title: The flavoprotein Cyc2p, a mitochondrial cytochrome c assembly factor, is a NAD(P)H-dependent haem reductase.
abstract: Cytochrome c assembly requires sulphydryls at the CXXCH haem binding site on the apoprotein and also chemical reduction of the haem co-factor. In yeast mitochondria, the cytochrome haem lyases (CCHL, CC(1) HL) and Cyc2p catalyse covalent haem attachment to apocytochromes c and c(1) . An in vivo indication that Cyc2p controls a reductive step in the haem attachment reaction is the finding that the requirement for its function can be bypassed by exogenous reductants. Although redox titrations of Cyc2p flavin (E(m) = -290 mV) indicate that reduction of a disulphide at the CXXCH site of apocytochrome c (E(m) = -265 mV) is a thermodynamically favourable reaction, Cyc2p does not act as an apocytochrome c or c(1) CXXCH disulphide reductase in vitro. In contrast, Cyc2p is able to catalyse the NAD(P)H-dependent reduction of hemin, an indication that the protein's role may be to control the redox state of the iron in the haem attachment reaction to apocytochromes c. Using two-hybrid analysis, we show that Cyc2p interacts with CCHL and also with apocytochromes c and c(1) . We postulate that Cyc2p, possibly in a complex with CCHL, reduces the haem iron prior to haem attachment to the apoforms of cytochrome c and c(1) .
authors.affiliation: Departments of Molecular Genetics and Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Vincent
authors.initials: V
authors.lastname: Corvest
authors.forename: Darren A
authors.initials: DA
authors.lastname: Murrey
authors.forename: Mazakazu
authors.initials: M
authors.lastname: Hirasawa
authors.forename: David B
authors.initials: DB
authors.lastname: Knaff
authors.forename: Bernard
authors.initials: B
authors.lastname: Guiard
authors.forename: Patrice P
authors.initials: PP
authors.lastname: Hamel
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D008190:Lyases / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: C083336:CYC2 protein, S cerevisiae; D002352:Carrier Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D009249:NADP; D045304:Cytochromes c; D008190:Lyases; C031581:cytochrome C synthetase