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.
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 nm. 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.
Department of Biochemistry, Queen's University, Kingston, ON K71 3N6, Canada.
Thomas R
TR
Cawthorn
Bradley E
BE
Poulsen
David E
DE
Davidson
Diann
D
Andrews
Bruce C
BC
Hill
D000042:Absorption; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001426:Bacterial Proteins; D008565:Membrane Proteins; C482909:Sco protein, Bacillus subtilis; D003300:Copper; D003576:Electron Transport Complex IV