Modulation of Redox Switches of Copper Chaperone Cox17 by Zn(ll) Ions Determined by New ESI MS-Based Approach

摘要

Coxl7, a copper chaperone for cytochrome-c oxidase, contains six conserved Cys residues and exists in three oxidative states, linked with two thiol-based redox switches. The first switch leads to formation of two disulfides and occurs upon transport of Coxl7 into mitochondrial intermembrane space (IMS). Coxl7(2S-S) is retained in the IMS and is also a functional form of the protein, which can be further oxidized to Coxl7 _(3S-S)- According to the midpoint redox potential values, Coxl7 can be partially oxidized in the cytosol, which might hinder its transport into IMS. We hypothesize that Zn(II) ions might protect cytosolic Coxl7 from oxidation. In order to get quantitative information about the modulatory effect of Zn(II) ions on redox switches in Coxl7, we have used ESI MS for determination of the midpoint potentials for redox couples of Coxl7: Coxl7_(3S-S)<->Coxl7_(2S-S) E_(m1) and Coxl7(2S-S)<-> Coxl7(0S-S) (E_(m2)) in the presence of Zn(II). 10 muM Zn(II) ions shift the E_(m2) by 21 mV and Eml by 15 mV to more positive values. Apparent dissociation constants for Zn(II) complexes of Coxl7(0S-S) and Coxl7_(2S-S), are 0.067 and 0.29 nM, respectively. The high affinity shows that metallation of Coxl7(0S-S) by Zn(II) might be significant in cellular conditions, which might protect Coxl7 from oxidation and enable its transport into IMS.