Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

Abstract

Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe(3+)←S ligand-to-metal charge transfer band from 700 to 650 nm. The observed UV-visible absorption bands for the Fe(3+)-nitrile intermediate species are similar to low spin Fe(3+)-enzyme and model complexes bound by NO or N3((-)). These data provide spectroscopic evidence for the direct coordination of the nitrile substrate to the nitrile hydratase active site low spin Fe(3+) center.