Spectroscopic Studies of Metal Binding and Metal Selectivity in Bacillus subtilis BSco, a Homologue of the Yeast Mitochondrial Protein Sco1p

Abstract

Sco1 is a mitochondrial membrane protein involved in the assembly of the CuA site of cytochrome c oxidase. The Bacillus subtilis genome contains a homologue of yeast Sco1, YpmQ (hereafter termed BSco), deletion of which leads to a phenotype lacking in caa3 (CuA-containing) oxidase activity but expressing normal levels of aa3 (quinol) oxidase activity. Here, we report the characterization of the metal binding site of BSco in its Cu(I)-, Cu(II)-, Zn(II)-, and Ni(II)-bound forms. Apo BSco was found to bind Cu(II), Zn(II), and Ni(II) at a 1:1 protein/metal ratio. The Cu(I) protein could be prepared by either dithionite reduction of the Cu(II) derivative or by reconstitution of the apo protein with Cu(I). X-ray absorption (XAS) spectroscopy showed that Cu(I) was coordinated by two cysteines at 2.22 +/- 0.01 A and by a weakly bound low-Z scatterer at 1.95 +/- 0.03 A. The Cu(II) derivative was reddish-orange and exhibited a strong type-2 thiolate to Cu(II) transition around 350 nm. Multifrequency electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) studies on the Cu(II) derivative provided evidence of one strongly coupled histidine residue, at least one strongly coupled cysteine, and coupling to an exchangeable proton. XAS spectroscopy indicated two cysteine ligands at 2.21 A and two O/N donor ligands at 1.95 A, at least one of which is derived from a coordinated histidine. The Zn(II) and Ni(II) derivatives were 4-coordinate with MS2N(His)X coordination. These results provide evidence that a copper chaperone can engage in redox chemistry at the metal center and may suggest interesting redox-based mechanisms for metalation of the mixed-valence CuA center of cytochrome c oxidase.

A hint for the function of human Sco1 from different structures

“…The ultraviolet͞visible (UV͞VIS) and EPR spectra of Cu(II)HSco1 are identical to those recently reported (15). Similar UV͞VIS spectra also were reported for bacterial Sco homologues (12,14,17) and Cu(II) nitrosocyanin (21,22). In addition, the UV͞VIS spectrum of Ni(II)HSco1 (Fig.…”

“…In addition, the UV͞VIS spectrum of Ni(II)HSco1 (Fig. 8, which is published as supporting information on the PNAS web site) is similar to that reported for the B. subtilis and Rhodobacter sphaeroides homologues (14,17), with two weak bands at 380 and 540 nm, respectively, correlating with the two low-energy bands of the Cu(II)HSco1 spectrum although shifted to lower energy (Fig. 8).…”

“…The presence of three ligands, one of each being a ''flexible'' His residue, also makes the site suitable for the binding of divalent metal ions. Indeed, Cu(II), Ni(II), and, presumably, Zn(II) (14) can bind at the same site of HSco1. In the case of a divalent metals, the coordination can be completed by an additional exogenous ligand, e.g., H 2 O, or by a protein carboxylate.…”

“…The structure revealed a potential metal binding site constituted by two Cys residues present in a conserved motif CPXXCP and a fully conserved His residue, in agreement with earlier extended x-ray absorption fine structure investigations of yeast Cu(I)Sco1 (13). Besides extended x-ray absorption fine structure data many other spectroscopic data on the human, yeast, and B. subtilis Sco proteins have confirmed that the protein binds Cu(I), Cu(II), and other metal ions (14)(15)(16)(17). The fold of apoSco1 protein, which contains four ␣-helices and seven ␤-strands organized in two ␤-sheets (12), is atypical for a metal chaperone given that it resembles the fold of thioredoxins, which are enzymes specialized for the reduction of protein disulfides (18).…”

Copper control of bacterial nitrous oxide emission and its impact on vitamin B 12 -dependent metabolism

“…The product of senC2 is a member of the bacterial homologs of Sco1, involved in the assembly of the Cu A center of cytochrome oxidases in mitochondria. The sensor of C ox (SenC)/Sco1 family, including SenC2, have a CXXXCP Cu-binding motif that provides for cysteinyl coordination of Cu, completed by an axial histidinyl ligand (19)(20)(21).…”