Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including human pathogens. Relatively recently it has become clear that dedicated assembly pathways and factors are crucial for cytochrome biogenesis. The assembly of c-type cytochromes occurs by one of three pathways, called systems I, II, and III. Systems I and II have nine and four assembly factors (membrane proteins) respectively, while system III uses a single enzyme called cytochrome c heme lyase. Defects in system III result in certain human genetic diseases. Because only prokaryotes, plants, and protozoa use systems I or 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, which must be reduced for attachment to the heme. Since assembly of c-type cytochromes occurs at the outer surface of the inner membrane, cells must deliver reduced heme, synthesized inside the cell, to the secreted unfolded apocytochrome. In this application we take advantage of our previous success for reconstituting all three systems in recombinant Escherichia coli to study the in vivo concentrations and features of heme that are optimal for each system. The heme delivery pathways will be established. Furthermore, the protein(s) required for the final cytochrome c synthetase (ligase) activity of each system will be purified and characterized for heme and apocytochrome c ligation properties, binding, and mechanisms. Based on our findings that certain metal porphyrins are specific inhibitors of the systems I and II pathways, other porphyrin analogs will be tested for their ability to be ligated to apocytochrome c (or to inhibit biogenesis), further delineating the properties of heme that are critical for ligation. In summary, this study will address which protein(s) constitute the cytochrome c synthetases, how heme is delivered, what concentrations and features of heme are required for each system. Results will help unravel why three very different pathways have emerged in nature and mechanisms to target them.

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
United States
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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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