Many microorganisms use c-type cytochromes for electron transport systems that are essential for growth. The assembly of these heme-proteins has recently been shown to require at least eight specific genes in gram-negative bacteria (called helABCDX, ccl1, ccl2, and cycH). Important genomic studies now indicate that although this complex biogenesis pathway is present in many prokaryotes, protozoa and plants, it is absent in yeast and probably vertebrates and invertebrates. This application is for support to study this new pathway in the model bacterium, Rhodobacter capsulatus, in which many of the biosynthetic genes were first discovered. Using genetics, immunological and biochemical approaches, the role of each protein in the biogenesis system will be investigated. All proteins of the pathway are tethered to the surface of the cytoplasmic membrane; it is proposed that reduced heme and reduced cysteine residues of the cytochromes are brought together at the surface by these proteins. Because this assembly in bacteria takes place at the outer surface of the cytoplasmic membrane, where all c-type cytochromes are located, the pathway comprises new and accessible targets for antimicrobial chemotherapeutics. However a more thorough understanding of the functions of individual assembly proteins and the underlying mechanisms involved in biogenesis are required. Reagents leading to the reconstitution of the system will be developed. Specifically, the following studies will be carried out: 1) Analyze proteins HelABCD and Ccl1, involved in heme delivery to the periplasmic surface, including specific amino acid residues and domains which are predicted to interact with heme. 2) Analyze the process whereby the two apocytochrome c cysteine residues are specifically reduced prior to covalent ligation to heme, the hallmark of all c-type cytochromes. The exact roles of Ccl2 and HelX, two periplasmic thioreactive proteins, will be determined, contributing to our understanding of thiol redox pathways in general. 3) Investigate further in vivo requirements for the pathway and develop key reagents required for in vitro reconstitution.
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