Conserved Residues of the Human Mitochondrial Holocytochrome c Synthase Mediate Interactions with Heme

Abstract

C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS–heme–cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical “correction” supports the proposed role of heme binding for the corresponding residues.

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

“…As expected, the complex with WT cyt c exhibited an ␣ peak maximum at 550.8 nm in the pyridine hemochrome spectrum (Fig. 3E), a value that is consistent with heme containing two covalent attachments (31,32,36). Collectively, these data suggest that, once bound to HCCS, cyt c variants containing deletions in ␣ helix-1 readily form only a single thioether with heme, although both reactive cysteines in the C 15 XXC 18 H 19 heme attachment motif are intact.…”

“…27, 31, and 32). Recombinant HCCS is purified with endogenous heme that is non-covalently bound (27,31,32), and the absence of any covalent linkages to the heme present in the ⌬M13/C18ЈA complex was confirmed by the reduced pyridine hemochrome ␣ absorption maximum value of 555.5 nm (Fig. 4E, red line).…”

“…Complexes with HCCS-Following coexpression, a fraction of cyt c can be co-purified in complex with membrane-bound HCCS (27,31,32), effectively trapping a transitional intermediate during the maturation process. For example, single cysteine or histidine (from the CXXCH motif) FIGURE 2.…”

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

“…Results from biochemical studies on human HCCS and cyt c, functionally expressed in recombinant Escherichia coli, have led to the proposal of a fourstep model for biosynthesis by HCCS ( Fig. 1) (3)(4)(5)(6).…”

“…Step 1 entails the binding of heme; certain HCCS mutants are defective in this step (5), including the E159K substitution that is responsible for micropthalmia with linear skin syndrome (MLS) (HCCS sequence in Fig. S1A, numbering from human HCCS).…”