This is facilitated by the electron-donating cofactors bound to the reductase domain. fHbs exhibit a nitric oxide dioxygenase (NOD) activity which render the microorganism resistant to NO˙ toxicity by converting it, at the heme active site, into nitrate (NO 3 −) in the presence of O 2 as co-substrate. fHb structure consists of the natural fusion of an α-helical heme-binding domain with a typical heme globin structure and a reductase domain which contains the FAD cofactor and the binding site of the co-substrate NADH. Introduction Flavohemoglobins (fHbs) belong to a family of heme proteins found in a large number of modern microbes. 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. These data are in agreement with the computational results that propose distinct binding sites for ketoconazole and miconazole on CO bound FHP. 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 HMP Sa does not impact the reaction volume. In FHP, the ligand photo-release is associated with Δ H = 26.2 ± 7.0 kcal mol −1 and Δ V = 25.0 ± 1.5 mL mol −1 while in HMP Sa, Δ H = 34.7 ± 8.0 kcal mol −1 and Δ V = 28.6 ± 17 mL mol −1 were observed, suggesting distinct structural changes associated with ligand escape from FHP and HMP Sa. 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 (HMP Sa) in the presence and absence of antibiotic azole compounds.
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. 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. They are involved in NO detoxification through an NO˙ dioxygenase mechanism. Flavohemoglobins (fHbs) are heme proteins found in prokaryotic and eukaryotic microbes.
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