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

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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 Å at pH 7.3; rav(Fe−N/O) = 1.99 Å] 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) FEBSLett.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 Å) 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) Biochemistry35, 10068−10077].

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