Departamento de Biología Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas (CSIC), Zaragoza, Spain.
Institute of Biochemistry, Department of Chemistry and Biochemistry, University of Cologne, Germany.
Physiol Plant. 2024 Jul-Aug;176(4):e14438. doi: 10.1111/ppl.14438.
Human mitochondria contain a molybdoprotein capable of reducing amidoximes using cytochrome b/cytochrome b reductase (Cb/CbR). This 'amidoxime reducing component' (ARC) also reduces nitrite to nitric oxide (NO). In the plant kingdom, distinct functions have been suggested for ARCs. Thus, the single ARC of Chlamydomonas reinhardtii (crARC) reduces nitrite to NO by taking electrons from nitrate reductase (NR). Therefore, it was proposed that a dual NR/crARC system can generate NO under physiological conditions and the crARC was renamed to 'NO-forming nitrite reductase' (NOFNiR). In contrast to this, the two ARC enzymes from Arabidopsis thaliana were not found to produce NO in vitro at physiological nitrite concentrations, suggesting a different, as yet unknown, function in vascular plants. Here, we have investigated the two ARCs of Lotus japonicus (LjARCs) to shed light on this controversy and to examine, for the first time, the distribution of ARCs in plant tissues. The LjARCs are localized in the cytosol and their activities and catalytic efficiencies, which are much higher than those of A. thaliana, are consistent with a role as NOFNiR. LjARCs are prone to S-nitrosylation in vitro by S-nitrosoglutathione and this post-translational modification drastically inhibits their activities. The enzymes are mainly expressed in flowers, seeds and pods, but are absent in nodules. LjARCs are active with NR and Cb/CbR as electron-transferring systems. However, the LjNR mRNA levels in seeds and pods are negligible, whereas our proteomic analyses show that pods contain the two ARCs, Cb and CbR. We conclude that LjARCs may play a role as NOFNiR by receiving electrons from the Cb/CbR system but do not act in combination with NR.
线粒体含有一种钼蛋白，能够利用细胞色素 b/细胞色素 b 还原酶（Cb/CbR）将酰胺肟还原。这个“酰胺肟还原成分”（ARC）也能将亚硝酸盐还原为一氧化氮（NO）。在植物王国中，ARC 具有不同的功能。因此，衣藻（Chlamydomonas reinhardtii）的单一 ARC（crARC）通过从硝酸还原酶（NR）获取电子将亚硝酸盐还原为 NO。因此，有人提出，在生理条件下，双 NR/crARC 系统可以产生 NO，并且 crARC 被重新命名为“形成 NO 的亚硝酸盐还原酶”（NOFNiR）。与此相反，在生理亚硝酸盐浓度下，拟南芥的两个 ARC 酶在体外未发现产生 NO，这表明在维管植物中存在不同的、目前未知的功能。在这里，我们研究了豆科植物（Lotus japonicus）的两个 ARC（LjARCs），以阐明这一争议，并首次研究 ARC 在植物组织中的分布。LjARCs 定位于细胞质中，其活性和催化效率比拟南芥高得多，与作为 NOFNiR 的作用一致。LjARCs 容易在体外通过 S-亚硝基谷胱甘肽发生 S-亚硝基化，这种翻译后修饰极大地抑制了它们的活性。这些酶主要在花、种子和豆荚中表达，但在根瘤中不存在。LjARCs 与 NR 和 Cb/CbR 作为电子转移系统一起发挥作用。然而，种子和豆荚中的 LjNR mRNA 水平可以忽略不计，而我们的蛋白质组学分析表明，豆荚中含有这两个 ARC 和 Cb 和 CbR。我们得出结论，LjARCs 可能通过从 Cb/CbR 系统接收电子来发挥作为 NOFNiR 的作用，但不与 NR 一起作用。
