Dispensable residues in the active site of the cytochrome c biogenesis protein CcmH

Abstract

CcmH functions in the assembly of c-type cytochromes in the Escherichia coli periplasm. The conserved cysteine pair in the N-terminal of its two membrane-anchored periplasmic domains is thought to reduce the CXXCH motif of cytochromes c. The recent structure of Pseudomonas aeruginosa CcmH identified conserved residues that might be functionally important. We replaced with alanine the active-site cysteines of E. coli CcmH, as well as R42, S54, R63, and tested the effects on cytochrome c production anaerobically and aerobically. Unexpectedly, replacement of the conserved non-cysteine active-site residues had little effect, whilst the cysteines were required under aerobic, but not anaerobic, conditions. We confirmed that removal of the C-terminal tetratricopeptidelike domain does not, surprisingly, abolish assembly of cytochromes c.

Cytochrome c biogenesis System I: An intricate process catalyzed by a maturase supercomplex?

“…E. coli CcmH is a fusion protein with a N-terminal domain corresponding to the redox-active CcmH and a C-terminal portion homologous to CcmI (see below) as found in other organisms, such as R. capsulatus and P. aeruginosa [103, 105]. Both Cys residues of E. coli CcmH are important for Ccm but not essential under all conditions [105, 106]. No thioredox compensation occurs between R. capsulatus mutants lacking both DsbA and CcmH, suggesting that the role of CcmH is not restricted to apocyts c reduction [93].…”

The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE

“…CcmG and CcmH each contain a conserved pair of thioredox-active cysteines that are required for cytochrome c synthesis in the context of the full system I (Fabianek et al, 1998;Robertson et al, 2008). To test whether holocytochrome c formation by CcmFGHonly required these thioredoxin functions, we engineered serine substitutions at the conserved cysteine pairs in CcmG or CcmH and assayed for cytochrome c formation.…”

A Pivotal Heme-transfer Reaction Intermediate in Cytochrome c Biogenesis

“…Subsequent holocytochrome (holo- c-b 562 ) formation ( boxed ) involves heme transfer from CcmE to the reduced apocytochrome; this requires the hemoprotein CcmF and CcmH ( light pink ) (10, 17, 18), but the mechanism is not known. E. coli CcmH consists of two functionally distinct domains that are found as separate proteins (CcmH and CcmI) in most Ccm-containing bacteria (38). Key amino acids for experiments in this work are highlighted.…”