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

Abstract

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.

Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future

“…Because the g ϭ 1.92 region contains only the contributions from overlapping individual g 2 or g 2,3 (g Ќ ) lines of distinct multiline rhombic or axial signals, the raw integral was multiplied by a factor of 7.41. This factor was derived from comparison of the integrated intensities of the g ʈ line and the entire spectrum of a rhombic S ϭ 1 ⁄ 2 Fe(III) signal from nitrile hydratase, with a spectral envelope of 450 G, that showed that 6% of the total spin density was reflected in the double integral of the single g ʈ line (114). The conversion factor was then scaled for the ratios of the squares of the spectral envelopes of the nitrile hydratase signal (450 G) and an averaged envelope for the FeS clusters (300 G) that contribute to the g ϭ 1.92 region (7).…”

The Effects of the Metal Ion Substitution into the Active Site of Metalloenzymes: A Theoretical Insight on Some Selected Cases

“…It is known that Fe 3+ −NHases shows higher affinity to aliphatic nitriles as substrates, while the Co 3+ −NHases to aromatic compounds. Experimental EPR measures are available [124] only for Fe 3+ -type NHase, because the d 6 low spin electronic configuration of Co 3+ −NHase enzymes does not allow their inspection by EPR spectroscopy. However, other spectroscopic data are available for Co 3+ −NHases [124].…”

“…Experimental EPR measures are available [124] only for Fe 3+ -type NHase, because the d 6 low spin electronic configuration of Co 3+ −NHase enzymes does not allow their inspection by EPR spectroscopy. However, other spectroscopic data are available for Co 3+ −NHases [124].…”

“…Despite the catalytic reaction of the nitrile hydrolysis by both Fe 3+ − and Co 3+ −NHases was extensively studied at the experimental level, [111,122,124,[128][129][130] its working mechanism remains poorly understood. At theoretical level, exhaustive studies exist only for the Fe 3+ −NHase enzyme [105,[114][115][116][117][118].…”

Cryogenic electron paramagnetic resonance spectroscopy of flash-frozen tissue for characterization of mitochondrial disease

“…Additional "fields" due to nuclear Zeeman and quadrupolar interactions can generally be neglected in the present context. EPR can, in principle, provide a wealth of information on the identity, chemical nature, chemical environment, electronic structure, and physical structure of the analyte from analysis of each of these interactions, typically employing computer analysis and simulations, and increasingly with quantum chemistry calculations (density functional theory, Taylor theory) [65][66][67][68][69][70][71][72][73][74][75][76] . However, in the case of biological tissues, the origins and spectroscopic parameters of many of the EPR signals have been well-characterized, as described in detail below.…”