Characterization of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturation

The CcmH protein of Escherichia coli is encoded by the last gene of the ccm gene cluster required for cytochrome c maturation. A mutant in which the e ntire ccmH gene was deleted failed to synthesize both indigenous and foreig n c-type cytochromes. However, deletion of the C-terminal hydrophilic domai n homologous to CycH of other gram-negative bacteria affected neither the b iogenesis of indigenous c-type cytochromes nor that of the Bradyrhizobium j aponicum cytochrome c(550). This confirmed that only the N-terminal domain containing a conserved CXXC motif is required in E. coli. PhoA fusion analy sis showed that this domain is periplasmic. Site-directed mutagenesis of th e cysteines of the CXXC motif revealed that both cysteines are required for cytochrome c maturation during aerobic growth, whereas only the second cys teine is required for cytochrome c maturation during anaerobic growth, The deficiency of the point mutants was complemented when 2-mercapto-ethanesulf onic acid was added to growing cells; other thiol compounds did not stimula te cytochrome c formation in these strains. We propose a model for the reac tion sequence in which CcmH keeps the heme binding site of apocytochrome c in a reduced form for subsequent heme ligation.