Oxygen activation by novel nonheme diiron enzymes

Abstract

Enzymes that utilize nonheme diiron centers to activate dioxygen and carry out various chemical transformations are of great fundamental and practical interest. The mechanisms of these enzymes can be studied using various spectroscopic and biochemical methods, which are greatly aided by parallel studies on relevant model complexes. This thesis work employs this approach to investigate three non-cannonical diiron enzymes, human deoxyhypusine hydroxylase (hDOHH), as well as CmlA and CmlI, a pair of oxygenases on the biosynthesis pathway of the antibiotic chloramphenicol. We first provide a summary of recent studies on enzymes that employ the canonical 4-helix-bundle motif to house a diiron cluster with a 2-His-4-carboxylate ligand set, as well as those that use a whole new range of structural motifs and/or exibit non-canonical reactivities (Chapter 1). We have successfully characterized an unusually stable, yet functional, peroxo intermediate of hDOHH (hDOHHperoxo), an enzyme which possesses a superhelical HEAT-repeat fold (Chapter 2). Detailed spectroscopic analysis of hDOHHperoxo and related model complexes reveals that hDOHHperoxo contains a (cis-&mu;-1,2-peroxo)diiron(III) unit (Chapter 2) with an additional &mu;-hydroxo ligand (Chapter 3). This &mu;-hydroxo ligand is proposed to contribute to the remarkable stability of hDOHHperoxo and its reactivity. The formation of hDOHHperoxo and its reaction with substrate are detailed in Chapter 4. hDOHH adopts various conformations in different forms, that significantly affect the formation and activation of hDOHHperoxo. Notably, we have observed the epoxidation of one of the unsaturated analogues of the native deoxyhypusine-containing eIF5A substrate. In line with the studies on the unusual hDOHHperoxo intermediate, we have also studied another unique and relatively stable peroxo intermediate of CmlI (CmlIperoxo). Resonance Raman spectroscopy suggests that CmlIperoxo possibly contains a (&mu;-&eta;<super>1</super>:&eta;<super>2</super>-peroxo)diiron(III) cluster instead of the more common (&mu;-1,2-peroxo)diiron(III) cluster found in canonical diiron enzymes and model complexes (Chapter 5). EXAFS analysis of as-isolated CmlI reveals that it contains about 3 His ligands, which is consistent with sequence homology analysis. The third His ligand in CmlI could be responsible for the shift in the peroxo binding geometry of CmlIperoxo. Finally, resonance Raman and EXAFS analysis of CmlA, a &beta;-hydroxylase, shows that this enzyme contains a (&mu;-oxo)(&mu;-1,3-carboxylato)diiron(III) cluster with about 4 His ligands, a feature similar to diiron reductases and hydrolases. As found for CmlI, the extra His ligands in CmlA may lead to alternative reactive oxygen species as opposed to those found for methane monooxygenase and other canonical diiron enzymes.