SPECIFIC ALTERATION OF THE OXIDATION POTENTIAL OF THE ELECTRON-DONOR IN REACTION CENTERS FROM RHODOBACTER-SPHAEROIDES

The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and histidine residue s in the reaction center from Rhodobacter sphaeroides have been invest igated. Site-directed mutations were designed to add or remove hydroge n bonds between the 2-acetyl groups of the dimer and histidine residue s at the symmetry-related sites His-L168 and Phe-M197, and between the 9-keto groups and Leu-L131 and Leu-M160. The addition of a hydrogen b ond was correlated with an increase in the dimer midpoint potential. M easurements on double and triple mutants showed that changes in the mi dpoint potential due to alterations at the individual sites were addit ive. Midpoint potentials ranging from 410 to 765 mV, compared with 505 mV for wild type, were achieved by various combinations of mutations. The optical absorption spectra of the reaction centers showed relativ ely minor changes in the position of the donor absorption band, indica ting that the addition of hydrogen bonds to histidines primarily desta bilized the oxidized state of the donor and had little effect on the e xcited state relative to the ground state. Despite the change in energ y of the charge-separated states by up to 260 meV, the mutant reaction centers were still capable of electron transfer to the primary quinon e. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quinone, and a fi t of these data using the Marcus equation indicated that the reorganiz ation energy for this reaction is approximate to 400 meV higher than t he change in free energy in wild type. The mutants were still capable of photosynthetic growth, although at reduced rates relative to the wi ld type. These results suggest a role for protein-cofactor interaction s-in particular, histidine-donor interactions-in establishing the redo x potentials needed for electron transfer in biological systems.