Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral and CONICET, S3000ZAA Santa Fe, Argentina.
Metallomics. 2020 Dec 23;12(12):2084-2097. doi: 10.1039/d0mt00177e.
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.
两种结构域的铜-亚硝酸盐还原酶（NirK）含有两种类型的铜中心，一种是 1 型（T1）电子转移（ET）中心，另一种是 2 型（T2）催化位点。NirK 的活性依赖于 pH 值，这被认为是由于某些催化相关残基在高 pH 值下的结构修饰产生的。为了研究 NirK 的 pH 值依赖动力学以及 T1 共价键在蛋白质内 ET 中的相关性，我们研究了 Bradyrhizobium japonicum NirK（BjNirK）及其电子供体细胞色素 c550（BjCycA）的生化、电化学和光谱特性，并辅以 QM/MM 计算。BjNirK 的吸收光谱主要由 S(Cys)3pπ → Cu2+配体到金属电荷转移（LMCT）跃迁决定。由于与 BjNirK/BjCycA 相互作用相关的蛋白质环的较高灵活性，该酶的活性较低。在高 pH 值下，亚硝酸盐以 T1 解耦的方式被还原，而没有 T1 → T2 ET，其中 T2 上的质子传递用于亚硝酸盐还原。我们的结果与我们在 Sinorhizobium meliloti NirK 中发现的先前结果进行了比较，其主要的紫外可见吸收特征由 S(Cys)3pσ/π → Cu2+ LMCT 跃迁决定。
