a b s t r a c tThe predicted Exigobacterium sibiricum bacterirhodopsin gene was amplified from an ancient Siberian permafrost sample. The protein bacteriorhodopsin from Exiguobacterium sibiricum (ESR) encoded by this gene was expressed in Escherichia coli membrane. ESR bound all-trans-retinal and displayed an absorbance maximum at 534 nm without dark adaptation. The ESR photocycle is characterized by fast formation of an M intermediate and the presence of a significant amount of an O intermediate. Proteoliposomes with ESR incorporated transport protons in an outward direction leading to medium acidification. Proton uptake at the cytoplasmic surface of these organelles precedes proton release and coincides with M decay/O rise of the ESR.

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.

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH4-). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH4- in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13(1)-deoxo-13(1)-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo- and P680+. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Qy and Qx transitions of this molecule were determined to be located at 5 degrees C at 679.5-680 nm and 542 nm, respectively.

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.

Isolated reaction centers of photosystem II with an altered pigment content were obtained by chemical exchange of the native pheophytin a molecules with externally added 13 1 -deoxo-13 1 -hydroxy-pheophytin a. Judged from a comparison of the absorption spectra and photochemical activities of exchanged and control reaction centers, 70^80% of the pheophytin molecules active in charge separation are replaced by 13 1 -deoxo-13 1 -hydroxy-pheophytin a after double application of the exchange procedure. The new molecule at the active branch was not active photochemically. This appears to be the first stable preparation in which a redox active chromophore of the reaction center of photosystem II was modified by chemical substitution. The data are compatible with the presence of an active and inactive branch of cofactors, as in bacterial reaction centers. Possible applications of the 13 1 -deoxo-13 1 -hydroxy-pheophytin a-exchanged preparation to the spectral and functional analysis of native reaction centers of photosystem II are discussed.z 1999 Federation of European Biochemical Societies.