Krzysztof Gibasiewicz

Modulation of Primary Radical Pair Kinetics and Energetics in Photosystem II by the Redox State of the Quinone Electron Acceptor QA

Time-resolved photovoltage measurements on destacked photosystem II membranes from spinach with the primary quinone electron acceptor Q(A) either singly or doubly reduced have been performed to monitor the time evolution of the primary radical pair P680(+)Pheo(-). The maximum transient concentration of the primary radical pair is about five times larger and its decay is about seven times slower with doubly reduced compared with singly reduced Q(A). The possible biological significance of these differences is discussed. On the basis of a simple reversible reaction scheme, the measured apparent rate constants and relative amplitudes allow determination of sets of molecular rate constants and energetic parameters for primary reactions in the reaction centers with doubly reduced Q(A) as well as with oxidized or singly reduced Q(A). The standard free energy difference DeltaG degrees between the charge-separated state P680(+)Pheo(-) and the equilibrated excited state (Chl(N)P680)* was found to be similar when Q(A) was oxidized or doubly reduced before the flash (approximately -50 meV). In contrast, single reduction of Q(A) led to a large change in DeltaG degrees (approximately +40 meV), demonstrating the importance of electrostatic interaction between the charge on Q(A) and the primary radical pair, and providing direct evidence that the doubly reduced Q(A) is an electrically neutral species, i.e., is doubly protonated. A comparison of the molecular rate constants shows that the rate of charge recombination is much more sensitive to the change in DeltaG degrees than the rate of primary charge separation.

Excitonic interactions in wild-type and mutant PSI reaction centers

Femtosecond excitation of the red edge of the chlorophyll a Q(Y) transition band in photosystem I (PSI), with light of wavelength > or = 700 nm, leads to wide transient (subpicosecond) absorbance changes: positive DeltaA between 635 and 665 nm, and four negative DeltaA bands at 667, 675, 683, and 695 nm. Here we compare the transient absorbance changes after excitation at 700, 705, and 710 nm at 20 K in several PSI preparations of Chlamydomonas reinhardtii where amino acid ligands of the primary donor, primary acceptor, or connecting chlorophylls have been mutated. Most of these mutations influence the spectrum of the absorbance changes. This supports the view that the chlorophylls of the electron transfer chain as well as the connecting chlorophylls are engaged in the observed absorbance changes. The wide absorption spectrum of the electron transfer chain revealed by the transient measurements may contribute to the high efficiency of energy trapping in photosystem 1. Exciton calculations, based on the recent PSI structure, allow an assignment of the DeltaA bands to particular chlorophylls: the bands at 675 and 695 nm to the dimers of primary acceptor and accessory chlorophyll and the band at 683 nm to the connecting chlorophylls. The subpicosecond transient absorption bands decay may reflect rapid charge separation in the PSI reaction center.

Mechanism of Recombination of the P(+)H(A)(-) Radical Pair in Mutant Rhodobacter Sphaeroides Reaction Centers with Modified Free Energy Gaps between P(+)B(A)(-) and P(+)H(A)(-).

The kinetics of recombination of the P(+)H(A)(-) radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, ΔG, between the charge separated states P(+)B(A)(-) and P(+)H(A)(-) was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller ΔG than in the wild-type, and was slowed in a mutant having a larger ΔG. The free energy difference between the state P(+)H(A)(-) and the PH(A) ground state was unaffected by most of these mutations. These observations were consistent with a model in which the P(+)H(A)(-) → PH(A) charge recombination is thermally activated and occurs via the intermediate state P(+)B(A)(-), with a mean rate related to the size of the ΔG between the states P(+)B(A)(-) and P(+)H(A)(-) and not the ΔG between P(+)H(A)(-) and the ground state. A more detailed analysis of charge recombination in the mutants showed that the kinetics of the reaction were multiexponential, and characterized by ~0.5, ~1-3, and 7-17 ns lifetimes, similar to those measured for wild-type reaction centers. The exact lifetimes and relative amplitudes of the three components were strongly modulated by the mutations. Two models were considered in order to explain the observed multiexponentiality and modulation, involving heterogeneity or relaxation of P(+)H(A)(-) states, with the latter model giving a better fit to the experimental results.

