Effect of Dehydrated Trehalose Matrix on the Kinetics of Forward Electron Transfer Reactions in Photosystem I

The effect of dehydration on the kinetics of forward electron transfer (ET) has been studied in cyanobacterial photosystem I (PS I) complexes in a trehalose glassy matrix by time-resolved optical and EPR spectroscopies in the 100 fs to 1 ms time domain. The kinetics of the flash-induced absorption changes in the subnanosecond time domain due to primary and secondary charge separation steps were monitored by pump–probe laser spectroscopy with 20-fs low-energy pump pulses centered at 720 nm. The back-reaction kinetics of P700 were measured by high-field time-resolved EPR spectroscopy and the forward kinetics of A∙−1A/A∙−1B→FX by time-resolved optical spectroscopy at 480 nm. The kinetics of the primary ET reactions to form the primary P∙+700A∙−0 and the secondary P∙+700A∙−1 ion radical pairs were not affected by dehydration in the trehalose matrix, while the yield of the P∙+700A∙−1 was decreased by ~20%. Forward ET from the phylloquinone molecules in the A∙−1A and A∙−1B sites to the iron–sulfur cluster FX slowed from ~220 ns and ~20 ns in solution to ~13 μs and ~80 ns, respectively. However, as shown by EPR spectroscopy, the ~15 μs kinetic phase also contains a small contribution from the recombination between A∙−1B and P∙+700. These data reveal that the initial ET reactions from P700 to secondary phylloquinone acceptors in the A- and B-branches of cofactors (A1A and A1B) remain unaffected whereas ET beyond A1A and A1B is slowed or prevented by constrained protein dynamics due to the dry trehalose glass matrix

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

AbstractThe 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 c6, it is likely that electrogenic reduction of P700+ in vivo occurs due to vectorial electron transfer within RC molecule rather than within the cytochrome c6-P700 complex

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 P700 and A1A occurs in the A-branch. However, it has been postulated that irreversible electron transfer from P700 through A1B to the terminal iron-sulfur clusters FA and FB 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 (Cl2NQ) into the A1A and A1B binding sites. The reduction midpoint potential of Cl2NQ 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

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

AbstractThe ultrafast (<100fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705nm) and the primary acceptor A0 (bleaching at 690nm) 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 720nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690nm 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 720nm. During the first ∼60fs the energy transfer from the chlorophyll (Chl) species bleaching at 690nm to the Chl bleaching at 705nm 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 ∼40fs the formation of a new broad band centered at ∼660nm (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→<100fs 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 705nm 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 (<100fs) charge separation with the formation of the P700+A0−A1 state in approximately one half of the RCs, the ∼5-ps energy transfer from antenna Chl* to P700A0A1 in the remaining RCs, and ∼25-ps formation of the secondary radical pair P700+A0A1−

Real-time kinetics of electrogenic Na+ transport by rhodopsin from the marine flavobacterium Dokdonia sp. PRO95

Discovery of the light-driven sodium-motive pump Na+-rhodopsin (NaR) has initiated studies of the molecular mechanism of this novel membrane-linked energy transducer. In this paper, we investigated the photocycle of NaR from the marine flavobacterium Dokdonia sp. PRO95 and identified electrogenic and Na+-dependent steps of this cycle. We found that the NaR photocycle is composed of at least four steps: NaR519 + hv -> K-585 -> (L-450 M-495) -> O-585 -> NaR519. The third step is the only step that depends on the Na+ concentration inside right-side-out NaR-containing proteoliposomes, indicating that this step is coupled with Na+ binding to NaR. For steps 2, 3, and 4, the values of the rate constants are 4x10(4) s(-1), 4.7 x 10(3) M-1 s(-1), and 150 s(-1), respectively. These steps contributed 15, 15, and 70% of the total membrane electric potential (Delta psi similar to 200 mV) generated by a single turnover of NaR incorporated into liposomes and attached to phospholipid-impregnated collodion film. On the basis of these observations, a mechanism of light-driven Na+ pumping by NaR is suggested.Peer reviewe

Trehalose matrix effects on electron transfer in Mn-depleted protein-pigment complexes of Photosystem II

The kinetics of flash-induced re-reduction of the Photosystem II (PS II) primary electron donor P-680 was studied in solution and in trehalose glassy matrices at different relative humidity. In solution, and in the re-dissolved glass, kinetics were dominated by two fast components with lifetimes in the range of 2-7 microsec, which accounted for &gt;85% of the decay. These components were ascribed to the direct electron transfer from the redox-active tyrosine YZ to P680+. The minor slower components were due to charge recombination between the primary plastoquinone acceptor QA- and P680+. Incorporation of the PS II complex into the trehalose glassy matrix and its successive dehydration caused a progressive increase in the lifetime of all kinetic phases, accompanied by an increase of the amplitudes of the slower phases at the expense of the faster phases. At 63% relative humidity the fast components contribution dropped to similar to 50%. A further dehydration of the trehalose glass did not change the lifetimes and contribution of the kinetic components. This effect was ascribed to the decrease of conformational mobility of the protein domain between YZ and P-680, which resulted in the inhibition of YZ -&gt; P680+ electron transfer in about half of the PS II population, wherein the recombination between QA- and P680+ occurred. The data indicate that PS II binds a larger number of water molecules as compared to PS I complexes. We conclude that our data disprove the "water replacement" hypothesis of trehalose matrix biopreservation

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

AbstractO2 reduction was investigated in photosystem I (PSI) 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. PSI 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 PSI

Trehalose matrix effects on charge-recombination kinetics in Photosystem i of oxygenic photosynthesis at different dehydration levels

Matrix effects on long-range electron transfer were studied in cyanobacterial Photosystem I (PS I) complexes, embedded into trehalose glasses at different hydration levels.W-band EPR studies demonstrated, via nitroxide spin probes, structural homogeneity of the dry PS I-trehalosematrix and no alteration of cofactors' distance and relative orientation under temperature andmatrix variation. In dry trehalose glasses at room temperature (RT), PS Iwas stable for months. Flash-induced charge recombination kinetics were examined by high-field time-resolved EPR and optical spectroscopies. The kinetics in hydrated PS I-trehalose glasses mostly reflected the reduction of the photooxidized primary donor P700 + by the reduced terminal iron-sulfur clusters. Upon dehydration, the P700 + decay accelerated and became more distributed. Continuous distributions of lifetimes \u3c0 were extracted from the kinetics by two numerical approaches: a maximum entropy method (MemExp program) and a constrained regularization method (CONTIN program). Both analyses revealed that upon dehydration the contribution of the two slowest components (lifetimes \u3c0 3c300 ms and 3c60 ms), attributed to P700 + [FA/FB]\u2022- recombination, decreased in parallel with the increase of the fastest component (\u3c0 3c 150 \u3bcs), and of additional distributed phaseswith intermediate lifetimes. Dehydration at RTmimicked the effects of freezingwater-glycerol PS I systems, suggesting an impairment of PS I protein dynamics in the dry trehalose glass. Similar effects were observed previously in bacterial reaction centers. The work presented for PS I provides new insights into the crucial issue of protein-matrix interactions for protein functionality as controlled by hydrogen-bond networks of the hydration shell