title: Structural and functional characterization of CcmG from Pseudomonas aeruginosa, a key component of the bacterial cytochrome c maturation apparatus.
abstract: The cytochrome c maturation process is carried out in the bacterial periplasm, where some specialized thiol-disulfide oxidoreductases work in close synergy for the correct reduction of oxidized apocytochrome before covalent heme attachment. We present a structural and functional characterization of the soluble periplasmic domain of CcmG from the opportunistic pathogen P. aeruginosa (Pa-CcmG), a component of the protein machinery involved in cyt c maturation in gram-negative bacteria. X-ray crystallography reveals that Pa-CcmG is a TRX-like protein; high-resolution crystal structures show that the oxidized and the reduced forms of the enzyme are identical except for the active-site disulfide. The standard redox potential was calculated to be E(0') = -0.213 V at pH 7.0; the pK(a) of the active site thiols were pK(a) = 6.13 +/- 0.05 for the N-terminal Cys74 and pK(a) = 10.5 +/- 0.17 for the C-terminal Cys77. Experiments were carried out to characterize and isolate the mixed disulfide complex between Pa-CcmG and Pa-CcmH (the other redox active component of System I in P. aeruginosa). Our data indicate that the target disulfide of this TRX-like protein is not the intramolecular disulfide of oxidized Pa-CcmH, but the intermolecular disulfide formed between Cys28 of Pa-CcmH and DTNB used for the in vitro experiments. This observation suggests that, in vivo, the physiological substrate of Pa-CcmG may be the mixed-disulfide complex between Pa-CcmH and apo-cyt.
authors.affiliation: Dipartimento di Scienze Biochimiche, Istituto di Biologia e Patologia Molecolari del CNR, Sapienza-Università di Roma, Piazzale A. Moro 5, 00185, Roma, Italy.
authors.forename: Adele
authors.initials: A
authors.lastname: Di Matteo
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Calosci
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
authors.forename: Maurizio
authors.initials: M
authors.lastname: Brunori
authors.forename: Carlo
authors.initials: C
authors.lastname: Travaglini-Allocatelli
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D045304:Cytochromes c / Q000096:biosynthesis*; D004795:Enzyme Stability; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013879:Thioredoxins / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D045304:Cytochromes c; D011490:Protein Disulfide Reductase (Glutathione)