P. M. Krasilnikov

Fluorescence quenching of the phycobilisome terminal emitter LCM from the cyanobacterium Synechocystis sp. PCC 6803 detected in vivo and in vitro

The fluorescence emission of the phycobilisome (PBS) core-membrane linker protein (L(CM)) can be directly quenched by photoactivated orange carotenoid protein (OCP) at room temperature both in vitro and in vivo, which suggests the crucial role of the OCP-L(CM) interaction in non-photochemical quenching (NPQ) of cyanobacteria. This implication was further supported (i) by low-temperature (77K) fluorescence emission and excitation measurements which showed a specific quenching of the corresponding long-wavelength fluorescence bands which belong to the PBS terminal emitters in the presence of photoactivated OCP, (ii) by systematic investigation of the fluorescence quenching and recovery in wild type and L(CM)-less cells of the model cyanobacterium Synechocystis sp. PCC 6803, and (iii) by the impact of dephosphorylation of isolated PBS on the quenching. The OCP binding site within the PBS and the most probable geometrical arrangement of the OCP-allophycocyanin (APC) complex was determined in silico using the crystal structures of OCP and APC. Geometrically modeled attachment of OCP to the PBS core is not at variance with the OCP-L(CM) interaction. It was concluded that besides being a very central element in the PBS to reaction center excitation energy transfer and PBS assembly, L(CM) also has an essential role in the photoprotective light adaptation processes of cyanobacteria.

Relaxation mechanism of molecular systems containing hydrogen bonds and free energy temperature dependence of reaction of charges recombination within Rhodobacter sphaeroides RC

The mechanism of protonic relaxation is shown to take place in molecular systems containing hydrogen bonds. The mechanism arises from the proton redistribution between two stable states on hydrogen bond lines. This redistribution occurs due to changes of hydrogen bond double well potential, brought about by changes of the electronic state of a molecular system. A characteristic of the relaxation process is that it takes place due to the proton tunneling along hydrogen bonds. The charge shift causes electrostatic potential variation in the electron localization area, which leads to the shift of molecular system energy levels and changes its redox potential. The characteristic time of the protonic relaxation is shown to depend essentially on hydrogen bond bending strain, which increases with the temperature rise and decreases abruptly the efficiency of proton redistribution. Hence, the rate of this process decreases with temperature, in contrast to the activation process. This relaxation process is shown to be responsible for energetic characteristics of recombination reaction P+QA--->PQA (free energy difference DeltaG and/or reorganization energy lambda), temperature dependence in Rhodobacter sphaeroides RC.

Cyanobacterial phycobilisomes and phycobiliproteins

In cyanobacteria, phycobilisomes (PBS) act as antenna of the photosynthetic pigment apparatus. They contain brightly colored phycobiliproteins (PBP) and form giant supramolecular complexes (up to 3000–7000 kDa) containing 200 to 500 phycobilin chromophores covalently bound to the proteins. There are over ten various PBP known, which falls into one of three groups: phycoerythrins, phycocyanins, and allophycocyanins. Hollow disks of PBP trimers and hexamers are arranged into cylinders by colorless linker proteins; the cylinders are then assembled into PBS. Typical semidiscoidal PBS consists of a central core formed by three allophycocyanin cylinders and of six lateral cylinders consisting of other PBP and attached as a fan to the nucleus. The PBS number, size, and pigment composition in cyanobacteria depend on light conditions and other ambient factors. While PBSs have certain advantages compared to other antennae, these pigment-protein complexes require more energy for their biosynthesis than the chlorophyll a/b and chlorophyll a/c proteins of oxygenic photosynthetic organisms.

Energetics and mechanisms of high efficiency of charge separation and electron transfer processes in Rhodobacter sphaeroides reaction centers

Effects of environmental changes due to D(2)O/H(2)O substitution and cryosolvent addition on the energetics of the special pair and the rate constants of forward and back electron transfer reactions in the picosecond-nanosecond time domain have been studied in isolated reaction centers (RC) of the anaxogenic purple bacterium Rhodobacter sphaeroides. The following results were obtained: (i). replacement of H(2)O by D(2)O or addition of either 70% (v/v) glycerol or 35% (v/v) DMSO do not influence the absorption spectra; (ii). in marked contrast to this invariance of absorption, the maxima of fluorescence spectra are red shifted relative to control by 3.5, 6.8 and 14.5 nm for RCs suspended in glycerol, D(2)O or DMSO, respectively; (iii). D(2)O/H(2)O substitution or DMSO addition give rise to an increase of the time constants of charge separation (tau(e)), and Q(A)(-) formation (tau(Q)) by a factors of 2.5-3.1 and 1.7-2.5, respectively; (iv). addition of 70% glycerol is virtually without effect on the values of tau(e) and tau(Q); (v). the midpoint potential E(m) of P/P(+) is shifted by about 30 and 45 mV towards higher values by addition of 70% glycerol and 35% DMSO, respectively, but remains invariant to D(2)O/H(2)O exchange; and (vi). an additional fast component with tau(1)=0.5-0.8 ns in the kinetics of charge recombination P(+)H(A)(-)-->P*(P)H(A) emerges in RC suspensions modified either by D(2)O/H(2)O substitution or by DMSO treatment. The results have been analysed with special emphasis on the role of deformations of hydrogen bonds for the solvation mechanism of nonequilibrium states of cofactors. Reorientation of hydrogen bonds provides the major contribution of the very fast environmental response to excitation of the special pair P. The Gibbs standard free energy gap between 1P* and P(+)B(A)(-) due to solvation is estimated to be approximately 70, 59 and 48 meV for control, D(2)O- and DMSO-treated RC samples, respectively.

