Probing the Kinetics and Thermodynamics of Copper(II) Binding to Bacillus subtilis Sco, a Protein Involved in the Assembly of the Cu-A Center of Cytochrome c Oxidase

作者：Cawthorn Thomas R; Poulsen Bradley E; Davidson David E; Andrews Diann; Hill Bruce C^*

来源：Biochemistry, 2009, 48(21): 4448-4454.

DOI：10.1021/bi802288m

BsSco is a member of the Sco protein family involved in the assembly of the Cu-A center within cytochrome c oxidase. BsSco forms a complex with Cu(II) that has properties consistent with dithiolate ligation. Stopped-flow UV-visible absorbance and fluorescence coupled with multiwavelength analysis reveal biphasic binding kinetics between BsSco and Cu(II). An initial species appears with absorbance centered at 382 nm at a copper concentration-dependent rate (2.9 x 10(4) M-1 s(-1)). The initial species decays at a first-order rate (1.5 s(-1)) to the equilibrium form with a maximum at 352 rim. Formation of the BsSco-Cu (II) complex is accompanied by quenching of protein fluorescence. The copper concentration-dependent phase gives 70% of the total quenching.. while the final 30% develops during the second phase of the absorbance change. The pH dependence of copper binding shows that the copper-dependent rate increases by 50-fold as the pH decreases from 8.5 to 5.5 with an apparent pK(a) of 6.7. The slower phase rate is independent of pH. Comparison of circular dichroism spectra between apo-BsSco and the BsSco-Cu(II) complex reveals a small change in the UV region consistent with a subtle conformational change upon copper binding. There is formation of a distinctive visible CD spectrum in the BsSco-Cu(II) complex. A model is presented in which the kinetic and thermodynamic stability of the BsSco-Cu(II) complex results from a two-step mechanism. Release of copper would be facilitated in the intermediate form of BsSco, and attaining such a low-Cu(II) affinity state may be important for BsSco's function in Cu-A assembly.

• 出版日期2009-6-2