Cytochromes c are electron transport proteins that contain a heme moiety covalently ligated to the protein at a conserved CXXCH motif. Organisms employ one of three biogenesis systems (system I, II, or III) to facilitate transmembrane delivery, reduction, and ligation of the apoprotein and heme. System II, employed by Gram positive bacteria, cyanobacteria, some proteobacteria, and chloroplasts, consists of four proteins: CcsB, CcsA, DsbD, and CcsX. Only CcsB and CcsA are absolutely required for function and represent the minimal system II synthetase. Naturally occurring fusions of the CcsB and CcsA proteins (CcsBA) from different bacteria were investigated for robustness of cytochrome c assembly in recombinant Escherichia coli and purification of CcsBA was optimized. The recombinant Helicobacter hepaticus CcsBA yielded the highest levels of diheme cytochrome c4. CcsBA can synthesize diverse cytochromes c, including those naturally assembled by systems I (monoheme cytochrome c2) and III (human cytochrome c). E. coli disulfide bond formation (Dsb) mutants and chemical reducing agents were employed to address the redox requirements for function of recombinant CcsBA. Under aerobic conditions DsbC and DsbD are required for function while under anaerobic conditions only DsbD is required. CcsBA contains a conserved motif called the WWD domain and is a member of the heme handling protein (HHP) superfamily. Detergent solubilized GST-tagged CcsBA purifies with heme trapped in the protein. Two conserved histidines in transmembrane domains (TMDs) are required for heme binding and mutants can be complemented for function by addition of the histidine side chain analogue imidazole to growth media. Results suggest the presence of a well defined heme binding site within a channel comprised of TMD3 and TMD8 where histidines in these TMDs function as axial ligands. To further refine the model of heme binding and trafficking by CcsBA, chemical complementation by imidazole analogues, low temperature spectral features, and binding of alternative porphyrins were investigated. The results and models presented in this thesis provide new insight into the mechanisms of system II heme trafficking and synthetase activity and will guide future investigations of the CcsBA protein and efforts to reconstitute cytochrome c biogenesis in vitro