Internal electrostatic control of the primary charge separation and recombination in reaction centers from Rhodobacter sphaeroides revealed by femtosecond transient absorption.

We report the observation of two conformational states of closed RCs from Rhodobacter sphaeroides characterized by different P(+)H(A)(-) --> PH(A) charge recombination lifetimes, one of which is of subnanosecond value (700 +/- 200 ps). These states are also characterized by different primary charge separation lifetimes. It is proposed that the distinct conformations are related to two protonation states either of reduced secondary electron acceptor, Q(A)(-), or of a titratable amino acid residue localized near Q(A). The reaction centers in the protonated state are characterized by faster charge separation and slower charge recombination when compared to those in the unprotonated state. Both effects are explained in terms of the model assuming modulation of the free energy level of the state P(+)H(A)(-) by the charges on or near Q(A) and decay of the P(+)H(A)(-) state via the thermally activated P(+)B(A)(-) state.

Primary Radical Pair P+H- Lifetime in Rhodobacter sphaeroides with Blocked Electron Transfer to QA. Effect of o-Phenanthroline

Transient absorption spectroscopy with a time resolution of approximately 1 ns was applied to study the decay of the primary radical pair P+H- in Rhodobacter sphaeroides R-26 reaction centers with blocked electron transfer from H- to QA. The block in the electron transfer was realized in two ways: by either reducing or removing QA. We found very different kinetics of the P+H- decay in these two cases. Convolution of the multiexponential decay with the instrument response function allowed resolution of as many as three kinetic components of <1-, 3-4-, and 9-12-ns lifetimes in chromatophores with QA reduced and in isolated reaction centers both with QA either reduced or removed (with or without o-phenanthroline) but with variable relative amplitudes. Removing QA or adding o-phenanthroline to isolated reaction centers increased the amplitude of the slowest decay phase relative to that of the fastest phase. On the basis of these observations, we propose that reaction centers adopt three conformational states characterized by different decay kinetics of P+H-. These conformational states appear to be controlled by the charges in the vicinity of the QA site as revealed by the effects of QA reduction and o-phenanthroline-mediated protonation of the sites close to QA.

Analysis of the temperature-dependence of P(+)HA(-) charge recombination in the Rhodobacter sphaeroides reaction center suggests nanosecond temperature-independent protein relaxation.

The temperature dependence of charge recombination of the pair P(+)HA(-) in isolated reaction centers from the purple bacterium Rhodobacter sphaeroides with prereduced quinone QA was studied by sub-nanosecond to microsecond time-scale transient absorption. Overall, the kinetics slowed down substantially upon cooling from room temperature to ∼200 K, and then remained virtually unchanged down to 77 K, indicating the coexistence of two competitive pathways of charge recombination, a thermally-activated pathway appearing only above ~200 K and a temperature-independent pathway. In our modelling, the thermally activated pathway includes an uphill electron transfer from HA(-) to BA(-) leading to transient formation of the state P(+)BA(-), whereas the temperature-independent pathway is due to direct downhill electron transfer from HA(-) to P(+). At all temperatures studied, the kinetics could be approximated by a four-component decay. Detailed analysis of the lifetimes and amplitudes of particular phases over the range of temperatures suggests that the kinetically resolved phases reveal the consecutive appearance of three conformational states characterized by an increasing free energy gap between the states P(+)BA(-) and P(+)HA(-). The initial gap between these states was estimated to be only ~8 meV, the intermediate gap being ~92 meV, and the final gap ~135 meV, with no dependence on temperature. It was also calculated through a very straightforward approach that the relaxation process from the initial to the intermediate state occurs within 0.6 ± 0.1 ns, whereas the second step of relaxation from the intermediate to the final state takes 11 ± 2 ns. Both phases of the protein relaxation process are essentially temperature-independent. Possible alternative models to describe the experimental data that cannot be definitely excluded are also discussed.

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells

Identical time-resolved fluorescence measurements with ~3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI-LHCI complexes and PSI-LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6-8ps, 25-30ps, 166-314ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI-LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12-15ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI-LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.

