An active-site model for nitrile hydratase: axially coordinate non-heme iron complexes in the low-spin ferric state

Abstract

Recently, nitrile hydratase isolated from Brevibacterium R3121 and Pseudomonas chlororaphis B232 was found to be a new iron-containing enzyme that catalyzes the hydration of aliphatic nitriles to the corresponding amides: RCN + H20 -* RCONH2. The active site of this enzyme was proposed to have a typical low-spin ferric coordination structure,3 similar to those of hemoproteins such as cytochrome P-450. The axial positions of the non-heme iron site in the resting state of the enzyme are concluded to be occupied by thiolato and aquo ligands.3During investigations on bis(thilato) metalloporphyrin complexes,4 we found that a non-heme iron(III) complex of a square-planar type binds thiolato ligands in the axial positions and forms bis(thiolato)-non-heme iron complexes that are similar to bis(thiolato) metalloporphyrin complexes. We report here the detection and characterization of the bis(thiolato)-non-heme iron(III) complexes by ESR (electron spin resonance) spectrometry and propose the active-site structure of native nitrile hydratase in the monomer form, postulating an axial thiolato-iron(III)imidazole coordination mode on the basis of analyses of the ESR parameters.Experimental Section. (7V,7V'-Disalicylideneethylenediaminato)iron(III) ¿-camphor-10-sulfonate (Fe(salen)(¿-camphor-10-sulfonate), FeC26H29N206S) was prepared and purified by a reported procedure.5 Ethyl thioglycolate (TGE), imidazole (Im), and tetrabutylammonium hydroxide (Bu4NOH (TBAH), 10% solution in methanol) were products of Wako Pure Chemicals. Acetone and methanol (MeOH) were distilled before use. All samples for analyses of ESR and electronic absorption spectra were prepared in air at 20 °C. ESR measurements were carried out in a JES-FE1XG spectrometer with 100-kHz field modulation at 77 K. Li-TCNQ (g = 2.002 52) and Mn(II) doped in MgO were used as standards. Electronic absorption spectra were re-

Synthetic Analogues of Cysteinate‐Ligated Non‐Heme Iron and Non‐Corrinoid Cobalt Enzymes

“…54,[63][64][65]69,[124][125][126][150][151][152][153][154][155][156][157][158][159][160][161][162][163][164][165] The first attempt at modeling NHase was reported in 1988 by Sakurai. 166 Figure 7, L = 1,4,7tris(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclononane), 156 which is intensely colored and low-spin (S = ½) at low temperatures (Table 2). A thermally accessible S = 5 / 2 ; state results in an S = ½ ↔ S = 5 / 2 ; spin-equilibrium and slightly longer Fe-S bond lengths (2.28 Å) in 1 relative to those reported (2.21 Å) for NHase ( Table 3).…”

Crystal structure of nitrile hydratase reveals a novel iron centre in a novel fold

“…Various spectroscopic methods have been applied to deduce the coordination of the NHase iron centre. Electron paramagnetic resonance (EPR) measurements [6,7] showed the iron to be the only example of a mononuclear non-heme low-spin ferric ion in a protein. On the basis of EPR [6,7], ENDOR [8,9], EXAFS [10,11] and resonance Raman [10,12] spectroscopy measurements the iron ion in the activated enzyme was suggested to have a pseudooctahedral N 3 S 2 O ligand field, the first example of a mixed NSO ligand field for a non-heme iron site.…”

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

“…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.…”