Impact of azole drugs on energetics, kinetics, and ligand migration pathways of CO photo-dissociation in bacterial flavohemoglobins.

Flavohemoglobins (fHbs) are heme proteins found in prokaryotic and eukaryotic microbes. They are involved in NO detoxification through an NO˙ dioxygenase mechanism. The N-terminal heme globin domain allows for binding of gaseous ligands whereas a C-terminal NADH/FADH binding domain facilitates association of redox cofactors necessary for ligand reduction. The NO˙ dioxygenase function is important in facilitating immune resistance by protecting the cell from nitrosative stress brought about by a host organism; as a result, bacterial flavoHbs have recently been considered as targets for the development of new antibiotics. Here, photoacoustic calorimetry and transient absorption spectroscopy have been used to characterize energetics, structural dynamics, and kinetics of CO migration within bacterial flavoHbs from Ralstonia eutropha (FHP) and Staphylococcus aureus (HMPSa) in the presence and absence of antibiotic azole compounds. In FHP, the ligand photo-release is associated with ΔH = 26.2 ± 7.0 kcal mol-1 and ΔV = 25.0 ± 1.5 mL mol-1 while in HMPSa, ΔH = 34.7 ± 8.0 kcal mol-1 and ΔV = 28.6 ± 17 mL mol-1 were observed, suggesting distinct structural changes associated with ligand escape from FHP and HMPSa. In the presence of ketoconazole, the CO escape leads to a more negative enthalpy change and volume change whereas association of miconazole to FHP or HMPSa does not impact the reaction volume. These data are in agreement with the computational results that propose distinct binding sites for ketoconazole and miconazole on CO bound FHP. Miconazole or ketoconazole binding to either protein has only a negligible impact on the CO association rates, indicating that azole drugs do not impact flavoHbs interactions with gaseous ligands but may inhibit the NOD activity through preventing the electron transfer between FAD and heme cofactors.

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.

Antimicrobial agents act differently on Staphyloccocus aureus and Ralstonia eutropha flavohemoglobins.

Flavohemoglobins (FlavoHb) play a key role in bacterial resistance to nitrosative stress and NO signaling modulation. In this study, we cloned, expressed, and characterized the flavoHb from the opportunistic pathogen, Staphylococcus aureus. The higher amino-acid sequence homology is shared with that from Saccharomyces cerevisiae which was therefore used to build a model structure by homology modeling. Interestingly, the high sequence homology with S. cerevisiae did not correlate with the enzymatic and kinetic properties which are much similar to those of Escherichia coli. In vitro and aerobically, we showed that S. aureus and Ralstonia eutropha flavoHbs accept cytochrome c and oxygen as substrates. Based on this feature, we investigated the preferences for both substrates depending on miconazole or econazole addition and found that the inhibitor chemical composition is determinant.