Laboratoire de Chimie Physique, Université Paris Sud, CNRS UMR 8000, 91405 Orsay Cedex France.
Institute of Biochemistry of Vilnius University, Saulėtekio 7, LT-10257 Vilnius, Lithuania.
Free Radic Biol Med. 2018 Aug 1;123:107-115. doi: 10.1016/j.freeradbiomed.2018.05.071. Epub 2018 May 21.
In microorganisms, flavohemoglobins (FHbs) containing FAD and heme (Fe, metHb) convert NO. into nitrate at the expense of NADH and O. FHbs contribute to bacterial resistance to nitrosative stress. Therefore, inhibition of FHbs functions may decrease the pathogen virulence. We report here a kinetic study of the reduction of quinones and nitroaromatic compounds by S. aureus FHb. We show that this enzyme rapidly reduces quinones and nitroaromatic compounds in a mixed single- and two-electron pathway. The reactivity of nitroaromatics increased upon an increase in their single-electron reduction potential (E), whereas the reactivity of quinones poorly depended on their E with a strong preference for a 2-hydroxy-1,4-naphthoquinone structure. The reaction followed a 'ping-pong' mechanism. In general, the maximal reaction rates were found lower than the maximal presteady-state rate of FAD reduction by NADH and/or of oxyhemoglobin (HbFeO) formation (~130 s, pH 7.0, 25 °C), indicating that the enzyme turnover is limited by the oxidative half-reaction. The turnover studies showed that quinones prefreqently accept electrons from reduced FAD, and not from HbFeO. These results suggest that quinones and nitroaromatics act as 'subversive substrates' for FHb, and may enhance the cytotoxicity of NO. by formation of superoxide and by diverting the electron flux coming from reduced FAD. Because quinone reduction rate was increased by FHb inhibitors such as econazole, ketoconazole, and miconazole, their combined use may represent a novel chemotherapeutical approach.
在微生物中，含有 FAD 和血红素（Fe、metHb）的黄素血红蛋白 (FHb) 将 NO.转化为硝酸盐，消耗 NADH 和 O。FHb 有助于细菌抵抗硝化应激。因此，抑制 FHb 的功能可能会降低病原体的毒力。我们在这里报告了金黄色葡萄球菌 FHb 还原醌和硝基芳烃化合物的动力学研究。我们表明，这种酶通过混合单电子和双电子途径快速还原醌和硝基芳烃化合物。随着单电子还原电位 (E) 的增加，硝基芳烃的反应性增加，而醌的反应性则较差，对 2-羟基-1,4-萘醌结构表现出强烈的偏好。反应遵循“乒乓”机制。一般来说，最大反应速率低于 NADH 和/或氧合血红蛋白 (HbFeO) 形成的 FAD 还原的最大准稳态速率 (~130 s, pH 7.0, 25°C)，表明酶周转率受氧化半反应限制。周转率研究表明，醌优先从还原的 FAD 接受电子，而不是从 HbFeO 接受电子。这些结果表明，醌和硝基芳烃作为 FHb 的“颠覆性底物”，可能通过形成超氧化物和从还原的 FAD 转移电子流来增强 NO 的细胞毒性。由于 FHb 抑制剂如酮康唑、克霉唑和咪康唑可增加醌的还原速率，因此联合使用这些抑制剂可能代表一种新的化疗方法。
