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 (HMP(Sa)) 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 HMP(Sa), 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 HMP(Sa). 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. 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.