The flavoprotein Cyc2p, a mitochondrial cytochrome c assembly factor, is a NAD(P)H-dependent haem reductase

Abstract

Summary: 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 1HL) 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=-290mV) indicate that reduction of a disulphide at the CXXCH site of apocytochrome c (E m=-265mV) 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. © 2012 Blackwell Publishing Ltd.