Abstract
Mitochondrial apocytochrome c and c1 are converted to their holoforms in the intermembrane space by attachment of heme to the cysteines of the CXXCH motif through the activity of assembly factors cytochrome c heme lyase and cytochrome c1 heme lyase (CCHL and CC1HL). The maintenance of apocytochrome sulfhydryls and heme substrates in a reduced state is critical for the ligation of heme. Factors that control the redox chemistry of the heme attachment reaction to apocytochrome c are known in bacteria and plastids but not in mitochondria. We have explored the function of Cyc2p, a candidate redox cytochrome c assembly component in yeast mitochondria. We show that Cyc2p is required for the activity of CCHL toward apocytochrome c and c1 and becomes essential for the heme attachment to apocytochrome c1 carrying a CAPCH instead of CAACH heme binding site. A redox function for Cyc2p in the heme lyase reaction is suggested from 1) the presence of a noncovalently bound FAD molecule in the C-terminal domain of Cyc2p, 2) the localization of Cyc2p in the inner membrane with the FAD binding domain exposed to the intermembrane space, and 3) the ability of recombinant Cyc2p to carry the NADPH-dependent reduction of ferricyanide. We postulate that, in vivo, Cyc2p interacts with CCHL and is involved in the reduction of heme prior to its ligation to apocytochrome c by CCHL.
Highlights
Diversity in terms of function is paralleled by a diversity in terms of assembly pathways and a surprising finding was the discovery that three pathways (Systems I–III) for the biogenesis of c-type cytochromes have evolved in bacteria, plastids, and mitochondria
We concluded that Cyc2p is required for the CCHL-dependent assembly of cytochrome c and cytochrome c1 but not for the assembly of cytochrome c1 when catalyzed by CC1HL
Numerous in organello and in vitro studies on the assembly of mitochondrial cytochrome c have established that the chemistry of thioether bond formation can only proceed when the appropriate redox conditions are provided (24, 29, 56 –58), yet the fact that no mitochondrial cytochrome c assembly proteins besides the so-called heme lyases have been identified led to the assumption that in vivo the redox chemistry of the heme lyase reaction was not under the control of dedicated factors and occurred spontaneously in the “reducing” environment of the IMS
Summary
Diversity in terms of function is paralleled by a diversity in terms of assembly pathways and a surprising finding was the discovery that three pathways (Systems I–III) for the biogenesis of c-type cytochromes have evolved in bacteria, plastids, and mitochondria (for reviews, see Refs. 1, 2, and 4 – 6). The biochemical requirements for holocytochrome c formation can be divided into functions needed for the transport and delivery of heme, the reduction of apoprotein cysteinyl thiol and heme co-factor prior to the heme ligation reaction, and the catalysis of thioether bond formation [4] This view was substantiated by the genetic and biochemical analysis of Systems I and II in bacteria and plastids, which led to the identification of multiple assembly factors with proposed or established activity in the transport and chaperoning of heme [4, 7,8,9], the provision of reducing equivalents for maintenance of reduced apocytochromes/ heme substrates (4, 10 –15), and the heme ligation reaction [8, 9, 16]. We discuss the implications of this unique factor in the control of the redox chemistry of the heme lyase reaction
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