Oxidative switches in functioning of mammalian copper chaperone Cox17

Abstract

Cox17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian Cox17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian Cox17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian Cox17(0S-S) binds co-operatively to four Cu+; Cox17(2S-S), with two disulfide bridges, binds to one of either Cu+ or Zn2+; and Cox17(3S-S), with three disulfide bridges, does not bind to any metal ions. The E(m) (midpoint redox potential) values for two redox couples of Cox17, Cox17(3S-S)<-->Cox17(2S-S) (E(m1)) and Cox17(2S-S)<-->Cox17(0S-S) (E(m2)), were determined to be -197 mV and -340 mV respectively. The data indicate that an equilibrium exists in the cytosol between Cox17(0S-S) and Cox17(2S-S), which is slightly shifted towards Cox17(0S-S). In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards Cox17(2S-S), enabling retention of Cox17(2S-S) in the IMS and leading to the formation of a biologically competent form of the Cox17 protein, Cox17(2S-S), capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4Cox17 contains a Cu4S6-type copper-thiolate cluster, which may provide safe storage of an excess of copper ions.

Functional Characterization of Mia40p, the Central Component of the Disulfide Relay System of the Mitochondrial Intermembrane Space

“…Consistent with our data, the NMR structure of Cox17p showed two interhelical disulfide bonds between the helices of its twin CX 9 C motif (25,46). These disulfide bonds stabilize the two helices in an antiparallel fold.…”

“…It also has two additional conserved cysteine residues and can form a labile third disulfide bond under oxidizing conditions (25,46). However, in contrast to the redox state of Mia40p, there is no evidence for the presence of a fully oxidized form of Cox17p in vivo, and the spacing of the additional cysteine residues is different.…”

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

“…UnfoldingMia40 substrates contain very stable disulfide bonds that require high concentration of reductants to be broken (16,18,19). We estimated the standard redox potential of Cox19 to be approximately Ϫ0.31 V (data not shown), very close to that reported for Cox17 (Ϫ0.34), Tim9 (Ϫ0.31), and Tim10 (Ϫ0.32), suggesting that Mia40 substrates have similar redox stability.…”

Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of Cu A in human cytochrome oxidase

“…Sco proteins have been suggested to act as thiol reductases of the Cys ligands of COX II based on their thioredoxin fold (27,37), which is indeed supported by in vitro experiments on the bacterial thermophilic Sco protein (32,37). Based on the reduction potentials of the S-S/2SH redox couple of the human proteins: Cox17 (−198 mV) > Sco1 (−277 mV) > COX II* (−288 mV) > Sco2 (less than −300 mV) (24,38,39), Sco2 would be the only protein able to reduce the disulfide bond in COX II*. We tested the redox reactivity of either human Sco1 or human Sco2 in their apo, reduced state with oxidized apo-COX II* S-S .…”