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 p rimary electron donor, P, in the reaction center (RC) from Rhodobacter (Rb.) sphaeroides were constructed and characterized in order to stud y 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 resid ues found in Rhodopseudomonas (Rps.) viridis and to, thus, probe the e ffects of nonconserved residues. The mutations were designed to change the nonconserved H-bonding interactions of P in Rb. sphaeroides, at t he level of a C2 acetyl, a C-9 keto, and a C-10 ester carbonyl of P, r espectively, to those present in Rps. viridis. The Fourier transform ( pre)resonance Raman (FTRR) spectra of P, in its reduced and oxidized s tates, from reaction centers of these mutants were studied to determin e 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 t ype, 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 com pared to that of wild type can unambiguously be interpreted in terms o f the formation of a new H-bond with an acetyl carbonyl of P, specific ally P(M). Correlating with the new H-bond, the Phe M197 --> Tyr mutan t exhibits an electronic absorption spectrum where the P absorption ba nd is significantly perturbed. Intact cell and chromatophore photoblea ching spectra of the same mutant indicate that the P absorption band h as red-shifted by ca. 10 nm; no such behavior is observed for the othe r 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 ty pe, 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-do nated 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 posi tion M197, only modestly modifies its spectral properties and is not a dominant determinant in its redox and triplet EPR properties.