title: A strategic protein in cytochrome c maturation: three-dimensional structure of CcmH and binding to apocytochrome c.
abstract: CcmH (cytochromes c maturation 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 1.7A 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 c(551) 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.
authors.affiliation: Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), La Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma Italy.
authors.forename: Adele
authors.initials: A
authors.lastname: Di Matteo
authors.affiliation: Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), La Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.affiliation: Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), La Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma Italy.
authors.forename: M Eugenia
authors.initials: ME
authors.lastname: Schininà
authors.affiliation: Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), La Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Giorgi
authors.affiliation: Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), La Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Altieri
authors.affiliation: Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), La Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Calosci
authors.affiliation: Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), La Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma Italy. Electronic address: maurizio.brunori@uniroma1.it.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Brunori
authors.affiliation: Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), La Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Travaglini-Allocatelli
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001665:Binding Sites; D003460:Crystallization; D045304:Cytochromes c / Q000737:chemistry* / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000737:chemistry*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D045304:Cytochromes c