title: Reactivity of ligand-swapped mutants of the SCO protein from Bacillus subtilis. Isomers of the CCH metal binding motif.
abstract: The Synthesis of Cytochrome Oxidase protein, or SCO protein, is required for the assembly of cytochrome c oxidase in many mitochondrial and bacterial respiratory chains. SCOs have been proposed to deliver copper to the CuA site of cytochrome c oxidase. We have reported that Bacillus subtilis SCO (i.e., BsSCO) binds Cu(II) with high-affinity via a two-step process mediated by three conserved residues (i.e., two cysteines and one histidine, or the CCH motif). A remarkable feature in the reaction of reduced (i.e., di-thiol) BsSCO with copper is that it does not generate any of the disulfide form of BsSCO. This molecular aversion is proposed to be a consequence of a binding mechanism in which the initial copper complex of BsSCO does not involve cysteine, but instead involves nitrogen ligands. We test this proposal here by constructing two isomers of BsSCO in which the conserved copper binding residues (i.e., the CCH-motif) are retained, but their positions are altered. In these variants the two cysteines are exchanged with histidine, and both react transiently with copper (II) with distinct kinetic profiles. The reaction generates Cu(I) and the protein is oxidized to its disulfide form. EPR analysis supports a copper binding model in which cysteine, which is at the "histidine position" in the mutant, is part of an initial encounter complex with copper. When cysteine is the initial ligating residue an oxidation reaction ensues. In contrast initial binding to native BsSCO uses nitrogen-based ligands, and thereby avoids the opportunity for thiol oxidation.
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L3N6, Canada.
authors.forename: Xin
authors.initials: X
authors.lastname: Yao
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L3N6, Canada.
authors.forename: Diann
authors.initials: D
authors.lastname: Andrews
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L3N6, Canada. Electronic address: hillb@queensu.ca.
authors.forename: Bruce C
authors.initials: BC
authors.lastname: Hill
keywords: BsSCO; Copper binding; Cu(II) EPR; Ligand binding mutants; Redox conversion