Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function

Abstract

Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.

Biosynthesis of artificial microperoxidases by exploiting the secretion and cytochrome c maturation apparatuses of Escherichia coli

“…Furthermore, the midpoint potential of the H 6 -tagged heme peptides in the absence of imidazole is close to the midpoint potential of acetylated MP-11 in the presence of the strong iron ligand imidazole. A requirement of the tag for heme insertion seems unlikely, because it is known that cytochromes c lacking a sixth heme ligand still have covalently bound heme (36,37). We therefore suspect that the H 6 tag protects the heme-binding peptide from degradation.…”

The activity of sigma E, an Escherichia coli heat-inducible sigma-factor, is modulated by expression of outer membrane proteins.

“…These protein fusions are exported to the E. coli periplasm, and the CycA portion is then rapidly degraded. This indicates that these hybrid proteins do not fold properly (Brandner et al 1991}. Expression of these misfolded periplasmic proteins did not alter Ea r activity (Fig.…”

“…The following plasmids were used to express the proteins indicated in parentheses and have been described previously: pKS 17 {DegP) (Strauch and Beckwith 1988), pMY111 (OmpC) {Mizuno et al 1983), pMY222 (OmpF) {Ramakrishnan et al 1985, pML21 (OmpT) {Grodberg et al 1988}, pC2A11, pC2L40, and pC2Q119 (CycA-AP fusions) (Brandner et al 1991), pGMC1 (Pt~c-OmpC] and pKMCtd (P,~c-OmpCtd) {Catron and Schnaitman 1987}, and pHtrA {DegP) (Lipinska et al 1988). To make pP3CAT, which was used in the multicopy selection, a 37-bp fragment of the P3 promoter of rpoH (Erickson and Gross 1989) was cloned into pKK232-8 {Pharmacia), resulting in pJM14.…”

The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment

“…PhoA hybrid proteins indicated that the signal peptide and first 29 residues of mature CycA are sufficient for covalent heme attachment (6), these experiments did not address whether synthesis as a higher-molecular-weight precursor was required for ligand association.…”

“…To prepare soluble and membrane fractions, cells were lysed by sonication (37) and extracts were separated by ultracentrifugation (11). To more precisely determine the location of cyt c2, cells were separated into cytoplasmic, periplasmic, CM, and outer membrane (OM) fractions (6,30,37). For these studies, malate and succinate dehydrogenase activities were measured as described previously (23).…”