Amicyanin is a type I copper protein that mediates electron transfer between methylamine dehydrogenase and cytochrome c-551i for energy production in Paracoccus denitrificans. Although the Met98 axial ligand of amicyanin has been shown to dictate metal selectivity and specificity during protein folding, the mechanism involved in copper-mediated amicyanin folding is unknown. Here, we kinetically and spectroscopically described reaction steps for incorporating copper into fully and less folded apo-amicyanin and established thermodynamic parameters for two amicyanin folding states. The rate constant for the incorporation of copper into fully folded apo-amicyanin at 25 °C was almost 1.5-fold lower than that for the initial phase of copper addition to the less folded apo-amicyanin. However, the rate constant was 10-fold higher than that of the second phase of copper addition to less folded apo-amicyanin at 25 °C. When overall binding energetic parameters (ΔH° and ΔS°) for the incorporation of copper into fully folded apo-amicyanin were measured by the van't Hoff method and isothermal titration calorimetry, the values were more positive than those determined for less folded apo-amicyanin. This indicates that during amicyanin biogenesis, copper rapidly binds to an unfolded apo-amicyanin active site, inducing protein folding and favorably influencing subsequent organization of copper ligands.

中文翻译：

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花青素在不同折叠状态下的铜结合能。

Amicyanin是一种I型铜蛋白，可介导甲胺脱氢酶和细胞色素c-551i之间的电子转移，从而在反硝化副球菌中产生能量。尽管已经显示出花青素的Met98轴向配体决定了蛋白质折叠过程中的金属选择性和特异性，但涉及铜介导的花青素折叠的机理尚不清楚。在这里，我们从动力学和光谱上描述了将铜结合到完全折叠的和折叠较少的载脂蛋白-花青素中的反应步骤，并建立了两个花色苷折叠状态的热力学参数。在25°C下将铜掺入完全折叠的脱辅基花青素中的速率常数比将铜添加到折叠较少的脱辅基花青素中初始阶段的速率常数几乎低1.5倍。然而，速率常数比第二阶段铜的速率常数高10倍，而在25°C时折叠的载脂蛋白花青素折叠较少。当通过van't Hoff方法和等温滴定量热法测量了将铜结合到完全折叠的脱辅基花青素中的总结合能参数（ΔH°和ΔS°）时，该值比未折叠的脱辅基-花青素所测定的值更正。花青素。这表明在花青素生物发生期间，铜迅速结合到未折叠的脱辅花青素活性位点，诱导蛋白质折叠并有利地影响铜配体的后续组织。用t Hoff法和等温滴定热法，该值比折叠较少的载脂蛋白-花青素所测得的值更正。这表明在花青素生物发生期间，铜迅速结合到未折叠的脱辅花青素活性位点，诱导蛋白质折叠并有利地影响铜配体的后续组织。用t Hoff法和等温滴定热法，该值比折叠少的载脂蛋白-花青素所测定的值更正。这表明在花青素生物发生期间，铜迅速结合到未折叠的脱辅花青素活性位点，诱导蛋白质折叠并有利地影响铜配体的后续组织。