Characterization of the Redox and Metal Binding Activity of BsSco, a Protein Implicated in the Assembly of Cytochrome c Oxidase

Abstract

Members of the Sco protein family are implicated in the assembly of the respiratory complex cytochrome c oxidase. Several possible roles have been proposed for Sco: a copper delivery agent, a site-specific thiol reductase, and an indicator of cellular redox status. Two cysteine residues (C45 and C49) in the sequence CXXXCP and a histidine (H135) approximately 90 residues toward the C-terminus are conserved in Sco from bacteria, yeast, and humans. The soluble domain of Sco has a thioredoxin fold that is suggestive of redox activity for this protein. We have characterized the soluble domain of the Sco protein from Bacillus subtilis (i.e., sBsSco) for its redox reactivity and metal binding capacity. In oxidized sBsSco, the cysteines are present as an intramolecular disulfide. Oxidized sBsSco does not bind metal, but can be reduced in vitro to a metal-binding form. Reduction of the disulfide in sBsSco is accompanied by increased intrinsic fluorescence. The reducibility of the cystine is unchanged when the conserved histidine is mutated to alanine. Tight binding by reduced sBsSco is observed for Cu(II) by electronic absorption, intrinsic fluorescence, and EPR spectroscopies, and isothermal titration calorimetry with an observed stoichiometry of one Cu(II) ion per sBsSco and a KD of approximately 50 nM. Tight binding of Cu(I) and Ag(I) is observed by quenching of intrinsic tryptophan fluorescence. Cobalt(II) exhibits weak binding, whereas Ni(II) and Zn(II) do not appear to bind. The high-affinity binding of metals by BsSco is triggered by its redox state, and this property could be important for its function in vivo.

H135A Controls the Redox Activity of the Sco Copper Center. Kinetic and Spectroscopic Studies of the His135Ala Variant of Bacillus subtilis Sco

“…We observe a remarkable increase in redox sensitivity when the H135 residue is replaced with a non-coordinating alanine residue traceable to a large decrease in the stability of the Cu(II) form. In contrast to previous studies (4, 12), this variant binds copper but is susceptible to autoreduction of Cu(II) to Cu(I) (coupled to cysteine autoxidation to a disulfide) and exhibits a 10 3 -fold increase in its reactivity towards hydrogen peroxide. Addition of imidazole causes EPR spectral changes consistent with rebinding of the imidazole ring to the Cu(II) center, yet this fails to fully restore the redox stability of the native Cu(II) form suggesting a critical conformational role of the native H135 in stabilizing the Cu(II) state.…”

Human Sco1 functional studies and pathological implications of the P174L mutant

“…The comprehension and description of these processes are still in their infancy, and only now knowledge about the structure and functional role of the assisting proteins has begun to become available. Many lines of evidence show that copper insertion into Cu A site of COXII subunit is not a simple, passive copper transfer from Cox17 to Sco1/2 proteins and finally to COXII, but redox reactions are interlinked with the process of copper transfer (12,15,16,27).…”

Disparate Pathways for the Biogenesis of Cytochrome Oxidases in Bradyrhizobium japonicum

“…UV-visible Spectroscopy of ScoI-The method was used to follow the binding of Cu(II) to reduced ScoI (52). Reduction of ScoI sol was achieved by incubation with 2 mM dithiothreitol for 4 h at 4°C.…”

“…The addition of CuCl 2 to the protein resulted in an absorbance peak at ϳ360 nm (Fig. 6), reflecting the binding of Cu(II) to the protein (52). Upon incremental copper addition, the peak increased and reached a plateau at a ratio of ϳ0.…”