Department of Chemistry, Institute of Biochemistry, University of Cologne, Cologne, Germany.
Department of Chemistry, Institute of Biochemistry, University of Cologne, Cologne, Germany.
J Biol Chem. 2022 Mar;298(3):101668. doi: 10.1016/j.jbc.2022.101668. Epub 2022 Feb 2.
Sulfite oxidase (SOX) is a homodimeric molybdoheme enzyme that oxidizes sulfite to sulfate at the molybdenum center. Following substrate oxidation, molybdenum is reduced and subsequently regenerated by two sequential electron transfers (ETs) via heme to cytochrome c. SOX harbors both metals in spatially separated domains within each subunit, suggesting that domain movement is necessary to allow intramolecular ET. To address whether one subunit in a SOX dimer is sufficient for catalysis, we produced heterodimeric SOX variants with abolished sulfite oxidation by replacing the molybdenum-coordinating and essential cysteine in the active site. To further elucidate whether electrons can bifurcate between subunits, we truncated one or both subunits by deleting the heme domain. We generated three SOX heterodimers: (i) SOX/Mo with two active molybdenum centers but one deleted heme domain, (ii) SOX/Mo_C264S with one unmodified and one inactive subunit, and (iii) SOX_C264S/Mo harboring a functional molybdenum center on one subunit and a heme domain on the other subunit. Steady-state kinetics showed 50% SOX activity for the SOX/Mo and SOX/Mo_C264S heterodimers, whereas SOX_C264S/Mo activity was reduced by two orders of magnitude. Rapid reaction kinetics monitoring revealed comparable ET rates in SOX/Mo, SOX/Mo_C264S, and SOX/SOX, whereas in SOX_C264S/Mo, ET was strongly compromised. We also combined a functional SOX Mo domain with an inactive full-length SOX R217W variant and demonstrated interdimer ET that resembled SOX_C264S/Mo activity. Collectively, our results indicate that one functional subunit in SOX is sufficient for catalysis and that electrons derived from either Mo or Mo follow this path.
亚硫酸氧化酶 (SOX) 是一种同二聚体钼血红素酶，在钼中心将亚硫酸盐氧化为硫酸盐。在底物氧化后，钼通过两个连续的电子转移 (ET) 被还原，并通过血红素随后再生到细胞色素 c。SOX 在每个亚基的空间分离结构域中都含有这两种金属，这表明结构域运动对于允许分子内 ET 是必要的。为了解决 SOX 二聚体中的一个亚基是否足以进行催化，我们通过替换活性位点中钼配位和必需的半胱氨酸，产生了具有阻止亚硫酸盐氧化作用的杂二聚体 SOX 变体。为了进一步阐明电子是否可以在亚基之间分叉，我们通过删除血红素结构域截短了一个或两个亚基。我们生成了三种 SOX 杂二聚体：(i) SOX/Mo 具有两个活性钼中心但一个缺失血红素结构域，(ii) SOX/Mo_C264S 具有一个未修饰和一个无活性的亚基，以及 (iii) SOX_C264S/Mo 在一个亚基上具有功能钼中心和另一个亚基上具有血红素结构域。稳态动力学显示 SOX/Mo 和 SOX/Mo_C264S 杂二聚体的 SOX 活性为 50%，而 SOX_C264S/Mo 的活性降低了两个数量级。快速反应动力学监测表明，SOX/Mo、SOX/Mo_C264S 和 SOX/SOX 中的 ET 速率相当，而在 SOX_C264S/Mo 中，ET 受到严重影响。我们还将功能 SOX Mo 结构域与无活性全长 SOX R217W 变体结合，并证明了类似于 SOX_C264S/Mo 活性的二聚体 ET。总的来说，我们的结果表明，SOX 中的一个功能亚基足以进行催化，并且来自 Mo 或 Mo 的电子遵循这条途径。
