, such as phenyldicarbaundecaborane, tetraphenylborate, tetrapentylammonium, and tetraphenylphosphonium. By measuring changes in the concentration of these ions in the suspension of proteoliposomes, chromatophores, mitochondria, or bacterial cells, one can follow the formation and dissipation of transmembrane potential differences in these systems. It is shown that the phospholipid-impregnated filters are much more reliable and handy than planar phospholipid membranes previously used for studying electrogenic activity of electric current-producing membrane proteins.

Electrogenic reduction of the secondary quinone acceptor in chromatophores of Rhodospirillum rubrum: Rapid kinetics measurements

Electron transfer QA → QB has been reconstituted with added Q‐10 in Rhodospirillum rubrum chromatophores associated with a phospholipid‐impregnated collodion film. Rapid kinetics measurements of laser flash‐induced ΔΨ generated in the chromatophores show that whereas electron transfer from Qa − to QB upon the first flash is not electrogenic in dark‐adapted chromatophores, reduction of Q− B to Qbh2 induced by the second flash gives rise to an electrogenic phase with τ = 250 μs at pH 7.5 which contributes about 10% to the total ΔΨ generated upon the flash. The electrogenic phase is ascribed to vectorial protonation of Q2− B.

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.

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.

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c6 ), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment–protein complex (from plastocyanin or cytochrome c6 through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.

Evidence that histidine forms a coordination bond to the A0A and A0B chlorophylls and a second H-bond to the A1A and A1B phylloquinones in M688HPsaA and M668HPsaB variants of Synechocystis sp. PCC 6803

The axial ligands of the acceptor chlorophylls, A(0A) and A(0B), in Photosystem I are the Met sulfur atoms of M688(PsaA) and M668(PsaB). To determine the role of the Met, His variants were generated in Synechocystis sp. PCC 6803. Molecular dynamics simulations on M688H(PsaA) show that there exist low energy conformations with the His coordinated to A(0A) and possibly H-bonded to A(1A). Transient EPR studies on M688H(PsaA) indicate a more symmetrical electron spin distribution in the A(1A) phyllosemiquinone ring consistent with the presence of an H-bond to the C1 carbonyl. Ultrafast optical studies on the variants show that the 150fs charge separation between P₇₀₀ and A(0) remains unaffected. Studies on the ns timescale show that 57% of the electrons are transferred from A(0A)(-) to A(1A) in M688H(PsaA) and 48% from A(0B)(-) to A(1B) in M668H(PsaB); the remainder recombine with P₇₀₀(+) with 1/e times of 25ns and 37ns, respectively. Those electrons that reach A(1A) and A(1B) in the branch carrying the mutation are not transferred to FX, but recombine with P₇₀₀(+) with 1/e times of ~15μs and ~5μs, respectively. Hence, the His is coordinated to A0 in all populations, but in a second population, the His may be additionally H-bonded to A(1). Electron transfer from A(0) to A(1) occurs only in the latter, but the higher redox potentials of A(0) and A(1) as a result of the stronger coordination bond to A(0) and the proposed second H-bond to A(1) preclude electron transfer to the Fe/S clusters.