Spectroscopic And Kinetic Investigation Of N-Oxygenation By Cmli, A Diiron-Cluster-Containing Oxygenase Involved In Antibiotic Biosynthesis

Abstract

Diiron-cluster-containing oxygenases catalyze a wide range of biological reactions. The chemical and biological breadth of the field is still being defined by discovery of new enzymes. The N-oxygenase CmlI, which plays a role in the biosynthesis of the broad-spectrum antibiotic chloramphenicol, is a new entry that brings two significant characteristics to this enzyme class. First, the active oxidant of CmlI is a diferric peroxo species (P), in contrast to the high-valent intermediates that often serve as oxidants in the cycles of nonheme iron oxygenases. P has unique spectroscopic features and a long lifetime in the absence of substrate (t1/2 ~ 3 h at 4 °C, pH 9), which facilitated the discovery of its novel η1- η1 or η1- η2 diferric peroxo geometry that distinguishes it from the common cis-μ-1,2 geometry and may account for its unique reactivity. Characterization of the structure and function of P has led to a new understanding of the role of peroxo species in oxygen activation and insertion chemistry. The second novel aspect of CmlI is the chemistry that it performs, the six-electron conversion of an aryl-amine precursor to the aryl-nitro group of the active antibiotic. Utilization of P in single turnover reactions allowed spectroscopic and kinetic characterization of each step of this conversion. The six-electron transformation begins when P converts the aryl-amine substrate into an aryl-hydroxylamine, which acts as a mid-cycle reductant to re-reduce and prime CmlI to regenerate P by reaction with O2 while itself being converted to the aryl-nitroso species. The regenerated P species then performs the last oxidation to convert the aryl-nitroso into the aryl-nitro group of chloramphenicol. Transient kinetic studies show that the substrate is likely to stay in a single active site during the entire, multistep reaction. Herein is described a new and highly efficient diiron-cluster-mediated N-oxygenase mechanism.