Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c₅₅₀ (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu²⁺ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu²⁺ LMCT transitions.

中文翻译：

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慢生根瘤菌含铜亚硝酸还原酶及其生理氧化还原伴侣细胞色素c550的异源生产及功能表征

两个域亚硝酸铜还原酶 (NirK) 包含两种类型的铜中心，一个是 1 型电子转移 (ET) 中心 (T1)，一个是 2 型催化位点 (T2)。NirK 活性是 pH 依赖性的，这被认为是由一些催化相关残基在高 pH 值下的结构修饰产生的。为了表征 NirK 的 pH 依赖性动力学和蛋白内 ET 中 T1 共价的相关性，我们研究了生化、电化学和光谱特性，并辅以日本慢生根瘤菌NirK ( Bj NirK) 及其电子供体细胞色素c 的QM/MM 计算₅₅₀ ( Bj CycA)。Bj NirK 表示主要由 S(Cys)3pπ → Cu 决定的吸收光谱²⁺配体到金属电荷转移 (LMCT) 跃迁。该酶表现出低活性，可能是由于与Bj NirK/ Bj CycA 相互作用相关的蛋白质环具有更高的灵活性。在没有 T1 → T2 ET 的情况下，高 pH 值下亚硝酸盐以 T1 解耦方式被还原，其中在 T2 时维持用于亚硝酸盐还原的质子传递。我们的结果与之前在苜蓿中华根瘤菌NirK 中发现的结果进行了比较分析，其主要的紫外-可见吸收特征由 S(Cys)3pσ/π → Cu ²⁺ LMCT 跃迁决定。