title: How periplasmic thioredoxin TlpA reduces bacterial copper chaperone ScoI and cytochrome oxidase subunit II (CoxB) prior to metallation.
abstract: Two critical cysteine residues in the copper-A site (Cu(A)) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu(2+) transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E'0 = -231 mV) as compared with that of TlpA (E'0 = -256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10(4) M(-1) s(-1) that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu(A)-CoxB complex may be bypassed in vivo by high environmental Cu(2+) concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.
authors.affiliation: From the Institute of Molecular Biology and Biophysics and Institute of Microbiology, ETH Zürich, CH-8093 Zürich and.
authors.forename: Helge K
authors.initials: HK
authors.lastname: Abicht
authors.affiliation: From the Institute of Molecular Biology and Biophysics and the Laboratory of Biomolecular Research, Paul Scherrer Institute, CH-5232 Villigen-PSI, Switzerland.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Schärer
authors.affiliation: From the Institute of Molecular Biology and Biophysics and.
authors.forename: Nick
authors.initials: N
authors.lastname: Quade
authors.affiliation: Institute of Microbiology, ETH Zürich, CH-8093 Zürich and.
authors.forename: Raphael
authors.initials: R
authors.lastname: Ledermann
authors.affiliation: From the Institute of Molecular Biology and Biophysics and.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mohorko
authors.affiliation: the Laboratory of Biomolecular Research, Paul Scherrer Institute, CH-5232 Villigen-PSI, Switzerland.
authors.forename: Guido
authors.initials: G
authors.lastname: Capitani
authors.affiliation: Institute of Microbiology, ETH Zürich, CH-8093 Zürich and hennecke@micro.biol.ethz.ch.
authors.forename: Hauke
authors.initials: H
authors.lastname: Hennecke
authors.affiliation: From the Institute of Molecular Biology and Biophysics and.
authors.forename: Rudi
authors.initials: R
authors.lastname: Glockshuber
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020369:Bradyrhizobium / Q000235:genetics / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D018832:Molecular Chaperones; C084726:TlpA protein, Bradyrhizobium; D013879:Thioredoxins; D003300:Copper; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV
keywords: Copper; Crystal Structure; Cytochrome Oxidase; Disulfide Bond Reduction; Metalloprotein; Oxidation-Reduction (Redox); Thioredoxin