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Use of metallopeptide based mimics demonstrates that the metalloprotein nitrile hydratase requires two oxidized cysteinates for catalytic activity.

ABSTRACT: Nitrile hydratases (NHases) are non-heme Fe(III) or non-corrin Co(III) containing metalloenzymes that possess an N(2)S(3) ligand environment with nitrogen donors derived from amidates and sulfur donors derived from cysteinates. A closely related enzyme is thiocyanate hydrolase (SCNase), which possesses a nearly identical active-site coordination environment as CoNHase. These enzymes are redox inactive and perform hydrolytic reactions; SCNase hydrolyzes thiocyanate anions while NHase converts nitriles into amides. Herein an active CoNHase metallopeptide mimic, [Co(III)NHase-m1] (NHase-m1 = AcNH-CCDLP-CGVYD-PA-COOH), that contains Co(III) in a similar N(2)S(3) coordination environment as CoNHase is reported. [Co(III)NHase-m1] was characterized by electrospray ionization-mass spectrometry (ESI-MS), gel-permeation chromatography (GPC), Co K-edge X-ray absorption spectroscopy (Co-S: 2.21 Å; Co-N: 1.93 Å), vibrational, and optical spectroscopies. We find that [Co(III)NHase-m1] will perform the catalytic conversion of acrylonitrile into acrylamide with up to 58 turnovers observed after 18 h at 25 °C (pH 8.0). FTIR data used in concert with calculated vibrational data (mPWPW91/aug-cc-TZVPP) demonstrates that the active form of [Co(III)NHase-m1] has a ligated SO(2) (? = 1091 cm(-1)) moiety and a ligated protonated SO(H) (? = 928 cm(-1)) moiety; when only one oxygenated cysteinate ligand (i.e., a mono-SO(2) coordination motif) or the bis-SO(2) coordination motif are found within [Co(III)NHase-m1] no catalytic activity is observed. Calculations of the thermodynamics of ligand exchange (B3LYP/aug-cc-TZVPP) suggest that the reason for this is that the SO(2)/SO(H) equatorial ligand motif promotes both water dissociation from the Co(III)-center and nitrile coordination to the Co(III)-center. In contrast, the under- or overoxidized motifs will either strongly favor a five coordinate Co(III)-center or strongly favor water binding to the Co(III)-center over nitrile binding.

SUBMITTER: Shearer J

PROVIDER: S-EPMC3570060 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Use of metallopeptide based mimics demonstrates that the metalloprotein nitrile hydratase requires two oxidized cysteinates for catalytic activity.

Shearer Jason J Callan Paige E PE Amie Justina J

Inorganic chemistry 20101001 19

Nitrile hydratases (NHases) are non-heme Fe(III) or non-corrin Co(III) containing metalloenzymes that possess an N(2)S(3) ligand environment with nitrogen donors derived from amidates and sulfur donors derived from cysteinates. A closely related enzyme is thiocyanate hydrolase (SCNase), which possesses a nearly identical active-site coordination environment as CoNHase. These enzymes are redox inactive and perform hydrolytic reactions; SCNase hydrolyzes thiocyanate anions while NHase converts nit ...[more]

PMID: 20831172

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Project description:BackgroundNitrile hydratases are enzymes involved in the conversion of nitrile-containing compounds into ammonia and organic acids. Although they are widespread in prokaryotes, nitrile hydratases have only been reported in two eukaryotes: the choanoflagellate Monosiga brevicollis and the stramenopile Aureococcus anophagefferens. The nitrile hydratase gene in M. brevicollis was believed to have arisen by lateral gene transfer from a prokaryote, and is a fusion of beta and alpha nitrile hydratase subunits. Only the alpha subunit has been reported in A. anophagefferens.Methodology/principal findingsHere we report the detection of nitrile hydratase genes in five eukaryotic supergroups: opisthokonts, amoebozoa, archaeplastids, CCTH and SAR. Beta-alpha subunit fusion genes are found in the choanoflagellates, ichthyosporeans, apusozoans, haptophytes, rhizarians and stramenopiles, and potentially also in the amoebozoans. An individual alpha subunit is found in a dinoflagellate and an individual beta subunit is found in a haptophyte. Phylogenetic analyses recover a clade of eukaryotic-type nitrile hydratases in the Opisthokonta, Amoebozoa, SAR and CCTH; this is supported by analyses of introns and gene architecture. Two nitrile hydratase sequences from an animal and a plant resolve in the prokaryotic nitrile hydratase clade.Conclusions/significanceThe evidence presented here demonstrates that nitrile hydratase genes are present in multiple eukaryotic supergroups, suggesting that a subunit fusion gene was present in the last common ancestor of all eukaryotes. The absence of nitrile hydratase from several sequenced species indicates that subunits were lost in multiple eukaryotic taxa. The presence of nitrile hydratases in many other eukaryotic groups is unresolved due to insufficient data and taxon sampling. The retention and expression of the gene in distantly related eukaryotic species suggests that it plays an important metabolic role. The novel family of eukaryotic nitrile hydratases presented in this paper represents a promising candidate for research into their molecular biology and possible biotechnological applications.

Project description:To determine how a substitutionally inert metal can play a catalytic role in the metalloenzyme nitrile hydratase (NHase), a reactive five-coordinate Co(III) thiolate complex ([Co(III)(S(2)(Me2)N(3)(Pr,Pr))](PF(6)) (1)) that resembles the active site of cobalt containing nitrile hydratase (Co NHase) was prepared. This was screened for reactivity, by using low-temperature electronic absorption spectroscopy, toward a number of biologically relevant "substrates". It was determined 1 will react with azide, thiocyanate, and ammonia, but is unreactive toward nitriles, NO, and butyrate. Substrate-bound 1 has similar spectroscopic and structural properties as [Co(III)(ADIT(2))](PF(6)) (2). Complex 2 is a six-coordinate Co(III) complex containing cis-thiolates and imine nitrogens, and has properties similar to the cobalt center of Co NHase. Substrate binding to 1 is reversible and temperature-dependent, allowing for the determination of the thermodynamic parameters of azide and thiocyanate binding and the rates of ligand dissociation. Azide and thiocyanate bind trans to a thiolate, and with similar entropies and enthalpies (thiocyanate: DeltaH = -7.5 +/- 1.1 kcal/mol, DeltaS = -17.2 +/- 3.2 eu; azide: DeltaH = -6.5 +/- 1.0 kcal/mol, DeltaS = -12.6 +/- 2.4 eu). The rates of azide and thiocyanate displacement from the metal center are also comparable to one another (k(d) = (7.22 +/- 0.04) x 10(-)(1) s(-)(1) for thiocyanate and k(d) = (2.14 +/- 0.50) x 10(-)(2) s(-)(1) for azide), and are considerably faster than one would expect for a low-spin d(6) six-coordinate Co(III) complex. These rates are comparable to those of an analogous Fe(III) complex, demonstrating that Co(III) and Fe(III) react at comparable rates when in this ligand environment. This study therefore indicates that ligand displacement from a low-spin Co(III) center in a ligand environment that resembles NHase is not prohibitively slow so as to disallow catalytic action in nonredox active cobalt metalloenzymes.

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Use of metallopeptide based mimics demonstrates that the metalloprotein nitrile hydratase requires two oxidized cysteinates for catalytic activity.

Publications

Use of metallopeptide based mimics demonstrates that the metalloprotein nitrile hydratase requires two oxidized cysteinates for catalytic activity.

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