6 research outputs found
Conserved Residues of the Human Mitochondrial Holocytochrome c Synthase Mediate Interactions with Heme
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