INTERACTION SITE FOR SOLUBLE CYTOCHROMES ON THE TETRAHEME CYTOCHROME SUBUNIT BOUND TO THE BACTERIAL PHOTOSYNTHETIC REACTION-CENTER MAPPED BY SITE-DIRECTED MUTAGENESIS
A. Osyczka et al., INTERACTION SITE FOR SOLUBLE CYTOCHROMES ON THE TETRAHEME CYTOCHROME SUBUNIT BOUND TO THE BACTERIAL PHOTOSYNTHETIC REACTION-CENTER MAPPED BY SITE-DIRECTED MUTAGENESIS, Biochemistry, 37(34), 1998, pp. 11732-11744
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.