Reaction of Mycobacterium tuberculosis Cytochrome P450 Enzymes with Nitric Oxide

During the initial growth infection stage of Mycobacterium tuberculosis (Mtb), (NO)-N-center dot produced by host macrophages inhibits heme-containing terminal cytochrome oxidases, inactivates iron/sulfur proteins, and promotes entry into latency. Here we evaluate the potential of (NO)-N-center dot as an inhibitor of Mtb cytochrome P450 enzymes, as represented by CYP130, CYP51, and the two previously uncharacterized. enzymes CYP125 and CYP142. Using UV-visible absorption, resonance Raman, and stopped-flow spectroscopy, we investigated the reactions of (NO)-N-center dot with these heme proteins in their ferric resting form. (NO)-N-center dot coordinates tightly to CYP125 and CYP142 (submicromolar) and with a lower affinity (micromolar) to CYP130 and CYP51. Anaerobic reduction of the ferric-NO species with sodium dithionite led to the formation of two spectrally distinct classes of five-coordinate ferrous-NO complexes. Exposure of these species to O-2 revealed that the ferrous-NO forms of CYP125 and CYP142 are labile and convert back to the ferric state within a few minutes, whereas ferrous CYP130 and CYP51 bind (NO)-N-center dot almost irreversibly. This work clearly indicates that, at physiological concentrations (approximate to 1 mu M), (NO)-N-center dot would impair the activity of CYP130 and CYP51, whereas CYP125 and CYP142 are more resistant. Selective P450 inhibition may contribute to the inhibitory effects of (NO)-N-center dot on Mtb growth.