Cawthorn Thomas R, Poulsen Bradley E, Davidson David E, Andrews Diann, Hill Bruce C
Department of Biochemistry, Queen's University, Kingston, ON K71 3N6, Canada.
Biochemistry. 2009 Jun 2;48(21):4448-54. 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 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.
BsSco是Sco蛋白家族的成员，参与细胞色素c氧化酶中Cu(A)中心的组装。BsSco与Cu(II)形成一种复合物，其性质与二硫醇盐配位一致。停流紫外可见吸收光谱和荧光光谱结合多波长分析揭示了BsSco与Cu(II)之间的双相结合动力学。在铜浓度依赖性速率（2.9×10⁴ M⁻¹ s⁻¹）下，出现一个初始物种，其吸收峰位于382 nm处。初始物种以一级速率（1.5 s⁻¹）衰减至平衡形式，其最大吸收峰位于352 nm处。BsSco-Cu(II)复合物的形成伴随着蛋白质荧光的淬灭。铜浓度依赖性阶段贡献了总淬灭的70%，而最后的30%在吸收变化的第二阶段产生。铜结合的pH依赖性表明，随着pH从8.5降至5.5，铜依赖性速率增加50倍，表观pKₐ为6.7。较慢阶段的速率与pH无关。脱辅基BsSco和BsSco-Cu(II)复合物的圆二色光谱比较显示，紫外区域有微小变化，这与铜结合后微妙的构象变化一致。BsSco-Cu(II)复合物中形成了独特的可见圆二色光谱。提出了一个模型，其中BsSco-Cu(II)复合物的动力学和热力学稳定性源于两步机制。在BsSco的中间形式中，铜的释放将更容易，而达到这种低Cu(II)亲和力状态可能对BsSco在Cu(A)组装中的功能很重要。
