Structural and functional characterization of CcmG from Pseudomonas aeruginosa,a key component of the bacterial cytochrome c maturation apparatus |
| |
Authors: | Adele Di Matteo Nicoletta Calosci Stefano Gianni Per Jemth Maurizio Brunori Carlo Travaglini‐Allocatelli |
| |
Institution: | 1. Dipartimento di Scienze Biochimiche, Istituto di Biologia e Patologia Molecolari del CNR, Sapienza‐Università di Roma, Piazzale A. Moro 5, 00185, Roma, Italy;2. Department of Medical Biochemistry and Microbiology, Uppsala University, SE‐75123 Uppsala, Sweden |
| |
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 E0′ = ?0.213 V at pH 7.0; the pKa of the active site thiols were pKa = 6.13 ± 0.05 for the N‐terminal Cys74 and pKa = 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. Proteins 2010. © 2010 Wiley‐Liss, Inc. |
| |
Keywords: | thiol‐disulfide oxidoreductases thioredoxin‐like protein pKa DTNB cytocrome c biogenesis cytochrome c heme binding motif |
|
|