Interaction Site for Soluble Cytochromes on the Tetraheme Cytochrome Subunit Bound to the Bacterial Photosynthetic Reaction Center Mapped by Site-Directed Mutagenesis

Abstract

The crystallographic structure of the Blastochloris (formerly called Rhodopseudomonas) viridis tetraheme cytochrome subunit bound to the photosynthetic reaction center (RC) suggests that all four hemes are located close enough to the surface of the protein to accept electrons from soluble cytochrome c2. 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 (mitochondrial horse cytochrome c, Blc. viridis cytochrome c2, and Rvi. gelatinosus cytochrome c8). In comparison with the wild-type, the mutants E79K (glutamate-79 substituted by lysine), E93K (glutamate-93 substituted by lysine), and E85K (glutamate-85 substituted by lysine) located near the solvent-exposed edge of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll, exhibited decreased second-order rate constants for the reaction between the tetraheme subunit and the soluble cytochromes. Double charge substitutions in this 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 soluble cytochromes. In light of the available structural information on Blc. viridis RC, these results indicate that the cluster of acidic residues immediately surrounding the distal heme 1 of the RC-bound tetraheme subunit forms an electrostatically favorable binding site for soluble cytochromes. Thus, all four hemes in the subunit seem to be directly 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 c2-RC transient complex for Blc. viridis.

A New Cytochrome Subunit Bound to the Photosynthetic Reaction Center in the Purple Bacterium, Rhodovulum sulfidophilum

“…Recently, we showed direct evidence through mutagenesis on the cytochrome subunit of Rubrivivax gelatinosus that electron transfer to the cytochrome subunit from soluble cytochromes occurred via electrostatic interactions between negatively charged amino acids surrounding heme-1 and positively charged amino acids on the soluble cytochromes (12), which is consistent with a suggestion by Knaff et al (13). These charged residues on the cytochrome subunit are well conserved among many purple bacteria so far examined, suggesting that the most distant heme-1 works as a direct electron acceptor from the soluble electron carriers (9).…”

“…It has been shown that electron transfer reactions from soluble electron donors to the cytochrome subunit are controlled by charge interactions (12,13). The study of site-directed mutagenesis in R. gelatinosus has shown that negatively charged amino acids (Glu) surrounding the heme-1 (positions 82, 113, and 129 in Fig.…”

“…The study of site-directed mutagenesis in R. gelatinosus has shown that negatively charged amino acids (Glu) surrounding the heme-1 (positions 82, 113, and 129 in Fig. 4), which are well conserved among purple bacteria, have a stimulative effect on the rate of electron transfer, suggesting that the heme-1 of the RC-bound cytochrome subunit is a direct electron acceptor from soluble electron donors in purple bacteria (9,12). The absence of a heme-1-binding domain in R. sulfidophilum suggests that the site of interaction with soluble cytochromes is different from that in usual purple bacteria.…”

“…These observations suggest that the electron transfer between the cytochrome subunit and soluble electron donors does not occur on the surface around the heme-1 but may occur around the other hemes of the cytochrome subunit in R. sulfidophilum. An unidentified interaction site on the cytochrome subunit will be revealed by the method of site-directed mutagenesis, as has been done in R. gelatinosus (12).…”

Tuning Heme Redox Potentials in the Cytochrome c Subunit of Photosynthetic Reaction Centers

“…Quinone oxidation taking place at the cyt bc 1 membrane protein complex establishes a proton gradient across the membrane, which is converted to chemical energy at the ATP synthase complex. The electrons are returned back to the RC by the periplasmic soluble electron carrier cyt c 2 that is supposed to bind to the C chain of the RC, probably close to the outermost heme group to reduce the oxidized hemes (4).…”