Trytophan Mutants Probe the Structural and Functional Status of a Copper Binding, Cytochrome C Oxidase Assembly Protein from Bacillus Subtilis (i.e., BsSCO)

Abstract

SCO protein family members aid in the assembly of the CuA center of cytochrome c oxidase. SCOs possess a thioredoxin-fold and are conserved in bacteria, yeast and humans. In Bacillus subtilis, BsSCO, is anchored to the plasma membrane via a covalently attached lipid with a soluble domain exposed on the outer cell surface. All SCOs contain a pair of cysteine residues within the soluble domain that ligate copper, and engage in redox processes. BsSCO has two tryptophan residues whose fluorescence is influenced by the redox and/or ligation status of the reactive cysteine pair. The two tryptophan residues of BsSCO (i.e., W36 and W101) are located about 22 A and 15 A from the cysteine pair. In wild-type BsSCO oxidation of the dithiol to the disulfide quenches fluorescence by 30%, whereas copper binding quenches fluorescence by 70%. Here, we alter the complement and positions of BsSCO's TRP residues so as to alter their sensitivity to different states of BsSCO. Addition of a tryptophan residue replacing F42, nearby the di-cysteine site, enhances the stability of the protein, but does not greatly alter its fluorescence response to redox, or copper binding. The double mutant W36A/F42W-BsSCO has reduced stability (TM from 53 to 37 oC), but its fluorescence is more acutely affected by copper binding than is wild-type BsSCO. Assays of BsSCO's ability to promote assembly of cytochrome c oxidase show that the double mutant (i.e., W36A/F42W-BsSCO) is inactive in B. subtilis grown at 37 oC, but is functional in cells grown at 25 oC. The sensitivity of the fluorophore tryptophan is changed by altering its positions in BsSCO's structure, but such changes can also undermine the protein's intrinsic stability.