Nitrile hydratases contain a novel iron centre with a structure not previously observed in proteins; it resembles a hybrid of the iron centres of heme and Fe-S proteins. The low-spin electronic configuration presumably results in part from two Fe-amide nitrogen bonds. The structure is consistent with the metal ion having a role as a Lewis acid in the catalytic reaction.

The iron K-edge X-ray absorption spectrum of Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) nitrile hydratase in frozen solutions at pH 7 and 9 has been analyzed to determine details of the iron coordination. EXAFS analysis implies two or three sulfur ligands per iron and overall six coordination; together with previous EPR and ENDOR results, this implies an N3S2O ligation sphere. The bond lengths from EXAFS analysis [rav(Fe-S) = 2.21 A at pH 7.3; rav(Fe-N/O) = 1.99 A] support cis coordination of two cysteine ligands and conclusively rule out nitric oxide coordination to the iron, a possibility proposed on the basis of an FTIR difference experiment [Noguchi, T., Honda, J., Nagamune, T., Sasabe, H., Inoue, Y., & Endo, I. (1995) FEBS Lett. 358, 9-12]. The higher-frequency EXAFS can be simulated well by inclusion of multiple scattering from two or three imidazole ligands; the fit to the data is improved if first-sphere multiple scattering pathways are also included. A slight shortening (by 0.02 +/- 0.01 A) of one or both Fe-S bonds when the pH is raised from 7.3 to 9.0 is consistent with shifts observed in the Raman spectrum [Brennan et al. (1996) Biochemistry 35, 10068-10077].

Analysis of the iron K-edge X-ray absorption spectrum (XAS) of the “dark”, inactive form of nitrile hydratase (NHdk) from Rhodococcus sp. R312 confirms a 1:1 stoichiometry of nitric oxide bound to low-spin iron(III). We also report XAS analyses of four iron complexes of the pentadentate ligands 2,3,13,14-tetramethyl-4,8,12-triaza-3,12-pentadecadiene-2,14-dithiolate (L2-, also denoted as S2 Me2N3(Pr,Pr)2-) and 2,12-dimethyl-3,7,11-triaza-2,11-tridecadiene-1,13-dithiolate (L‘2-, also denoted as S2N3(Pr,Pr)2-):  five-coordinate Fe II L‘ and FeL + and low-spin six-coordinate FeL(N 3 ) and FeL(NO) + (cationic species are PF6 - salts). The XAS of FeL(N 3 ) and FeL(NO) + closely mimic the spectra of butyrate-stabilized active nitrile hydratase (NHlt) and NHdk, respectively. The 1s → 3d pre-edge peak is about twice as intense in five-coordinate FeL + than for the remaining samples, suggesting that the iron in both NHlt and NHdk is six-coordinate. This peak and other edge features are 1 eV higher in energy for NHdk and FeL(NO) + , consistent with a {FeNO}6 electron count for both the enzyme and the model. Analysis of the EXAFS (including multiple scattering effects) for NHdk and FeL(NO) + gives the following identical results:  a single NO bound per iron with r FeN = 1.68 ± 0.03 Å and ∠FeNO ≈ 165°. In NHdk, the presence of the NO ligand lengthens at least one of the Fe−S bonds relative to those in NHlt. These data show that synthetic inorganic complexes can be designed to assume iron coordination geometries very similar to those of the iron center in nitrile hydratase and confirm results from EPR spin-trapping experiments (Odaka, M.; Fujii, K.; Hoshino, M.; Noguchi, T.; Tsujimura, M.; Nagashima, S.; Yohda, M.; Nagamune, T.; Inoue, Y.; Endo, I. J. Am. Chem. Soc. 1997, 119, 3785−3791) that photoactivation of nitrile hydratase includes loss of a single NO ligand from the iron.