Metalloproteins catalyze nitric oxide (NO) chemistries in bioenergetics pathways, natural product biosynthesis pathways, and to protect against cell damage caused by nitrosative stress. Our lab is interested in the mechanisms of oxidative NO-dependent metalloenzymes involved in natural product biosynthesis and nitrosative stress protection. Understanding the mechanisms of these enzymes will enable the engineering of nitration biocatalysts and provide insight into how human pathogens evade the human immune response.
The first enzyme discussed will be TxtE, a cytochrome P450 (CYP) homolog that mediates a nitric oxide (NO)-dependent direct nitration of L-tryptophan (L-Trp) to form 4- nitrotryptophan (4-NO2-L-Trp). This nitration activity differs from the canonical CYP activity of substrate hydroxylation. We have recently shown that the TxtE ferric-superoxo intermediate resists reduction. By contrast, canonical CYPs reduce this intermediate to form the active oxidant compound I. This observation combined with available structural data suggests that outer sphere interactions influence the ferric-superoxo reactivity, thereby enabling efficient nitration activity in TxtE. Evidence for this conclusion will be discussed in addition to more recent data probing the influences on the reactivity of the ferric-superoxo intermediate.
The second enzyme discussed will be a hemerythrin-like protein (HLP) found in pathogenic Mycobacteria. In Mycobacterium tuberculosis the rv2663c gene that codes for this HLP is associated with known virulence operons; however, the activity of this HLP was unknown. We showed that HLP from Mycobacterium kansasii exhibited reductive nitrosylation to form nitrite and NO dioxygenation to form nitrate, albeit only in stoichiometric amounts. Addition of H2O2 enabled NO peroxidase turnover. These reactivities are novel for a non-heme diiron enzyme and are instead associated with heme proteins. Evidence for these novel non-heme metalloenzymes chemistries will be presented.