Excitation and electron transfer in reaction centers from Rhodobacter sphaeroides probed and analyzed globally in the 1-nanosecond temporal window from 330 to 700 nm

Global analysis of a set of room temperature transient absorption spectra of Rhodobacter sphaeroides reaction centers, recorded in wide temporal and spectral ranges and triggered by femtosecond excitation of accessory bacteriochlorophylls at 800 nm, is presented. The data give a comprehensive review of all spectral dynamics features in the visible and near UV, from 330 to 700 nm, related to the primary events in the Rb. sphaeroides reaction center: excitation energy transfer from the accessory bacteriochlorophylls (B) to the primary donor (P), primary charge separation between the primary donor and primary acceptor (bacteriopheophytin, H), and electron transfer from the primary to the secondary electron acceptor (ubiquinone). In particular, engagement of the accessory bacteriochlorophyll in primary charge separation is shown as an intermediate electron acceptor, and the initial free energy gap of approximately 40 meV, between the states P(+)B(A)(-) and P(+)H(A)(-) is estimated. The size of this gap is shown to be constant for the whole 230 ps lifetime of the P(+)H(A)(-) state. The ultrafast spectral dynamics features recorded in the visible range are presented against a background of results from similar studies performed for the last two decades.

Fluorescence hole-burning and site-selective studies of LHCII

We report the observation of two types of changes in fluorescence spectra of LHCII at 4.2 K following intense illumination of the sample with a spectrally narrow laser beam at wavelengths between 678 and 686 nm. Nonspecific changes (burning-wavelength independent) are characterized by two relatively broad bands: a positive one at ∼ 678.7 nm and a negative one at ∼ 680.8 nm. These changes reveal a ∼1.3-nm blue shift of the distribution of final emitters in LHCII, from 680.3 nm to ∼ 679.0 nm independent of the excitation wavelength. Specific fluorescence changes (burning-wavelength dependent) are characterized by a sharp hole exactly at the burning wavelength, and positive changes directly to the shorter-and longer-wavelength side of the narrow hole. The negative changes are interpreted as zero-phonon holes, while the positive features are assigned to non-photochemical products. In the low-burning intensity experiment, in addition to the zero-phonon holes, we observed also the holes to the longer wavelength of the zero-phonon hole, which were assigned to a sum of phonon and pseudo-phonon side bands. The shapes of these extra holes are identical to the shapes of the holes revealed in the fluorescence line narrowing experiment. On the basis of the low-burning intensity experiment we estimated the upper limit of the electron-phonon coupling strength for LHCII, characterized by a Huang-Rhys factor of 1.5.

Unified Model of Nanosecond Charge Recombination in Closed Reaction Centers from Rhodobacter sphaeroides: Role of Protein Polarization Dynamics

Ongoing questions surround the influence of protein dynamics on rapid processes such as biological electron transfer. Such questions are particularly addressable in light-activated systems. In Rhodobacter sphaeroides reaction centers, charge recombination or back electron transfer from the reduced bacteriopheophytin, HA(-), to the oxidized dimeric bacteriochlorophyll, P(+), may be monitored by both transient absorption spectroscopy and transient fluorescence spectroscopy. Signals measured with both these techniques decay in a similar three-exponential fashion with lifetimes of ∼0.6-0.7, ∼2-4, and ∼10-20 ns, revealing the complex character of this electron transfer reaction. In this study a single kinetic model was developed to connect lifetime and amplitude data from both techniques. The model took into account the possibility that electron transfer from HA(-) to P(+) may occur with transient formation of the state P(+)BA(-). As a result it was possible to model the impact of nanosecond protein relaxation on the free energy levels of both P(+)HA(-) and P(+)BA(-) states relative to that of the singlet excited state of P, P*. Surprisingly, whereas the free energy gap between P* and P(+)HA(-) increased with time in response to protein reorganization, the free energy gap between P* and P(+)BA(-) decreased. This finding may be accounted for by a gradual polarization of the protein environment which stabilizes the state P(+)HA(-) and destabilizes the state P(+)BA(-), favoring productive charge separation over unproductive charge recombination.