Role of inter-domain cavity in the attachment of the orange carotenoid protein to the phycobilisome core and to the fluorescence recovery protein

Using molecular modeling and known spatial structure of proteins, we have derived a universal 3D model of the orange carotenoid protein (OCP) and phycobilisome (PBS) interaction in the process of non-photochemical PBS quenching. The characteristic tip of the phycobilin domain of the core-membrane linker polypeptide (LCM) forms the attachment site on the PBS core surface for interaction with the central inter-domain cavity of the OCP molecule. This spatial arrangement has to be the most advantageous one because the LCM, as the major terminal PBS-fluorescence emitter, accumulates energy from the most other phycobiliproteins within the PBS before quenching by OCP. In agreement with the constructed model, in cyanobacteria, the small fluorescence recovery protein is wedged in the OCP’s central cavity, weakening the PBS and OCP interaction. The presence of another one protein, the red carotenoid protein, in some cyanobacterial species, which also can interact with the PBS, also corresponds to this model.

Structural modeling of the phycobilisome core and its association with the photosystems

The phycobilisome (PBS) is a major light-harvesting complex in cyanobacteria and red algae. To obtain the detailed structure of the hemidiscoidal PBS core composed of allophycocyanin (APC) and minor polypeptide components, we analyzed all nine available 3D structures of APCs from different photosynthetic species and found several variants of crystal packing that potentially correspond to PBS core organization. Combination of face-to-face APC trimer crystal packing with back-to-back APC hexamer packing suggests two variants of the tricylindrical PBS core. To choose one of these structures, a computational model of the PBS core complex and photosystem II (PSII) dimer with minimized distance between the terminal PBS emitters and neighboring antenna chlorophylls was built. In the selected model, the distance between two types of pigments does not exceed 37 Å corresponding to the Förster mechanism of energy transfer. We also propose a model of PBS and photosystem I (PSI) monomer interaction showing a possibility of supercomplex formation and direct energy transfer from the PBS to PSI.

Effect of hydrogen bonds on the energetics of macromolecules in the course of electron transfer

For a model system consisting of a bacteriochlorophyll dimer (P) and a primary quinone with the nearest environment (QA), which are the electron donor and acceptor in the recombination reaction in the Rhodobacter spheroides reaction center, the energies of states P+QA− and PQA have been calculated at several stable conformations of QA that differ in the positions of the proton involved in the hydrogen bond. It is shown that the position of the proton has a considerable influence on the energy of vertical transition P+QA− → PQA.

Influence of the energy of H-bond protons on the electron transfer rate in photosynthetic reaction centers

We present here a theoretical interpretation of the temperature dependence of the rate of dark recombination between a primary quinone (QA) and a bacteriochlorophyll dimer in the reaction center of Rhodobacter sphaeroides. We were able to describe qualitatively the nonmonotonous character of this dependence using the energy of interaction between an excess electron and H-bond protons. We considered a molecular model of QA and two reaction center fragments that make H-bonds with QA: His(M219) and Asn(M259)-Ala(M260). We used the two-center approach with regard for electron-phonon interaction in order to calculate the characteristic time of electron tunneling during the recombination reaction. The energy of the phonon emitted/ absorbed during the electron tunneling was determined by the relative shift of donor and acceptor energy levels, the detuning of levels. The detuning was shown to depend on temperature nonmonotonously for H-bonds with double-well potential energy surface. The characteristic time (or the reaction rate) depended on temperature parametrically. The computed dependence was in qualitative agreement with the experimental one.

The spectral-kinetic indicators of relaxation processes following the electron stabilization into the acceptor compartment of photosynthetic RCs of bacteria

Longterm temporary stabilization of the electron in the acceptor part of the photosynthetic reaction center (RC) is essential for subsequent efficient trans� fer of reducing equivalents to the photosynthetic membrane. Comparative study of the kinetics of redox reactions of photoactive bacteriochlorophyll of the RC of purple bacteria and quinone acceptors in their indi� vidual absorption bands is an informative approach to study the mechanisms of this stabilization. The analy� sis of the revealed kinetic differences makes it possible to estimate the activation energy and the characteristic time of the transition relaxation process associated with the stabilization of the electron on the final quinone acceptor and establish the hydrogen bonds in the quinone environment that are involved in this pro� cess. Conformational dynamics of protein-pigment complexes of photosynthetic RCs plays the crucial role in all stages of light energy conversion, including the temporary stabilization of electrons in the quinone acceptor component of the RCs of purple bacteria (1, 2). The important functional role of effective oper� ation of this component of the electron transport chain (ETC) is determined primarily by the fact that it couples the very fast initial stages of the lightinduced charge separation with the significantly slower diffu� sioncontrolled reactions of transferring the reducing equivalents to the membrane. Ultimately, two quinone molecules, QA and QB, integrated into the RC struc� ture, are actively involved in the coupled electron- proton transport processes that lead to the formation of a proton gradient across the photosynthetic mem� brane, required for the synthesis of ATP. These two quinones have different redox potentials, providing the driving force for the vector electron transfer from the primary to the secondary quinone acceptor. Since

Theory of Intermolecular Electron Transfer in Nanoscale Biological Structures

Macromolecular biological systems performing directed electron transfer are nano-sized structures. The distance between carrier molecules (cofactors), which represent practically isolated electron localization centers, reaches tens of angstroms. The electron transfer theory based on the concept of delocalized electron states, which is conventionally used in biophysics, is unable to adequately interpret the results of concrete observations in many cases. On the basis of the theory of electronic transitions in the case of localized states, developed in the physics of disordered matter, a mechanism of long-distance electron transfer in biological systems is suggested. The molecular relaxation of the microenvironment of electron localization centers that accompanies the electron transfer process is also considered.