Structure, spectroscopic, and redox properties of Rhodobacter sphaeroides reaction centers bearing point mutations near the primary electron donor.
Single mutations of three amino acid residues in the vicinity of the primary electron donor, P, in the reaction center (RC) from Rhodobacter (Rb.) sphaeroides were constructed and characterized in order to study the effects of hydrogen-bonding on the physical properties of P. The mutations, Phe M197-->Tyr, Met L248-->Thr, and Ser L244-->Gly, represent single amino acid changes near P designed to introduce residues found in Rhodopseudomonas (Rps.) viridis and to, thus, probe the effects of nonconserved residues. The mutations were designed to change the nonconserved H-bonding interactions of P in Rb. sphaeroides, at the level of a C2 acetyl, a C9 keto, and a C10 ester carbonyl of P, respectively, to those present in Rps. viridis. The Fourier transform (pre)resonance Raman (FTRR) spectra of P, in its reduced and oxidized states, from reaction centers of these mutants were studied to determine modifications of H-bond interactions of the pi-conjugated C2 acetyl and C9 keto carbonyl groups and the C10 carbomethoxy ester carbonyl groups of P. The vibrational spectra of reduced P in the Met L248-->Thr and Ser L244-->Gly mutants reveal no evidence for changes in the H-bonding pattern of P; this suggests that for Rb. sphaeroides wild type, Ser L244 is not H-bonded to the C10 ester carbonyl of PL. The vibrational spectrum of reduced P from the Phe M197-->Tyr mutant compared to that of wild type can unambiguously be interpreted in terms of the formation of a new H-bond with an acetyl carbonyl of P, specifically PM. Correlating with the new H-bond, the Phe M197-->Tyr mutant exhibits an electronic absorption spectrum where the P absorption band is significantly perturbed. Intact cell and chromatophore photobleaching spectra of the same mutant indicate that the P absorption band has red-shifted by ca. 10 nm; no such behavior is observed for the other mutants. As well, the P-->BPheL electron transfer rate does not seem to strongly depend on the H-bonding of the C2 acetyl carbonyl of PM to a tyrosine residue. The EPR zero-field splitting parameters, E and D, of the primary donor triplet are only slightly modified in the mutant reaction centers, on the order of 1%.(ABSTRACT TRUNCATED AT 400 WORDS)
Wachtveitl
J
J
Department of Membrane Biochemistry, Max-Planck-Institut für Biochemie, München, Germany.
Farchaus
J W
JW
Das
R
R
Lutz
M
M
Robert
B
B
Mattioli
T A
TA
D001429:Bacteriochlorophylls / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005583:Fourier Analysis; D006860:Hydrogen Bonding; D045342:Light-Harvesting Protein Complexes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics; D012241:Rhodopseudomonas / Q000737:chemistry; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D017186:Titrimetry
D001429:Bacteriochlorophylls; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins