Monooxygenase/arylamine N-oxygenase activity within a single non-heme diiron enzyme (MiaE)

University Of Alabama Tuscaloosa, Tuscaloosa AL

Investigators

Abstract

With this award granted by the National Science Foundation-Chemistry Division, Chemistry of Life Processes Program, Dr. Brad S. Pierce at The University of Texas at Arlington will investigate the factors influencing the extent of biological oxidations. The proposed activities provide an opportunity to better understand structural factors driving biological oxidations and thus provide a framework for the rational design of biologically-inspired or bioengineered oxidation catalysts. Oxidations result in a net transfer of electrons from the initial molecule (or substrate) to molecular oxygen. From an accounting point of view, the "extent" of an oxidation can be defined as the number of electrons transferred. It has been previously noted that changes in structure around iron dramatically influence the extent of substrate oxidation by catalysts. The proposed biochemical studies utilize an iron catalyst to compare the factors directing 2- and 6-electron oxidations within a single, simplified platform. Beyond training for graduate/undergraduate students in biochemistry, biophysics and chemical catalysis, broader impact activities for this award include training and practical experience in scientific communication for the PI and students (undergraduate and graduate) participating in NSF-supported activities. Non-heme diiron enzymes are a ubiquitous family of enzymes capable of catalyzing an amazing diversity of biological oxidations. Among these enzymes, it has been observed that single amino acid perturbations within the first Fe-coordination sphere have a profound impact on the nature of transient intermediates produced following O2-activation and the extent of substrate oxidation. For example, within the bacterial multicomponent monooxygenase (BMM) superfamily, the hydroxylase component diiron site is coordinated by 2-histidine and 4-carboxylate (Asp or Glu) residues [2-His/4-carboxylate]. The reduced (diferrous) active sites of these enzymes are capable of reductively activating molecular oxygen to catalyze the 2-electron oxidation of its specific substrate. By contrast, a small number of enzymes have been identified which contain an additional His-residue coordinated to one of the two Fe-sites; resulting in a [3-His/4-carboxlate] diiron cluster. Among these are the arylamine N-oxygenase class of non-heme diiron enzymes AurF and CmlI. These enzymes catalyze a remarkable 6-electron oxidation of arylamine substrates to yield a nitroaryl product. The proposed studies utilize an unusual non-heme tRNA-hydroxylase isolated from S. typhimurium (St MiaE) to compare the relevant factors directing (6-electron) arylamine N-oxygenase and (2-electron) monooxygenase chemistry within a single, enzymatic platform. This comparison is made possible by a single amino acid substitution (L199H) within the St MiaE active site which yields a [3-His/4-carboxylate] first Fe-coordination sphere and imparts 6-electron arylamine N-oxygenase activity. Beyond training for graduate/undergraduate students in biochemistry, biophysics and chemical catalysis, broader impact activities for this award include training and practical experience in scientific communication for the PI and students (undergraduate and graduate) participating in NSF-supported activities.