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Using site-directed mutagenesis, we obtained the mutant of the purple bacterium Rhodobacter sphaeroides with Ile to His substitution at position 177 in the L-subunit of the photosynthetic reaction center (RC). The mutant strain forms stable and photochemically active RC complexes. Relative to the wild type RCs, the spectral and photochemical properties of the mutant RC differ significantly in the absorption regions corresponding to the primary donor P and the monomer bacteriochlorophyll (BChl) absorption. It is shown that the RC I(L177)H contains only three BChl molecules compared to four BChl molecules in the wild type RC. Considering the fact that the properties of both isolated and membrane-associated mutant RCs are similar, we conclude that the loss of a BChl molecule from the mutant RC is caused by the introduced mutation but not by the protein purification procedure. The new mutant missing one BChl molecule but still able to perform light-induced reactions forming the charge-separated state P+QA- appears to be an interesting object to study the mechanisms of the first steps of the primary electron transfer in photosynthesis.

In this work, we report the unique case of bacteriochlorophyll (BChl) -protein covalent attachment in a photosynthetic membrane complex caused by a single mutation. The isoleucine L177 was substituted by histidine in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Pigment analysis revealed that one BChl molecule was missing in the acetone-methanol extract of the I(L177)H RCs. SDS-PAGE demonstrated that this BChl molecule could not be extracted with organic solvents apparently because of its stable covalent attachment to the mutant RC L-subunit. Our data indicate that the attached bacteriochlorophyll is one of the special pair BChls, P A . The chemical nature of this covalent interaction remains to be identified.

During photosynthesis light energy is converted into energy of chemical bonds through a series of electron and proton transfer reactions. Over the first ultrafast steps of photosynthesis that take place in the reaction center (RC) the quantum efficiency of the light energy transduction is nearly 100%. Compared to the plant and cyanobacterial photosystems, bacterial RCs are well studied and have relatively simple structure. Therefore they represent a useful model system both for manipulating of the electron transfer parameters to study detailed mechanisms of its separate steps as well as to investigate the common principles of the photosynthetic RC structure, function, and evolution. This review is focused on the research papers devoted to chemical and genetic modifications of the RCs of purple bacteria in order to study principles and mechanisms of their functioning. Investigations of the last two decades show that the maximal rates of the electron transfer reactions in the RC depend on a number of parameters. Chemical structure of the cofactors, distances between them, their relative orientation, and interactions to each other are of great importance for this process. By means of genetic and spectral methods, it was demonstrated that RC protein is also an essential factor affecting the efficiency of the photochemical charge separation. Finally, some of conservative water molecules found in RC not only contribute to stability of the protein structure, but are directly involved in the functioning of the complex.

The nuclear wave packet formed by 20 fs excitation on the P* potential energy surface in native and mutant (YM210W and YM210L) reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides and in Chloroflexus (C.) aurantiacus RCs was found to be reversibly transferred to the P + B A -surface at 120, 380, etc. fs delays (monitored by measurements of B A -absorption at 1020-1028 nm). The YM210W(L) mutant RCs show the most simple pattern of femtosecond oscillations with a period of 230 fs in stimulated emission from P* and with the initial amplitude comparable to that in plant pheophytin a (Pheo)-modified Rb. sphaeroides R-26 RCs. Similar reversible oscillations are observed in the 1020 nm band of the mutants, the initial amplitude of which is smaller by a factor of ∼10 with respect to Pheo-modified Rb. sphaeroides R-26 RCs. In contrast to native and Pheo-modified Rb. sphaeroides R-26 RCs, irreversible quasi-exponential stabilization of P + B Ais considerably suppressed in the mutant RCs in the picosecond time domain. The water rotational mode with a frequency of 32 cm -1 and its overtones, described earlier et al. Biochemistry 2002 et al. Biochemistry , 41, 2667 et al. Biochemistry -2674, are decreased in the YM210W(L) mutants and strongly suppressed in dry films of the mutant RCs. In the dry film of both YM210W and YM210L RCs neither reversible nor irreversible P + B A -formation monitored at 1020 nm is observed despite the preservation of fs oscillations with a frequency of 144 cm -1 in the 935 nm kinetics of stimulated emission from P * . Furthermore, the 1020 nm band is not formed inside of P*. In C. aurantiacus RCs, containing leucine instead of tyrosine at the M208 position, the P* decay is slowed to ∼5 ps at 90 K (1.5 ps in Rb. sphaeroides RCs) and characterized by fs oscillations with the amplitude comparable to that measured in native Rb. sphaeroides R-26 RCs. The B A -absorption band development at 1028 nm is observed at 90 K with fs oscillations similar to those described for native Rb. sphaeroides R-26 RCs at 293 K but with the amplitude being smaller by a factor of ∼6. The kinetics of absorbance changes in the 1028 nm band in C. aurantiacus RCs includes the stabilization of P + B A -within ∼5 ps with subsequent decay due to electron transfer to H A within ∼1 ps. The mechanisms of the electron-transfer between P* and B A and of the stabilization of the state P + B A -in bacterial RCs are discussed.