Effects of Azole Drugs on Conformational Dynamics and Energetics Associated with Diatomic Ligand Photorelease and Migration within Bacterial Flavohemoglobins

Abstract

Bacterial flavohemoglobins (fHbs) are a widely distributed class of proteins which are though to protect microbes from nitrosative stress due to the immune response of a host organism via nitric oxide dioxygenase (NOD) activity. There has been interest lately in developing fHbs as targets for antibiotics due to their ubiquity in bacteria and the discovery of inhibition of NOD activity of fHbs by azole-based drugs which bind with high affinity (<12 μM). To determine how azole drugs affect fHbs we have used photoacoustic calorimetry (PAC) and transient absorption spectroscopy (TA) to calculate enthalpy and volume changes (ΔH and ΔV) and kinetic parameters for CO photorelease and rebinding to fHbs from Cupriavidus necator (FHP) and Staphylococcus aureus (HMPSa) in the presence and absence of the drugs ketoconazole and miconazole. We observe that binding of drugs to fHbs results in a more exothermic ΔH for CO photorelease and faster bimolecular rebinding of CO. We also use classical molecular dynamics (cMD) to characterize ligand migration pathways present in FHP. We have conducted cMD simulations of CO-FHP WT and FHP WT containing free CO molecules using a locally enhanced sampling (LES) algorithm. We have carried out a 10 ns simulation of CO-FHP WT and a 30 ns simulation of the LES-enabled CO:FHP WT. We observe dissociation of 18 of 20 LES-enhanced CO molecules from the distal heme pocket. The primary escape pathways are between the CD-loop and heme-propionate groups and through a pathway exiting the protein matrix near the N-terminal end of the A-helix. These pathways are close to the locations of ketoconazole and miconazole binding predicted by AutoDock Vina, suggesting that drug binding inhibits NOD activity by closing migration pathways for O2 and NO.