Coordination sphere of the ferric ion in nitrile hydratase

Hu, Jin; Turner, Ivan M.; Nelson, Mark J.; Gurbiel, Ryszard J.; Doan, Peter E.; Hoffman, Brian M.

doi:10.1021/ja00065a048

Cited by 78 publications

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“…The other two ligands remain unknown. Previous spectroscopic studies of the active enzyme suggested the coordination of His imidazole ligands at these sites (14,20 (38). Consistently with the crystal structure, the fact that IW11, a minimum peptide segment, does not contain a His residue as well as potential side chains nitrogen donors such as Asn, Gln, and Arg, suggests the coordination of backbone amide nitrogen atoms in IW11.…”

Section: Structure Of the Iron Center Of The Nhase In The Inactive Fosupporting

confidence: 79%

“…The structure of the iron center in the active form has been studied by various spectroscopies including ESR (3), resonance Raman (13), extended x-ray absorption fine structure (13) and electron nuclear double resonance (14), and the ligand-donor set of N 3 OS 2 has been proposed (14), which is supported by model complexes of the iron center (15)(16)(17). Recently, the metal site structure has been studied in detail by means of electron nuclear double resonance (18), resonance Raman (19), and x-ray absorption (20) spectroscopies.…”

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confidence: 99%

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Structure of the Photoreactive Iron Center of the Nitrile Hydratase from Rhodococcus sp. N-771

Tsujimura

Dohmae

Odaka

et al. 1997

Journal of Biological Chemistry

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Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the ␣ subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn 105 -Val-Ile-Val-CysSer-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu-Gly-LeuPro-Pro-Thr-Trp-Tyr-Lys 128 . The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from ␣Ile 107 to ␣Trp 117 . Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the ␣ subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO 2 H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.Nitrile hydratase (NHase; EC 4.2.1.84) 1 is a bacterial metalloenzyme catalyzing the hydration of nitriles to corresponding amides (1, 2). NHase consists of two kinds of subunits (␣ and ␤ with the molecular mass values of 23 kDa) and contains non-heme iron (3) or non-corrinoid cobalt (4) atoms. The NHase from Rhodococcus sp. N-771, which is a ␣␤ heterodimer containing a non-heme iron, is inactivated by aerobic incubation in the dark for a half-day (dark-inactivation), whereas the enzyme purified from the dark-inactivated cells is immediately converted to the active form by light irradiation (photoactivation) (5, 6). Recently, it has been revealed that dark-inactivation and photoactivation are controlled by association and photodissociation of nitric oxide (NO) with the non-heme iron center (7-9). Similar photosensitivity is observed in the NHases from Rhodococcus sp. N-774 (10) and R312.2 These NHases seem to be identical to the one from Rhodococcus sp. N-771 because of the same nucleotide sequences (11, 12). 3The iron-containing NHase is the first enzyme with a mononuclear low-spin non-heme iron(III) which is thought to be involved in the catalysis (3). The structure of the iron center in the active form has been studied by various spectroscopies including ESR (3), resonance Raman (13), extended x-ray absorption fine structure (13) and electron nuclear double resonance (14), and the ligand-donor set of N 3 OS 2 has been proposed (14), which is supported by model complexes of the iron center (15-17). Recently, the metal site structure has been studied in detail by means of electro...

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Section: Structure Of the Iron Center Of The Nhase In The Inactive Fosupporting

confidence: 79%

mentioning

confidence: 99%

Structure of the Photoreactive Iron Center of the Nitrile Hydratase from Rhodococcus sp. N-771

Tsujimura

Dohmae

Odaka

et al. 1997

Journal of Biological Chemistry

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“…The same procedure was used previously to identify EPR-silent ferrous sites in lipoxygenase (17,58) and catecholic dioxygenases (5,76). Only three other enzymes that contain monomeric iron sites, phenylalanine monooxygenase (25), superoxide dismutase (8,70), and nitrile hydratase (31,67), are known to exist. The latter two enzymes and intradiol-type catecholic dioxygenases (37) contain a ferric center, while extradiol dioxygenases (76), lipoxygenases (17), phenylalanine monooxygenase (72), and ADH of Z. mobilis (6,69) contain high-spin ) and the dithionitereduced enzyme (predominantly Fe 2ϩ ) were equally active.…”

