Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including human pathogens. Recently it has become clear that dedicated assembly factors are crucial for cytochrome biogenesis. The biogenesis of c-type cytochromes occurs by one of three pathways, systems I, II, or III. System I has eight (CcmABCDEFGH) and system II has two (CcsBA) dedicated assembly factors (membrane proteins), while system III uses a single enzyme called cytochrome c heme lyase. Because only prokaryotes, plants, and protozoa use systems I and II, these pathways represent potential targets for antimicrobial agents. The c-type cytochromes possess heme that is covalently ligated to the apocytochrome at two cysteines. The cysteines and heme (to Fe2+) must be reduced for attachment to occur. Moreover, all cytochromes c are assembled and function outside of the inner membrane. This study examines how proteins in systems I and II deliver heme, synthesized inside the cell, and attach it to the secreted unfolded apocytochrome c. The proposal takes advantage of our previous success in purifying all proteins of systems I and II from recombinant Escherichia coli. For most of these purified components, endogenous heme has been trapped, facilitating analyses of heme transport, red-ox control, and attachment mechanisms.
Three aims are proposed, the first two for system I, and the third for system II. System I is described in two steps. Step 1 is the CcmABCD-mediated synthesis and release of periplasmic holoCcmE (heme in the oxidized Fe3+ state).
Aim 1 analyzes this step, establishing residues in CcmC and CcmE that directly interact with heme and the chemical mechanisms to form the oxidized holoCcmE. The holoCcmE release step is reconstituted with purified CcmABCDE proteins. Step 2, analyzed in Aim 2, includes the CcmF/H-mediated reduction of holoCcmE (to Fe2+) and ligation of this heme to apocytochrome. The mechanism of reduction in vivo (e.g., quinone-based) is analyzed, as well as residues in CcmF for interacting with CcmH, holoCcmE, and a novel b heme we discovered.
Aim 3 analyzes the recombinant system II CcsBA integral membrane protein that we demonstrated has heme export and cytochrome c synthetase functions. The following hypothesis will be tested: CcsBA binds heme via two conserved histidines in a """"""""channel"""""""" and protects the heme from oxidation as it moves to an external heme binding site. The external apocytochrome c binding site and attachment to heme will be studied in vitro. Prokaryotes have evolved hundreds of different cytochromes c, ranging from the mitochondrial-like cytochrome c with a single heme to extraordinary c-type cytochromes with over ten heme molecules attached to a single polypeptide. Results here will unravel the mechanisms underlying heme transport, heme red-ox control, and heme attachment to all prokaryotic and plant c-type cytochromes.

Public Health Relevance

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National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Gindhart, Joseph G
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Washington University
Schools of Arts and Sciences
Saint Louis
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Sutherland, Molly C; Tran, Nathan L; Tillman, Dustin E et al. (2018) Structure-Function Analysis of the Bifunctional CcsBA Heme Exporter and Cytochrome c Synthetase. MBio 9:
Sutherland, Molly C; Jarodsky, Joshua M; Ovchinnikov, Sergey et al. (2018) Structurally Mapping Endogenous Heme in the CcmCDE Membrane Complex for Cytochrome c Biogenesis. J Mol Biol 430:1065-1080
Babbitt, Shalon E; Hsu, Jennifer; Mendez, Deanna L et al. (2017) Biosynthesis of Single Thioether c-Type Cytochromes Provides Insight into Mechanisms Intrinsic to Holocytochrome c Synthase (HCCS). Biochemistry 56:3337-3346
Mendez, Deanna L; Babbitt, Shalon E; King, Jeremy D et al. (2017) Engineered holocytochrome c synthases that biosynthesize new cytochromes c. Proc Natl Acad Sci U S A 114:2235-2240
Mendez, Deanna L; Akey, Ildikó V; Akey, Christopher W et al. (2017) Oxidized or Reduced Cytochrome c and Axial Ligand Variants All Form the Apoptosome in Vitro. Biochemistry 56:2766-2769
Babbitt, Shalon E; Hsu, Jennifer; Kranz, Robert G (2016) Molecular Basis Behind Inability of Mitochondrial Holocytochrome c Synthase to Mature Bacterial Cytochromes: DEFINING A CRITICAL ROLE FOR CYTOCHROME c ? HELIX-1. J Biol Chem 291:17523-34
Sutherland, Molly C; Rankin, Joel A; Kranz, Robert G (2016) Heme Trafficking and Modifications during System I Cytochrome c Biogenesis: Insights from Heme Redox Potentials of Ccm Proteins. Biochemistry 55:3150-6
Babbitt, Shalon E; Sutherland, Molly C; San Francisco, Brian et al. (2015) Mitochondrial cytochrome c biogenesis: no longer an enigma. Trends Biochem Sci 40:446-55
Babbitt, Shalon E; San Francisco, Brian; Bretsnyder, Eric C et al. (2014) Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme. Biochemistry 53:5261-71
San Francisco, Brian; Kranz, Robert G (2014) Interaction of holoCcmE with CcmF in heme trafficking and cytochrome c biosynthesis. J Mol Biol 426:570-85

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