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 C-9 keto, and a C-10 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 C-9 keto carbonyl groups and the C-10 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 C-10 ester carbonyl of P(L). 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 P(M). 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 --> BPhe(L) electron transfer rate does not seem to strongly depend on the H-bonding of the C2 acetyl carbonyl of P(M) 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%. The FT Raman spectrum of the oxidized primary donor, P+, of the Phe M197 --> Tyr mutant shows a 3 cm-1 upshift of the C-9 keto carbonyl band, as compared to wild type, which may indicate that the + charge is slightly more localized on the P(L) component in P.+. Optical redox titrations of the Met L248 --> Thr mutant indicate that the redox midpoint potential is unchanged (within +/- 10 mV) compared to that of wild type; the Phe M197 --> Tyr and Ser L244 --> Gly mutants showed a small but significant increase of +20-30 and +30-40 mV, respectively, indicating that the tyrosine-donated H-bond to the C2 acetyl carbonyl of P(M) in the Phe M197 --> Tyr mutant plays no dominant role in modulating the redox properties of P. This study suggests that the introduction of a new H-bond on the C2 acetyl carbonyl of P in Rb. sphaeroides, by a tyrosine residue at position M197, only modestly modifies its spectral properties and is not a dominant determinant in its redox and triplet EPR properties.