Quinones and nitroaromatic compounds as subversive substrates of Staphylococcus aureus flavohemoglobin

In microorganisms, flavohemoglobins (FHbs) containing FAD and heme (Fe³⁺, metHb) convert NO. into nitrate at the expense of NADH and O₂. 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 (E¹₇), whereas the reactivity of quinones poorly depended on their E¹₇ 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 (HbFe²⁺O₂) formation (~130 s^-1, 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 HbFe²⁺O₂. 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.