A strategic protein in cytochrome c maturation: three-dimensional structure of CcmH and binding to apocytochrome c.

CcmH ((c) under bar ytochromes (c) under bar (m) under bar aturation protein H) is an essential component of the assembly line necessary for the maturation of c-type cytochromes in the periplasm of Gram-negative bacteria. The protein is a membrane-anchored thiol-oxidoreductase that has been hypothesized to be involved in the recognition and reduction of apocytochrome c, a prerequisite for covalent heme attachment. Here, we present the 1.7 angstrom crystal structure of the soluble periplasmic domain of CcmH from the opportunistic pathogen Pseudomonas aeruginosa (Pa-CcmH*). The protein contains a three-helix bundle, i.e. a fold that is different from that of all other thiol-oxidoreductases reported so far. The catalytic Cys residues of the conserved LRCXXC motif (Cys(25) and Cys(28)), located in a long loop connecting the first two helices, form a disulfide bond in the oxidized enzyme. We have determined the pK(a) values of these 2 Cys residues of Pa-CcmH* ( both > 8) and propose a possible mechanistic role for a conserved Ser(36) and a water molecule in the active site. The interaction between Pa-CcmH* and Pa-apocyt c(551) ( where cyt c(551) represents cytochrome c(551)) was characterized in vitro following the binding kinetics by stopped-flow using a Trp-containing fluorescent variant of Pa-CcmH* and a dansylated peptide, mimicking the apocytochrome c551 heme binding motif. The kinetic results show that the protein has a moderate affinity to its apocyt substrate, consistent with the role of Pa-CcmH as an intermediate component of the assembly line for c-type cytochrome biogenesis.