INTERACTION SITE FOR SOLUBLE CYTOCHROMES ON THE TETRAHEME CYTOCHROME SUBUNIT BOUND TO THE BACTERIAL PHOTOSYNTHETIC REACTION-CENTER MAPPED BY SITE-DIRECTED MUTAGENESIS

The crystallographic structure of the Blastochloris (formerly called R hodopseudomonas) viridis tetraheme cytochrome subunit bound to the pho tosynthetic reaction center (RC) suggests that all four hemes are loca ted close enough to the surface of the protein to accept electrons fro m soluble cytochrome c(2). TO identify experimentally the site of this reaction we prepared site-directed mutants of Rubrivivax gelatinosus RCs with surface charge substitutions in the bound cytochrome subunit and studied the kinetics of their reduction by soluble cytochromes (mi tochondrial horse cytochrome c, Blc. viridis cytochrome c(2), and Rvi. gelatinosus cytochrome cs). Ln comparison with the wild-type, the mut ants E79K (glutamate-79 substituted by lysine), E93K (glutamate-93 sub stituted by lysine), and E85K (glutamate-85 substituted by lysine) loc ated near the solvent-exposed edge of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll, exhibited decrease d second-order rate constants for the reaction between the tetraheme s ubunit and the soluble cytochromes, Double charge substitutions in thi s region: E79K/E85K (glutamate-79 and -85 both replaced by lysine) and E93K/E85K (glutamate-93 and -85 both replaced by lysine) appeared to show an additive inhibitory effect. Mutations in other charged regions did not alter the kinetics of electron transfer between bound and sol uble cytochromes. In light of the available structural information on Blc, viridis RC, these results indicate that the cluster of acidic res idues immediately surrounding the distal heme 1 of the RC-bound tetrah eme subunit forms an electrostatically favorable binding site for solu ble cytochromes, Thus, all four hemes in the subunit seem to be direct ly involved in the electron transfer toward the photo-oxidized special pair of bacteriochlorophyll. On the basis of these findings, a model is proposed for the hypothetical cytochrome c(2)-RC transient complex for Blc. viridis.