Recombinant cytochromes c biogenesis systems I and II and analysis of haem delivery pathways in Escherichia coli

• Published in: Molecular microbiology 2006-05, Vol.60 (3), p.563-577
• Main Authors: Feissner, Robert E., Richard‐Fogal, Cynthia L., Frawley, Elaine R., Loughman, Jennifer A., Earley, Keith W., Kranz, Robert G.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial proteins; Bacteriology; Biological and medical sciences; Bordetella pertussis; Bordetella pertussis - enzymology; Bordetella pertussis - genetics; Cytochrome c Group - chemistry; Cytochrome c Group - genetics; Cytochrome c Group - metabolism; Cytochromes c1 - biosynthesis; Cytochromes c1 - genetics; Cytochromes c2 - biosynthesis; Cytochromes c2 - genetics; E coli; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Ferrochelatase - antagonists & inhibitors; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genetics; Heme - metabolism; Microbiology; Miscellaneous; Molecular Sequence Data; Protoporphyrins - pharmacology; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/j.1365-2958.2006.05132.x

Summary Genetic analysis has indicated that the system II pathway for c‐type cytochrome biogenesis in Bordetella pertussis requires at least four biogenesis proteins (CcsB, CcsA, DsbD and CcsX). In this study, the eight genes (ccmA–H) associated with the system I pathway in Escherichia coli were deleted. Using B. pertussis cytochrome c4 as a reporter for cytochromes c assembly, it is demonstrated that a single fused ccsBA polypeptide can replace the function of the eight system I genes in E. coli. Thus, the CcsB and CcsA membrane complex of system II is likely to possess the haem delivery and periplasmic cytochrome c‐haem ligation functions. Using recombinant system II and system I, both under control of IPTG, we have begun to study the capabilities and characteristics of each system in the same organism (E. coli). The ferrochelatase inhibitor N‐methylprotoporphyrin was used to modulate haem levels in vivo and it is shown that system I can use endogenous haem at much lower levels than system II. Additionally, while system I encodes a covalently bound haem chaperone (holo‐CcmE), no covalent intermediate has been found in system II. It is shown that this allows system I to use holo‐CcmE as a haem reservoir, a capability system II does not possess.