R. A. Khatypov

Predicted bacteriorhodopsin from Exiguobacterium sibiricum is a functional proton pump

The 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.

SPECTRAL AND PHOTOCHEMICAL PROPERTIES OF BOROHYDRIDE-TREATED D1-D2-CYTOCHROME B-559 COMPLEX OF PHOTOSYSTEM II

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 (BH− 4). 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 BH− 4 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 131‐deoxo‐131‐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°C at 679.5–680 nm and 542 nm, respectively.

Reaction centers of photosystem II with a chemically-modified pigment composition: exchange of pheophytins with 131-deoxo-131-hydroxy-pheophytin a

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 131‐deoxo‐131‐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 131‐deoxo‐131‐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 131‐deoxo‐131‐hydroxy‐pheophytin a‐exchanged preparation to the spectral and functional analysis of native reaction centers of photosystem II are discussed.

Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 13(1)-deoxo-13(1)-hydroxy-pheophytin a and comparison of their 6 K absorption spectra.

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 131-deoxo-131-hydroxy-pheophytin a (131-OH-Pheo). The absorption bands of 131-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC1×) or twice (RC2×). HPLC analysis and Pheo QX absorption at 543 nm show that in RC1× about 50% of Pheo is replaced and in RC2× about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo− is unmodified in RC1× and decreased only in RC2×, suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the QY region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC1× or RC2×, showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength.

Substitution of Isoleucine L177 by Histidine Affects the Pigment Composition and Properties of the Reaction Center of the Purple Bacterium Rhodobacter sphaeroides

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+QAA− appears to be an interesting object to study the mechanisms of the first steps of the primary electron transfer in photosynthesis.

Primary charge separation within P870* in wild type and heterodimer mutants in femtosecond time domain☆

Primary charge separation dynamics in the reaction center (RC) of purple bacterium Rhodobacter sphaeroides and its P870 heterodimer mutants have been studied using femtosecond time-resolved spectroscopy with 20 and 40fs excitation at 870nm at 293K. Absorbance increase in the 1060-1130nm region that is presumably attributed to P(A)(δ+) cation radical molecule as a part of mixed state with a charge transfer character P*(P(A)(δ+)P(B)(δ-)) was found. This state appears at 120-180fs time delay in the wild type RC and even faster in H(L173)L and H(M202)L heterodimer mutants and precedes electron transfer (ET) to B(A) bacteriochlorophyll with absorption band at 1020nm in WT. The formation of the P(A)(δ+)B(A)(δ-) state is a result of the electron transfer from P*(P(A)(δ+)P(B)(δ-)) to the primary electron acceptor B(A) (still mixed with P*) with the apparent time delay of ~1.1ps. Next step of ET is accompanied by the 3-ps appearance of bacteriopheophytin a(-) (H(A)(-)) band at 960nm. The study of the wave packet formation upon 20-fs illumination has shown that the vibration energy of the wave packet promotes reversible overcoming of an energy barrier between two potential energy surfaces P* and P*(P(A)(δ+)B(A)(δ-)) at ~500fs. For longer excitation pulses (40fs) this promotion is absent and tunneling through an energy barrier takes about 3ps. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Substitution of isoleucine L177 by histidine in Rhodobacter sphaeroides reaction center results in the covalent binding of PA bacteriochlorophyll to the L subunit

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, PA. The chemical nature of this covalent interaction remains to be identified.

The site-directed mutation I(L177)H in Rhodobacter sphaeroides reaction center affects coordination of P(A) and B(B) bacteriochlorophylls.

To explore the influence of the I(L177)H single mutation on the properties of the nearest bacteriochlorophylls (BChls), three reaction centers (RCs) bearing double mutations were constructed in the photosynthetic purple bacterium Rhodobacter sphaeroides, and their properties and pigment content were compared with those of the correspondent single mutant RCs. Each pair of the mutations comprised the amino acid substitution I(L177)H and another mutation altering histidine ligand of BChl P(A) or BChl B(B). Contrary to expectations, the double mutation I(L177)H+H(L173)L does not bring about a heterodimer RC but causes a 46nm blue shift of the long-wavelength P absorbance band. The histidine L177 or a water molecule were suggested as putative ligands for P(A) in the RC I(L177)H+H(L173)L although this would imply a reorientation of the His backbone and additional rearrangements in the primary donor environment or even a repositioning of the BChl dimer. The crystal structure of the mutant I(L177)H reaction center determined to a resolution of 2.9Å shows changes at the interface region between the BChl P(A) and the monomeric BChl B(B). Spectral and pigment analysis provided evidence for β-coordination of the BChl B(B) in the double mutant RC I(L177)H+H(M182)L and for its hexacoordination in the mutant reaction center I(L177)H. Computer modeling suggests involvement of two water molecules in the β-coordination of the BChl B(B). Possible structural consequences of the L177 mutation affecting the coordination of the two BChls P(A) and B(B) are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Properties of mutant reaction centers of Rhodobacter sphaeroides with substitutions of histidine L153, the axial Mg2+ ligand of bacteriochlorophyll BA

Mutant reaction centers (RC) from Rhodobacter sphaeroides have been studied in which histidine L153, the axial ligand of the central Mg atom of bacteriochlorophyll BA molecule, was substituted by cysteine, methionine, tyrosine, or leucine. None of the mutations resulted in conversion of the bacteriochlorophyll BA to a bacteriopheophytin molecule. Isolated H(L153)C and H(L153)M RCs demonstrated spectral properties similar to those of the wild-type RC, indicating the ability of cysteine and methionine to serve as stable axial ligands of the Mg atom of bacteriochlorophyll BA. Because of instability of mutant H(L153)L and H(L153)Y RCs, their properties were studied without isolation of these complexes from the photosynthetic membranes. The most prominent effect of the mutations was observed with substitution of histidine by tyrosine. According to the spectral data and the results of pigment analysis, the BA molecule is missing in the H(L153)Y RC. Nevertheless, being associated with the photosynthetic membrane, this RC can accomplish photochemical charge separation with quantum yield of approximately 7% of that characteristic of the wild-type RC. Possible pathways of the primary electron transport in the H(L153)Y RC in absence of photochemically active chromophore are discussed.

Properties of Rhodobacter sphaeroides photosynthetic reaction center with double amino acid substitution I(L177)H+H(M182)L

Histidine M182 in the reaction center (RC) of Rhodobacter sphaeroides serves as the fifth ligand of the bacterio-chlorophyll (BChl) BB Mg atom. When this His is substituted by an amino acid that is not able to coordinate Mg, bacterio-pheophytin appears in the BB binding site instead of BChl (Katilius, E., et al. (1999) J. Phys. Chem. B, 103, 7386–7389). We have shown that in the presence of the additional mutation I(L177)H the coordination of the BChl BB Mg atom in the double mutant I(L177)H+H(M182)L RC still remains. Changes in the double mutant RC absorption spectrum attributed to BChl absorption suggest that BChl BB Mg atom axial ligation might be realized not from the usual α-side of the BChl macrocycle, but from the opposite, β-side. Weaker coordination of BChl BB Mg atom compared to the other mutant RC BChl molecules suggests that not an amino acid residue but a water molecule might be a possible ligand. The results are discussed in the light of the structural changes that occurred in the RC upon Ile/His substitution in the L177 position.