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 g1 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.

Abstract:The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page.Biochemical Journal, Vol 474, No. 2 (January 15, 2017): pg. 247-258. DOI. This article is © Portland Press Limited and permission has been granted for this version to appear in e-Publications@Marquette. Portland Press Limited does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from Portland Press Limited. 2GTP to GDP with a kcat value of 1.2 × 10 −3 s −1 and a Km value of 40 μM in the presence of 5 mM MgCl2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a Kd of 2.9 μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes.

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Background:No direct evidence exists for the direct coordination of nitrile to the Fe 3ϩ active site in nitrile hydratases. Results: The first Fe 3ϩ -nitrile intermediate species is reported using stopped-flow spectroscopy. Conclusion: These data establish that the direct ligation of the nitrile substrate occurs during catalytic turnover. Significance: Understanding the catalytic mechanism of nitrile hydratases is critical to harness their bioremediation and industrial potential.