Human mitochondrial holocytochrome c synthase’s heme binding, maturation determinants, and complex formation with cytochrome c

Abstract

Proper functioning of the mitochondrion requires the orchestrated assembly of respiratory complexes with their cofactors. Cytochrome c , an essential electron carrier in mitochondria and a critical component of the apoptotic pathway, contains a heme cofactor covalently attached to the protein at a conserved CXXCH motif. Although it has been known for more than two decades that heme attachment requires the mitochondrial protein holocytochrome c synthase (HCCS), the mechanism remained unknown. We purified membrane-bound human HCCS with endogenous heme and in complex with its cognate human apocytochrome c . Spectroscopic analyses of HCCS alone and complexes of HCCS with site-directed variants of cytochrome c revealed the fundamental steps of heme attachment and maturation. A conserved histidine in HCCS (His154) provided the key ligand to the heme iron. Formation of the HCCS:heme complex served as the platform for interaction with apocytochrome c . Heme was the central molecule mediating contact between HCCS and apocytochrome c . A conserved histidine in apocytochrome c (His19 of CXXCH) supplied the second axial ligand to heme in the trapped HCCS:heme:cytochrome c complex. We also examined the substrate specificity of human HCCS and converted a bacterial cytochrome c into a robust substrate for the HCCS. The results allow us to describe the molecular mechanisms underlying the HCCS reaction.

A heme-binding domain controls regulation of ATP-dependent potassium channels

“…In the case of the Slo1 channels (12), a cytochrome c-like CXXCH motif has been implicated (12,28). However, this raises immediate questions that do not chime with established patterns of behavior in other heme proteins, because most proteins bind heme reversibly (i.e., noncovalently), whereas cytochrome c uses complex and specialized biosynthetic machinery to bind heme irreversibly (i.e., covalently) through thioether bonds from the Cys residues of the CXXCH motif to the heme vinyl groups (29,30). There is as yet no evidence that ion channels proteins use similarly specialized biosynthetic machinery; thus, it seems unlikely that their heme is covalently attached.…”

Molecular Basis Behind Inability of Mitochondrial Holocytochrome c Synthase to Mature Bacterial Cytochromes

“…Previous genetic studies have shown that bacterial cytochromes are incompatible substrates for HCCS-mediated maturation (25,27,28,30). However, it has also been shown by two independent groups that making three mutations in the N-terminal ␣ helix-1 region of bacterial cytochromes can convert them into a substrate that is recognized and matured by HCCS (27,28).…”

“…However, it has also been shown by two independent groups that making three mutations in the N-terminal ␣ helix-1 region of bacterial cytochromes can convert them into a substrate that is recognized and matured by HCCS (27,28). c-type cytochromes from the alphaproteobacteria Rhodobacter and Paracoccus have glutamate residues at the sequence positions equivalent to Lys 8 and Ile 10 found in human cyt c (see Fig.…”

“…2A, blue asterisks). Furthermore, following an important Phe residue found in human (Phe 11 ) and some bacterial cytochromes (21,27), a residue at the sequence position corresponding to Met 13 in human cyt c (see Fig. 2A) is missing in certain bacterial cytochromes.…”

“…By contrast, the eukaryotic HCCS appears to require a more extended recognition domain than just the CXXCH motif for cytochrome maturation, specifically sequence features or residues located in the cyt c N terminus (see Fig. 1) (21,(25)(26)(27)(28)(29).…”