The Influence of Copper Binding on the Stability of the SCO Protein from Bacillus subtilis

Abstract

Every aerobic organism expresses cytochrome c oxidase to catalyze reduction of molecular oxygen to water, and takes advantage of this energy releasing reaction to produce an electrochemical gradient used in cellular energy production. The protein SCO (Synthesis of cytochrome c oxidase) is a required assembly factor for the oxidase, conserved across many species. SCO is implicated in the assembly of one of two copper centres (ie., CuA) of cytochrome oxidase. The exact mechanism of SCO’s participation in CuA assembly is not known. SCO has been proposed to bind and deliver copper, or alternatively to act in reductive preparation of the CuA site within the oxidase. In this body of work, the strength and stability of Cu(II) binding to Bacillus subtilis SCO is explored via electronic absorption and fluorescence spectroscopies and by calorimetric methods. An equilibrium dissociation constant (Kd) of 3.5x10-12 M was determined as an upper limit for the BsSCO-Cu(II) interaction, via differential scanning calorimetry. In the first reported case for a SCO homolog, dissociation kinetics of Cu(II) from BsSCO were characterized, and found to be dependent on both ionic strength and the presence of free Cu(II) in solution. Further differential scanning calorimetry experiments performed at high ionic strength support a two-step model of BsSCO and Cu(II) binding. The implications of this model for the BsSCO-Cu(II) interaction are presented in relation to the mechanism of interaction between SCO and the CuA site of cytochrome c oxidase.