Mitochondrial cytochrome c biogenesis: no longer an enigma

Abstract

Cytochromes c (cyt c) and c1 are heme proteins that are essential for aerobic respiration. Release of cyt c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for the import of apocytochrome c (apocyt c). Thus, HCCS affects cellular levels of cyt c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles of heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways.

Keywords: cytochrome c; heme attachment; holocytochrome c synthase; mitochondria.

Figure 1

HCCS-mediated cytochrome c heme attachment. (A) Schematic of the biogenesis and functions of mitochondrial cytochrome c discussed in the text. Components involved in cellular respiration (ETC) and apoptosis represent simplified models of each pathway. FECH, ferrochelatase; Fe²⁺, reduced iron; HCCS, holocytochrome c synthase; H⁺, proton; e⁻, electron; CoQ, coenzyme Q; Apaf-1, Apoptotic protease activating factor 1; TOM, transporter outer membrane (B) Pymol-generated structure depicting stereospecific attachment of heme vinyl groups to thiol side chains of Cys15 and Cys18 residues in crystallized human cytochrome c. (For Pymol rendering, PDB 3ZCF chain D was used.) The selected peptide fragment displays residues shown to be important for heme attachment and interaction with HCCS. Residue numbering represents that of the full length sequence of human cytochrome c, starting from the initiating methionine (iMet). However, in most cases iMet is cleaved, such that residue numbering is often reduced by 1 (e.g. Cys15 is Cys14). The rotated orientation of heme is shown in the bottom right, showing the distance disparity between the α-carbons of each vinyl group.

Figure 2

Mechanisms of cytochrome c biogenesis by mitochondrial HCCS. The schematic depicts the ordered steps of HCCS-mediated cytochrome c biogenesis discussed in the text as they are proposed to take place in the mitochondrial intermembrane space (IMS). HCCS is represented as a blue oval with its heme ligand His154 (H154); heme is in red with a grey iron (Fe²⁺) atom; apocytochrome c is depicted in its unfolded state with the conserved C15XXC18H19 motif and its N and C termini. Selected key features for each step are shown as described in the text.

Figure 3

HCCS protein sequence alignment. HCCS sequences from diverse eukaryotic phyla are displayed with conserved residues colored in shades of blue. Functional/structural domains I – IV are discussed in the text. The axial heme ligand residue His154 (numbering of the human HCCS protein) is marked with a yellow star in domain II. For simplification, sequences with extensive gaps were condensed, with the number of removed residues enclosed by brackets. Alignment was generated by ClustalW. The sequences represent nearly all groups (from Box 2) using HCCS, as follows: Human HCCS – Homo sapiens(UniProt P53701); Green alga HCCS – Volvox coli(NCBI XP_002952003); Red alga HCCS – Chondrus crispus(NCBI XP_005713760); Choanoflagellate HCCS – Monosiga brevicollis(NCBI XP_001749606); Fungi HCCS – Saccharomyces cerevisiae(UniProt P06182); Amoeba HCCS – Polysphondylium pallidum(NCBI EFA80147); Apicomplexan HCCS – Plasmodium falciparum(UniProt Q8I5H1); Phytoplankton HCCS – Thalassiosira oceanica(NCBI EJK64518).

Box 1 Figure I

Evolutionary tree with distribution of cytochrome c biogenesis pathways across eukaryotic kingdoms. The format of the tree, including timelines, represents an adaptation of the work from Cavalier-Smith [98 and 99] with the inclusion of new analyses regarding the presence or absence of the identified cytochrome c biogenesis system in the indicated organisms. Those with System I (e.g. CcmF) or System III (i.e.HCCS) are denoted with (I) or (III: HCCS) following the representative organism name. Organisms that lack mitochondria, and thus have neither system, are denoted with “no mito”. The complete scientific names of the designated organisms (from top left to bottom right) are as follows: Saccharomyces cerevisiae, Homo sapiens, Monosiga brevicollis, Capsapora owczarzaki, Entamoeba histolytica, Dictyostelium discoideum, Reticulomyxa filosa, Plasmodium falciparum, Tetrahymena thermophila, Thalassiosira oceanica, Dinenympha exilis, Giardia lamblia, Trichomonas vaginalis, Reclinomonas americana, Naegleria fowleri, Percolomonas cosmopolitus, Malawimonas jakobiformis, Cyanophora paradoxa, Arabidopsis thaliana, Chlamydomonas reinhardtii, Chondrus crispus, Cyanidioschyzon merolae, Trypanosoma brucei, Leishmania major, Euglena gracilis; bya, billion years ago. Filled-in circles mark events in evolution relevant to this review.

Box 2 Figure I

The cytochrome c synthetases of System I (CcmF) and System II (CcsBA). (A) Schematics of CcmF [95] and (B) CcsBA [96] integral membrane proteins are shown. For simplicity, the apocytochrome c and CcmH (which normally complexes with CcmF) are not shown. Four conserved histidine residues have been identified in each synthetase, two in the transmembrane domain (TM-His, yellow stars) and two in the periplasmic domain (P-His, orange stars). The P-His ligands the heme (dashed lines) in the WWD domain (highlighted in green) to allow for proper positioning of the heme vinyl groups for attachment to apocytochrome c. (A) In the case of CcmF, it is proposed that heme in the WWD domain is reduced by an associated b-heme (solid wavy arrow). This b-heme is liganded by the TM-His residues. (B) In contrast, CcsBA is proposed to transport heme across the inner membrane (dashed arrow) to the WWD domain in a reduced state, initially bound by the TM-His residues.

Box 3 Figure I

Conservation of the WWD domain in “heme handling” proteins. The conserved sequence of System III HCCS domain II is displayed above the conserved region of the WWD domains from CcsA in System II and CcmC and CcmF in System I. The WWD domain is shaded in light green. Residue numbering at the top represents that of the human HCCS sequence discussed in the text. PHis1 and PHis2 represent the periplasmic histidine residues found in the representative System I and II proteins that are proposed to ligand the heme bound to the WWD domains in these proteins (see Box 2).