Multiple States of Nitrile Hydratase from Rhodococcus equi TG328-2: Structural and Mechanistic Insights from Electron Paramagnetic Resonance and Density Functional Theory Studies

Iron-type nitrile hydratases (NHases) contain an Fe(III) ion coordinated in a characteristic “claw setting” by an axial cysteine thiolate, two equatorial peptide nitrogens, the sulfur atoms of equatorial cysteine-sulfenic and cysteine-sulfinic acids, and an axial water/hydroxyl moiety. The cysteine-sulfenic acid is susceptible to oxidation, and the enzyme is traditionally prepared using butyric acid as an oxidative protectant. The as-prepared enzyme exhibits a complex electron paramagnetic resonance (EPR) spectrum due to multiple low-spin ( S = ¹ / ₂ ) Fe(III) species. Four distinct signals can be assigned to the resting active state, the active state bound to butyric acid, an oxidized Fe(III)–bis(sulfinic acid) form, and an oxidized complex with butyric acid. A combination of comparison with earlier work, development of methods to elicit individual signals, and design and application of a novel density functional theory method for reproducing g tensors to unprecedentedly high precision was used to assign the signals. These species account for the previously reported EPR spectra from Fe-NHases, including spectra observed upon addition of substrates. Completely new EPR signals were observed upon addition of inhibitory boronic acids, and the distinctive g ₁ features of these signals were replicated in the steady state with the slow substrate acetonitrile. This latter signal constitutes the first EPR signal from a catalytic intermediate of NHase and is assigned to a key intermediate in the proposed catalytic cycle. Earlier, apparently contradictory, electron nuclear double resonance reports are reconsidered in the context of this work.