X-ray Spectroscopy of Nitrile Hydratase at pH 7 and 9

Abstract

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].

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

“…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.…”

“…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. It was demonstrated that the coordination sphere of the metal site consisted of two cis-coordinated sulfhydryl ligands, three His imidazole ligands, and an exchangeable solvent ligand, probably an hydroxo group.…”

EXAFS studies of the novel iron(III) complexes with an N/S(Se) chromophore simulating ligand environment of the active site of nitrile hydratase

“…Iron-sulfur bond lengths, Fe-S~=2.227(2) A and Fe-$2 = 2.230(2) ~,, are similar to that reported for nitrile hidratase (Fe-S = 2.21 A from the EXAFS data (Scarrow et al, 1996). Ironnitrogen bond lengths in 1, Fe-Nav=l.99,~, are also identical to those in the enzyme.…”

How does cyanide inhibit superoxide reductase? Insight from synthetic Fe III N 4 S model complexes

“…The bond valence sum calculated on the basis of fits shown in Table 2 is in the range (3.7-4.5) found for low-spin ferric complexes (32) (2), which is blue in MeCN and displays a single LMCT band centered at 585(1,975) nm (Table 3). For comparison, azide-bound SOR is blue and has a LMCT band centered at 660(2,300) nm (16).…”