Comparing substrate specificity between cytochrome c maturation and cytochromecheme lyase systems for cytochrome c biogenesis

Abstract

Hemes c are characterized by their covalent attachment to a polypeptidevia a widely conserved CXXCH motif. There are multiple biological systems that facilitate hemec biogenesis. System I, the cytochrome c maturation (CCM) system, is found in many bacteria and is commonly employed in the maturation of bacterial cytochromesc in Escherichia coli-based expression systems. System III, cytochrome cheme lyase (CCHL), is an enzyme found in the mitochondria of many eukaryotes and is used for heterologous expression of mitochondrial holocytochromes c. To test CCM specificity, a series of Hydrogenobacter thermophiluscytochrome c₅₅₂ variants was successfully expressed and matured by the CCM system with CX_nCH motifs where n = 1–4, further extending the known substrate flexibility of the CCM system by successful maturation of a bacterial cytochrome c with a novel CXCH motif. Horse cytochrome c variants with both expanded and contracted attachment motifs (n = 1–3) were also tested for expression and maturation by both CCM and CCHL, allowing direct comparison of CCM and CCHL substrate specificities. Successful maturation of horse cytochrome c by CCHL with an extended CXXXCH motif was observed, demonstrating that CCHL shares the ability of CCM to mature hemesc with extended heme attachment motifs. In contrast, two single amino acid mutants were found in horse cytochrome c that severely limit maturation by CCHL, yet were efficiently matured with CCM. These results identify potentially important residues for the substrate recognition of CCHL.