A Model for the Low-Spin, Non-Heme, Thiolate-Ligated Iron Site of Nitrile Hydratase

Shoner, Steven C.; Barnhart, David M.; Kovacs, Julie A.

doi:10.1021/ic00122a001

Cited by 56 publications

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“…45,56 Like the NHase active site, 7,11 complex 3 is low-spin (S = ½) over a wide temperature range, stabilized in the 3+ oxidation state (E 1/2 = −1.01 V vs SCE), and intense green in color (λ max = 718 (2500) nm). A low spin state is unexpected for non-heme iron, especially when ligated by π-donors such as RS − , which tend to decrease 10Dq.…”

Section: Synthesis and Structure Of Singly Oxygenated [Fe III -(Adit)mentioning

confidence: 99%

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

et al. 2006

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Nitrile hydratase (NHase) is one of a growing number of enzymes shown to contain posttranslationally modified cysteine sulfenic acids (Cys-SOH). Cysteine sulfenic acids have been shown to play diverse roles in cellular processes, including transcriptional regulation, signal transduction, and the regulation of oxygen metabolism and oxidative stress responses. The function of the cysteine sulfenic acid coordinated to the iron active site of NHase is unknown. Herein we report the first example of a sulfenate-ligated iron complex, [Fe III (ADIT)(ADIT-O)] + (5), and compare its electronic and magnetic properties with those of structurally related complexes in which the sulfur oxidation state and protonation state have been systematically altered. Oxygen atom addition was found to decrease the unmodified thiolate Fe-S bond length and blue-shift the ligand-to-metal charge-transfer band (without loss of intensity). S K-edge X-ray absorption spectroscopy and density functional theory calculations show that, although the modified RS-O − fragment is incapable of forming a π bond with the Fe III center, the unmodified thiolate compensates for this loss of π bonding by increasing its covalent bond strength. The redox potential shifts only slightly (75 mV), and the magnetic properties are not affected (the S = ½ spin state is maintained). The coordinated sulfenate S-O bond is activated and fairly polarized (S + -O − ). Addition of strong acids at low temperatures results in the reversible protonation of sulfenateligated 5. An X-ray structure demonstrates that Zn 2+ binds to the sulfenate oxygen to afford [Fe III HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript unmodified NHase thiolate, involving its ability to "tune" the electronics in response to protonation of the sulfenate (RS-O − ) oxygen and/or substrate binding, is discussed.Nitrile hydratases (NHases) are non-heme iron enzymes that convert nitriles to less toxic amides cleanly, and rapidly, under mild conditions. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] It is unusual for a hydrolytic metalloenzyme to incorporate iron, as opposed to zinc, 17,18 presumably because, unlike other metal ions, Zn 2+ is not complicated by redox chemistry. Iron, on the other hand, can promote unwanted side reactions with dioxygen (i.e., Fenton chemistry involving OH · radicals) upon reduction to the Fe 2+ oxidation state. 19,20 The NHase iron site is, however, redox inactive and stabilized in the 3+ oxidation state. The stabilization of Fe 3+ is accomplished by placing the iron in an electron-rich environment consisting of five anionic ligands-two deprotonated peptide amides and three cysteinates (Figure 1). Two of the three coordinated cysteinate sulfurs are oxidized (post-translationally modified) in NHase -one to a sulfenic acid ( 114 Cys-S-(OH)) and the other to a sulfinate 112 CysSO 2 −.3,21 The third cysteinate sulfur, which is trans to the inhibitor/substrate binding site and less accessible to solvent, remains unmodifie...

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Section: Synthesis and Structure Of Singly Oxygenated [Fe III -(Adit)mentioning

confidence: 99%

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

et al. 2006

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“…The structure of monocationic 3 was described previously. 23 Metrical parameters for 1-4 are summarized in Table 2. 29 The iron in structures 1-4 is contained in a pseudo-octahedral bis(tris-chelated) environment, ligated by two trans-imines, two cisthiolates, and either terminal pyridines (py) (1), imidazoles (Im) (2), or amines (NH 2 ) (3 and 4).…”

Section: X-ray Crystallographic Structure Determinationsmentioning

confidence: 99%

Probing the Influence of Local Coordination Environment on the Properties of Fe-Type Nitrile Hydratase Model Complexes

et al. 2001

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A series of four structurally related cis-dithiolate-ligated Fe(III) complexes, [Fe III (DITpy) (3 and 4). The crystallographically determined mean Fe-S bond distances in 1-4 range from 2.196 to 2.232 Å and are characteristic of low-spin Fe(III)-thiolate complexes. The low-spin S = ½ ground state was confirmed by both EPR and magnetic susceptibility measurements. The electronic spectra of these complexes are characterized by broad absorption bands centered near ~700 nm that are consistent with ligand-to-metal chargetransfer (CT) bands. The complexes were further characterized by cyclic voltammetry measurements, and all possess highly negative Fe(III)/Fe(II) redox couples (~ −1 V vs SCE, saturated calomel electrode) indicating that alkyl thiolate donors are effective at stabilizing Fe(III) centers. Both the redox couple and the 700 nm band in the visible spectra show solvent-dependent shifts that are dependent upon the H-bonding ability of the solvent. The implications of these results with respect to the active site of the iron-containing nitrile hydratases are also discussed.

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“…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.…”

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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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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A Model for the Low-Spin, Non-Heme, Thiolate-Ligated Iron Site of Nitrile Hydratase

Cited by 56 publications

References 0 publications

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

Probing the Influence of Local Coordination Environment on the Properties of Fe-Type Nitrile Hydratase Model Complexes

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

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