Human mitochondrial holocytochrome c synthase’s PNAS PLUS 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 AUTHOR SUMMARY

Mitochondria are organelles responsible for aerobic , which is the use of oxygen to derive energy from nutrients. Disruption of cellular respiration has been implicated in many human dis- eases, including cancer. As such, there has been a renewed interest in understanding how the components of the mitochondrial respiratory chain are assembled. C-type cytochromes are heme that are involved in multiple stages of cellular res- piration, but the details of their assembly remain unknown. The c-type cytochromes

are unique in that they possess a heme BIOCHEMISTRY cofactor that is covalently attached via a posttranslational process that requires a called holocytochrome c synthase (HCCS). Although the encoding HCCS was discovered more than 25 y ago, research aimed at understanding how HCCS works has been limited by an in- ability to purify this . Here, we purify and characterize the human HCCS enzyme in complex with heme and its substrate, cytochrome c. Our results sug- gest detailed mechanisms for how HCCS covalently attaches heme to cytochrome c (Fig. P1, Upper). The first observation linking cellular Fig. P1. The covalent attachment of heme to apocytochrome c to form holocytochrome c in respiration to the was made mitochondria requires HCCS. HCCS catalyzes the formation of thioether linkages between the precisely one century ago (1). Today the two vinyl groups of heme and the two cysteines of the CysXxxXxxCysHis motif of apocytochrome mechanisms underlying cellular respiration c.(Upper) Four fundamental steps in the holocytochrome c synthase reaction: heme binding, are understood at the atomic level, with apocytochrome c recognition, thioether formation, and holocytochrome c release. (Lower)A 3D crystal structures available for nearly model of the HCCS:heme:apocytochrome c ternary complex. In this complex heme is coordinated all the proteins in the mitochondrial re- by His154 of HCCS and His19 of apocytochrome c. Additional designated residues in the N spiratory chain. The first structures for the terminus of apocytochrome c are important for various steps in the pathway. c-type cytochromes were determined in the 1970s (2), confirming that two cysteine residues of a conserved CysXxxXxxCysHis heme-binding motif efforts to address questions on how HCCS binds heme and how it were linked to the two heme vinyl groups via covalent bonds recognizes, interacts with, and attaches heme to cytochrome c. known as “thioether linkages.” The histidine residue of this motif Our results suggest that the inability to purify recombinant serves as a ligand to the heme iron. The posttranslational HCCS likely reflects its proteolytic susceptibility and presence mechanism by which these thioether linkages were formed did at low levels only in membrane fractions, despite the absence not become apparent until 1987, when it was shown that the gene of predicted transmembrane helices in the protein. We optimized encoding cytochrome c heme lyase (also known as HCCS) was required for synthesis of holocytochrome c (i.e., with heme) (3). Subsequent studies indicated that HCCS was present in the mitochondrial (IMS), the subcellular Author contributions: B.S.F. and R.G.K. designed research; B.S.F. and E.C.B. performed compartment in which cytochrome c functions. Classic work research; B.S.F., E.C.B., and R.G.K. analyzed data; and B.S.F. and R.G.K. wrote the paper. by Neupert and colleagues (4) and Sherman and colleagues The authors declare no conflict of interest. (5) further demonstrated that HCCS plays a critical role in This article is a PNAS Direct Submission. importing the unfolded apocytochrome c (i.e., without heme) 1To whom correspondence should be addressed. E-mail: [email protected]. into the IMS from the cytosol. However, the lack of biochemical See full research article on page E788 of www.pnas.org. or structural information on HCCS has severely hampered Cite this Author Summary as: PNAS 10.1073/pnas.1213897109.

www.pnas.org/cgi/doi/10.1073/pnas.1213897109 PNAS | February 26, 2013 | vol. 110 | no. 9 | 3221–3222 Downloaded by guest on October 1, 2021 the expression and purification of the human HCCS from molecule mediating the interaction between HCCS and the cy- Escherichia coli membranes. Purified HCCS contains endoge- tochrome c. These findings offer insight into our understanding nous heme as one of its two substrates, the other substrate of posttranslational cofactor attachment and heme biology in c being apocytochrome . Using various approaches, we showed general. Furthermore, our elucidation of the molecular mech- that histidine residue 154 (His154) in HCCS is the key ligand in anisms underlying HCCS function advances our knowledge HCCS responsible for binding heme. Additionally, we engi- of mitochondrial respiratory chain assembly and thus has im- neered in the cognate human apocytochrome c, plications for our understanding of mitochondrial diseases and defining residues within the CysXxxXxxCysHis heme-binding motif that are required for heme attachment and maturation abnormalities. (Fig. P1, Lower). With this knowledge, we also converted a c 1. Ernster L, Schatz G (1981) Mitochondria: A historical review. JCellBiol91(3 Pt 2): bacterial cytochrome , which is not a natural substrate for – HCCS, into one that is recognized and matured by the human 227s 255s. fi 2. Dickerson RE, et al. (1971) Ferricytochrome c. I. General features of the horse and HCCS. Finally, we were able to isolate puri ed complexes be- bonito proteins at 2.8 A resolution. J Biol Chem 246(5):1511–1535. tween the human HCCS and each variant human cytochrome c. 3. Dumont ME, Ernst JF, Hampsey DM, Sherman F (1987) Identification and sequence Further examination of each ternary complex (HCCS:heme: of the gene encoding cytochrome c heme lyase in the yeast Saccharomyces cerevisiae. cytochrome c) provided insight into the putative HCCS active EMBO J 6(1):235–241. site where heme is coordinated by His154 in HCCS and by 4. Nicholson DW, Hergersberg C, Neupert W (1988) Role of cytochrome c heme lyase in c the import of cytochrome c into mitochondria. J Biol Chem 263(35):19034–19042. His19 of the cytochrome (Fig. P1). Our results demonstrate 5. Dumont ME, Ernst JF, Sherman F (1988) Coupling of heme attachment to import of that heme, in addition to being a substrate for HCCS and cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria the essential cofactor for c-type cytochromes, is the central and altered apocytochromes c. J Biol Chem 263(31):15928–15937.

3222 | www.pnas.org/cgi/doi/10.1073/pnas.1213897109 San Francisco et al. Downloaded by guest on October 1, 2021