Section: Discussionmentioning

confidence: 99%

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

et al. 1995

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The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91؇C by the fermentation of peptides and reduces elemental sulfur (S 0 ) to H 2 S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S 0 in the medium, and no growth was observed in the absence of S 0 . The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S 0 concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S 0 limitation. This was accompanied by increased amounts of H 2 and alcohol (ethanol and butanol) from cultures grown with limiting S 0 . Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M r of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under S 0 limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.Hyperthermophiles are a recently discovered group of microorganisms that have the remarkable property of growing at temperatures of 90ЊC and above (1, 2, 64, 65). They have been isolated from a variety of geothermally heated environments, and almost all are classified as Archaea (formerly Archaeobacteria [75]). The majority are strict anaerobes, and most are obligately dependent upon the reduction of elemental sulfur (S 0 ) to H 2 S for optimal growth. Various organic compounds and molecular H 2 serve as electron donors for the apparent respiration of S 0 . The mechanism of S 0 reduction in one autotrophic hyperthermophile, Pyrodictium brockii, an obligate S 0 -reducing species which grows optimally at 105ЊC, has been investigated. This organism was shown to have a primitive membrane-bound electron transport chain for coupling H 2 oxidation and H 2 S production (52), although the S 0 -reducing entity was not purified.On the other hand, some of the heterotrophic hyperthermophiles are able to grow well in the absence of S 0 . These have fermentative-type metabolisms and produce H 2 during growth, although they also produce H 2 S if S 0 is added to the growth medium. S 0 reduction was thought to be...

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“…The observed pK ES1 value cannot be the metal-bound water molecule because ENDOR data recorded in both 1 H 2 O and 2 H 2 O as well as in 17 O-labeled water on the iron-type NHase from Brevibacterium sp. strain R312 indicate that a water molecule is bound to the metal center at pH 7.5 (45). The observed pK ES2 value may be due to the deprotonation of a conserved activesite arginine residue (␤Arg 52 or ␤Arg 157 ) that forms a hydrogen bond with both the sulfenic and sulfinic acid ligands of the active site (20).…”

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confidence: 99%

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Mitra

Holz

2007

Journal of Biological Chemistry

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To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k cat and K m for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK ES1 and pK ES2 values of 5.9 ؎ 0.1 and 9.2 ؎ 0.1 (k cat ‫؍‬ 130 ؎ 1 s ؊1 ), respectively, and pK E1 and pK E2 values of 5.8 ؎ 0.1 and 9.1 ؎ 0.1 (k cat /K m ‫؍‬ (6.5 ؎ 0.1) ؋ 10 3 s ؊1 mM ؊1 ), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60°C, an Arrhenius plot was constructed by plotting ln(k cat ) versus 1/T, providing E a ‫؍‬ 23.0 ؎ 1.2 kJ/mol. The thermal stability of PtNHase also allowed ⌬H 0 ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK ES1 and pK ES2 values. Based on ⌬H ion 0 data, pK ES1 is assigned to ␤Tyr 68 , whereas pK ES2 is assigned to ␤Arg 52 , ␤Arg 157 , or ␣Ser 112 (NHases are ␣ 2 ␤ 2 -heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron-and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.Nitrile hydratase (NHase 2 ; EC 4.2.1.84), one of the enzymes in the nitrile degradation pathway, catalyzes the hydrolysis of nitriles to their corresponding higher value amides in a chemo-, regio-, and/or enatioselective manner at ambient pressures and temperatures at physiological pH (Scheme 1) (1-6). NHases have attracted substantial interest as biocatalysts for industrial applications such as the large-scale production of acrylamide (3, 7-9) and nicotinamide (10). Acrylamide production utilizing the bacterium Rhodococcus rhodochrous J1 has increased to Ͼ30,000 tons/year (3), whereas Ͼ3500 tons of nicotinamide are produced per year (11). Yields of Ͼ99% are achieved, and the formation of by-products such as acrylic acid, which plagues traditional methodology, is completely avoided. However, one of the most attractive features of nitrile-metabolizing enzymes is their ability to selectively hydrolyze one cyano group of a dinitrile to its corresponding amine, something that is virtually impossible using conventional chemical methods (12-14). Therefore, the potential use of nitrile-hydrolyzing enzymes for the production of several fine chemicals is increasingly recognized.NHases are metalloenzymes that contain either a non-heme Fe(III) ion (iron-type) or a non-corrin Co(III) ion (cobalt-type) in their active site and are typically ␣ 2 ␤ 2 -heterotetramers (5, 6, 15, 16). In all known NHases, each ␣-subunit has a highly homologous amino acid sequence (CXYCSCX) that forms the metal-binding site. Cobalt-type NHases contain threonine and tyrosine in the -C(T/S)YCSC(Y/T)-se...

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Coordination sphere of the ferric ion in nitrile hydratase

Cited by 78 publications

References 0 publications

Structure of the Photoreactive Iron Center of the Nitrile Hydratase from Rhodococcus sp. N-771

Structure of the Photoreactive Iron Center of the Nitrile Hydratase from Rhodococcus sp. N-771

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

Unraveling the Catalytic Mechanism of Nitrile Hydratases

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