Influence of Asn/His L166 on the hydrogen-bonding pattern and redox potential of the primary donor of purple bacterial reaction centers.
The primary electron donor (P) of the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter (Rb.) sphaeroides is constituted of two bacteriochlorophyll molecules in excitonic interaction. The C2 acetyl carbonyl group of one of the two bacteriochlorophyll molecules (PL), the one more closely associated with the L polypeptide subunit, is engaged in a hydrogen bond with histidine L168, while the other pi-conjugated carbonyl groups of P are free from such hydrogen-bonding interactions. The three-dimensional X-ray crystal structures of the RC from several strains of Rb. sphaeroides reveal that asparagine L166 probably interacts indirectly with P through His L168. Such an interaction is expected to modulate the hydrogen bond between P and His L168, a residue which is highly conserved in purple bacteria. RC mutants of Rb. sphaeroides where asparagine L166 was genetically replaced by leucine [NL(L166)], histidine [NH(L166)], and aspartate [ND(L166)] were studied using Fourier transform (FT) Raman spectroscopy. All of these mutations resulted in an increase in the strength of the hydrogen bond between His L168 and the acetyl carbonyl group of P(L), as observed in the FT Raman spectrum, by the 2-4 cm(-1) decrease in vibrational frequency of the 1620 cm(-1) band which has been assigned to this specific acetyl carbonyl group [Mattioli, T. A., Lin, X., Allen, J. P., & Williams, J. C. (1995) Biochemistry 34, 6142-6152]. At pH 8, the NH(L166) mutation showed the greatest change in the P0/P.+ redox midpoint potential (515 mV), increasing it by ca. 30 mV compared to that of wild type (485 mV). A similar increase in P0/P.+ redox midpoint potential for NH(L166) compared to that of wild type is also observed at pH 5, 6, and 9.5. The p0/P.+ midpoint potential of the NL(L166) mutant was comparable to that of wild type at all pH values. In contrast, for the ND(L166) mutant, the midpoint potential shows a markedly different pH dependency, being 25 mV higher than wild type at pH 5 but 20 mV lower than wild type at pH 9.5. The hydrogen bond interactions of the primary electron donor from Rhodospirillum (Rsp.) centenum were determined from the FT Raman vibrational spectrum which exhibits a 1616 cm(-1) band similar to what is seen in the NH(L166) and ND(L166) Rb. sphaeroides mutants. Comparison of the sequence of the L subunit determined for the Rsp. centenum RC with that of other species indicates that positions L166 and L168 are occupied by His residues. The stronger hydrogen bond between the conserved His L168 and the acetyl carbonyl group of P(L), observed in the primary donor of Rsp. centenum and of several bacterial species which are known to possess a histidine residue at the analogous L166 position, is proposed to be due to interactions between these two histidine residues.
Ivancich
A
A
Departement de Biologie Cellulaire et Moleculaire, CEA and CNRS URA 2096, CEA/Saclay, Gif-sur-Yvette, France.
Mattioli
T A
TA
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D004563:Electrochemistry; D004579:Electron Transport; D006639:Histidine / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D045342:Light-Harvesting Protein Complexes; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000737:chemistry*; D012241:Rhodopseudomonas / Q000737:chemistry; D016415:Sequence Alignment; D017421:Sequence Analysis; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D006639:Histidine; D001216:Asparagine