Institute of Biochemistry, Department of Chemistry, University of Cologne, Cologne, 50674, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, 50931, Germany.
Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, the Netherlands.
Nitric Oxide. 2019 Aug 1;89:22-31. doi: 10.1016/j.niox.2019.04.004. Epub 2019 Apr 17.
The oxygen-independent nitrate-nitrite-nitric oxide (NO) pathway is considered as a substantial source of NO in mammals. Dietary nitrate/nitrite are distributed throughout the body and reduced to NO by the action of various enzymes. The intermembrane spaced (IMS), molybdenum cofactor-dependent sulfite oxidase (SO) was shown to catalyze such a nitrite reduction. In this study we asked whether the primary function of SO - sulfite oxidation - and its novel function - nitrite reduction - impact each other. First, we utilized benzyl viologen as artificial electron donor to investigate steady state NO synthesis by SO and found fast (k = 14 s) nitrite reduction of SO full-length and its isolated molybdenum domain at pH 6.5. Next, we determined the impact of nitrite on pre-steady state kinetics in SO catalysis and identified nitrite as a pH-dependent inhibitor of SO reductive and oxidative half reaction. Finally, we report on the time-dependent formation of the paramagnetic Mo(V) species following nitrite reduction and demonstrate that sulfite inhibits nitrite reduction. In conclusion, we propose a pH-dependent reciprocal regulation of sulfite oxidation and nitrite reduction by each substrate, thus facilitating quick responses to hypoxia induced changes in the IMS, which may function in protecting the cell from reactive oxygen species production.
氧非依赖型硝酸盐-亚硝酸盐-一氧化氮（NO）途径被认为是哺乳动物中 NO 的主要来源。膳食中的硝酸盐/亚硝酸盐分布于全身，并在各种酶的作用下还原为 NO。跨膜间隙（IMS）中，钼辅因子依赖的亚硫酸盐氧化酶（SO）被证明可以催化这种亚硝酸盐还原。在这项研究中，我们询问了 SO 的主要功能——亚硫酸盐氧化——及其新功能——亚硝酸盐还原——是否相互影响。首先，我们利用苄基紫精作为人工电子供体来研究 SO 的稳态 NO 合成，并在 pH 值为 6.5 时发现 SO 全长及其分离的钼结构域快速（k=14 s）还原亚硝酸盐。接下来，我们确定了亚硝酸盐对 SO 催化中预稳态动力学的影响，并将亚硝酸盐鉴定为 SO 还原和氧化半反应的 pH 依赖性抑制剂。最后，我们报告了亚硝酸盐还原后顺磁 Mo(V)物种的时变形成，并证明了亚硫酸盐抑制亚硝酸盐还原。总之，我们提出了一种 pH 依赖性的亚硫酸盐氧化和亚硝酸盐还原的相互调节，从而促进了对 IMS 中缺氧诱导变化的快速响应，这可能有助于保护细胞免受活性氧的产生。
