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Human Mitochondrial Holocytochrome C Synthasets Heme Binding, Maturation Determinants, and Complex Formation with Cytochrome C

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Human Mitochondrial Holocytochrome C Synthasets Heme Binding, Maturation Determinants, and Complex Formation with Cytochrome C Human mitochondrial holocytochrome c synthase’s PNAS PLUS heme binding, maturation determinants, and complex formation with cytochrome c Brian San Francisco, Eric C. Bretsnyder, and Robert G. Kranz1 Department of Biology, Washington University in St. Louis, St. Louis, MO 63130 Edited by Sabeeha Sabanali Merchant, University of California, Los Angeles, CA, and approved October 16, 2012 (received for review August 10, 2012) Proper functioning of the mitochondrion requires the orchestrated cytochromes in mitochondria: cytochrome c maturation (CCM) assembly of respiratory complexes with their cofactors. Cyto- (12–15) and cytochrome c heme lyase, also called “holocytochrome chrome c, an essential electron carrier in mitochondria and a critical c synthase” (HCCS, the term used in this paper) (16, 17). The component of the apoptotic pathway, contains a heme cofactor CCM system is composed of eight or nine integral membrane covalently attached to the protein at a conserved CXXCH motif. proteins and functions in the mitochondrial inner membrane Although it has been known for more than two decades that heme of plants and some protozoa and in the cytoplasmic membranes of attachment requires the mitochondrial protein holocytochrome c alpha- and gamma-proteobacteria (18). Most mitochondria (e.g., fi synthase (HCCS), the mechanism remained unknown. We puri ed those of fungi, invertebrates, vertebrates, and some protozoa) use membrane-bound human HCCS with endogenous heme and in HCCS for synthesis of cytochrome c. In fungi, two related c complex with its cognate human apocytochrome . Spectroscopic homologs, HCCS and HCC S, are dedicated to maturation of analyses of HCCS alone and complexes of HCCS with site-directed 1 cytochrome c and cytochrome c , respectively (19, 20), whereas in variants of cytochrome c revealed the fundamental steps of heme 1 animals a single HCCS enzyme is active toward both cytochrome c attachment and maturation. A conserved histidine in HCCS (His154) and cytochrome c (17, 21). Additionally, in yeast and other fungi, provided the key ligand to the heme iron. Formation of the HCCS: 1 the FAD-containing protein Cyc2p is required for heme attach- heme complex served as the platform for interaction with apocy- c – BIOCHEMISTRY tochrome c. Heme was the central molecule mediating contact ment to apocytochrome (22 24). between HCCS and apocytochrome c. A conserved histidine in The human HCCS has been increasingly implicated in disease. apocytochrome c (His19 of CXXCH) supplied the second axial li- For example, chromosomal mutations in the gene encoding “ gand to heme in the trapped HCCS:heme:cytochrome c complex. HCCS can lead to a condition called microphthalmia with linear We also examined the substrate specificity of human HCCS and skin defects syndrome” (25, 26). Additionally, a role for HCCS in converted a bacterial cytochrome c into a robust substrate for the apoptosis (separate from that of cytochrome c) has been de- HCCS. The results allow us to describe the molecular mechanisms scribed in injured motor neurons (27). Despite the identification underlying the HCCS reaction. of HCCS as the gene product responsible for heme attachment to cytochrome c in Saccharomyces cerevisiae more than 25 y ago (19), CCHL | microphthalmia | biogenesis | apoptosis | post-translational the enzyme has never been purified or characterized, and the modification mechanism of covalent heme attachment is unknown (16, 17). In yeast, HCCS is nuclear encoded and is imported directly into the enewed interest in mitochondria stems from recent associa- mitochondrial IMS from the cytosol via the translocase of the Rtions of malfunctioning mitochondria with many cancers (1), outer membrane complex (20, 28, 29). Studies in S. cerevisiae have neurological diseases (2–4), and even reduced life span (5). The shown that HCCS is membrane associated in mitochondria and basis for these associations lies in the respiratory chains that is exposed to the IMS (28–30). The apparent absence of trans- power aerobic life. 3D structures for many respiratory complexes membrane helices suggests that membrane association is likely and carriers (6–8) have elucidated the detailed mechanisms of peripheral. Pioneering studies by Sherman and colleagues (30, electron transport, proton pumping, the reduction of oxygen to 31) and by Neupert and colleagues (32, 33) have demonstrated water, and ATP formation. Less is known about the biogenesis of that HCCS also plays an essential role in the import of the these respiratory chain components. The synthesis and insertion apocytochrome c from the cytosol