Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase.
Cox17, a copper chaperone for cytochrome c oxidase, is an essential and highly conserved protein. The structure and mechanism of functioning of Cox17 are unknown, and even its metalbinding stoichiometry is elusive. In the present study, we demonstrate, using electrospray ionization-MS, that porcine Cox17 binds co-operatively four Cu+ ions. Cu4Cox17 is stable at pH values above 3 and fluorescence spectra indicate the presence of a solvent-shielded multinuclear Cu(I) cluster. Combining our results with earlier EXAFS results on yeast CuCox17, we suggest that Cu4Cox17 contains a Cu4S6-type cluster. At supramillimolar concentrations, dithiothreitol extracts metals from Cu4Cox17, and an apparent copper dissociation constant KCu=13 fM was calculated from these results. Charge-state distributions of different Cox17 forms suggest that binding of the first Cu+ ion to Cox17 causes a conformational change from an open to a compact state, which may be the rate-limiting step in the formation of Cu4Cox17. Cox17 binds non-co-operatively two Zn2+ ions, but does not bind Ag+ ions, which highlights its extremely high metal-binding specificity. We further demonstrate that porcine Cox17 can also exist in partly oxidized (two disulphide bridges) and fully oxidized (three disulphide bridges) forms. Partly oxidized Cox17 can bind one Cu+ or Zn2+ ion, whereas fully oxidized Cox17 does not bind metals. The metal-binding properties of Cox17 imply that, in contrast with other copper chaperones, Cox17 is designed for the simultaneous transfer of up to four copper ions to partner proteins. Metals can be released from Cox17 by non-oxidative as well as oxidative mechanisms.
Palumaa
Peep
P
Institute of Gene Technology, Tallinn Technical University, Akadeemia tee 23, EE-12018 Tallinn, Estonia. peepp@staff.ttu.ee
Kangur
Liina
L
Voronova
Anastassia
A
Sillard
Rannar
R
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079922:Copper Transport Proteins; D004229:Dithiothreitol / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011489:Protein Denaturation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment / Q000379:methods; D017386:Sequence Homology, Amino Acid; D012834:Silver / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013552:Swine; D015032:Zinc / Q000378:metabolism
C100359:COX17 protein, S cerevisiae; C491006:COX17 protein, human; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; C491005:Cox17 protein, mouse; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D012834:Silver; D003300:Copper; D003576:Electron Transport Complex IV; D005978:Glutathione; D015032:Zinc; D004229:Dithiothreitol