Cytochromes are heme proteins essential for aerobic and anaerobic electron transport in most organisms. While the structures and functions of many of these proteins have been studied for over fifty years, only relatively recently has it become clear that cytochromes often require assembly factors. Such assembly factors are defective in certain human disorders, and essential for growth in many pathogens. Biogenesis of the c-type cytochromes requires a large number of factors and can proceed by any one of three systems. Prokaryotes, plant mitochondria, and chloroplasts use either system I or II, which are each predicted to require dedicated mechanisms for heme delivery and apocytochrome thiolreduction. In system III, which has specifically evolved in the mitochondna of fungi, invertebrates, and vertebrates, a pivotal role is played by a single enzyme called cytochrome c heme lyase in the mitochondrial intermembrane space. Cytochrome c biogenesis requires nine dedicated, integral membrane proteins for System I and at least three for System II. The goals of this proposal are to understand the molecular mechanisms by which Systems I and II operate using three model proteobacteria, Rhodobacter capsulatus (System I), Bordetella pertussis (System II), and Escherichia coli (System I and recombinant System II). Specifically, aims are to (1) identify and characterize novel System II genes in B. pertussis; (2) reconstitute in vitro biogenesis using System I and System II; (3) determine the substrate recognition requirements (ie. apocytocbrome and heme) and the physiological conditions essential for System I and II; (4) carry out a functional analysis of key proteins involved in System I and II.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Shapiro, Bert I
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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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