to the mitochondrion. It is of cofactors (e.g., heme and metals) into large, multisubunit unknown how heme enters the IMS from its site of synthesis in the membrane complexes represents a new frontier in the study of mitochondrial matrix, although early studies showed that reduced c mitochondrial function. The -type cytochromes, among the best- heme (Fe2+) is necessary for covalent attachment to cytochrome studied players in mitochondrial electron transport (9, 10), are c (34, 35). Preliminary genetic results suggested that heme bind- redox-active heme proteins whose biosynthesis is only now be- ing by HCCS occurred at partially conserved cysteine–proline ginning to be understood. Cytochrome c is a soluble electron sequences (36), which serve as heme-regulatory motifs in several carrier in the intermembrane space (IMS) of mitochondria that – functions in electron transport between the quinol:cytochrome c other proteins. However, neither of the cysteine proline sequences in S. cerevisiae HCCS is required for heme attachment to cyto- oxidoreductase (complex III, or cytochrome bc1) and the cyto- chrome c oxidase (complex IV, or cytochrome a/a3). In addition to its role in mitochondrial respiration, cytochrome c plays a crucial role in apoptotic signaling (11). A second, membrane- bound c-type cytochrome, cytochrome c1,isanintegralpartof Author contributions: B.S.F. and R.G.K. designed research; B.S.F. and E.C.B. performed complex III. research; B.S.F., E.C.B., and R.G.K. analyzed data; and B.S.F. and R.G.K. wrote the paper. C-type cytochromes differ from other cytochromes in that the The authors declare no conflict of interest. heme is attached to the protein covalently via two thioether link- This article is a PNAS Direct Submission. ages between the heme vinyls and two cysteine residues of a con- 1To whom correspondence should be addressed. E-mail: [email protected]. served CysXxxXxxCysHis (CXXCH) heme-binding motif. Two This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. major pathways have been identified for the biogenesis of c-type 1073/pnas.1213897109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1213897109 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 chrome c (37), and several HCCS proteins lack cysteine–proline tion, and a requirement for release of mature holocytochrome sequences entirely. c from HCCS. Both the mechanism by which HCCS mediates covalent heme attachment to the apocytochrome and the specificity determi- Results nants for recognition of heme and the apocytochrome c are Purified Human HCCS Contains Heme. Despite longstanding interest poorly understood. Recombinant systems for production of in HCCS, the enzyme has remained refractile to successful pu- mitochondrial cytochromes c in Escherichia coli, developed by rification and biochemical characterization (e.g., refs. 43–45). To Mauk and colleagues (38), have facilitated some progress in this address this problem, we engineered the cDNA for the human regard (SI Appendix, Table S1) (39–41). The N-terminal region HCCS in three different vectors (pET Blue-2 with an N-terminal of the apocytochrome c substrate (including the CXXCH motif) His6 tag, pTXB1 with a C-terminal Intein fusion, and pGEX is important for recognition by S. cerevisiae HCCS (reviewed in with an N-terminal GST fusion) for expression and purification ref. 16), and a few residues in this region have been identified as in E. coli. Early attempts at purifying HCCS from cytoplasmic important for holocytochrome c maturation (42, 43). However, fractions were largely unsuccessful for each of these constructs. the features of the cytochrome c substrate that are recognized by However, upon fractionation of E. coli expressing GST-HCCS, the human HCCS have never been examined. Here, we report we observed that the membrane fraction appeared to be enriched successful purification and characterization of the human HCCS for a polypeptide of 57 kDa, the predicted size for the GST- from recombinant E. coli. The human HCCS is membrane as- HCCS fusion protein (SI Appendix,Fig.S1). Thus, we directed sociated and is purified with endogenous heme coordinated by our efforts toward purifying human HCCS from membranes by conserved His154. We define the amino acids in the human cy- solubilization in N-dodecyl β-D-maltopyranoside (DDM) (Fig. 1 tochrome c that are required for holocytochrome c formation by A–C) or Triton X-100. Purified full-length GST-HCCS (∼57 HCCS, and we successfully convert a nonsubstrate cytochrome c, kDa) and three minor proteolytic products each reacted with B cytochrome c2 from the alpha-proteobacterium Rhodobacter GST antisera (Fig. 1 , lane 9). Note that soluble (cytoplasmic) capsulatus, into a robust substrate for the human HCCS by in- fractions contained mostly the degraded products and very low troducing three sequence alterations. Finally, we report purifica- levels of full-length GST-HCCS
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