title: 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].
authors: Department of Chemistry, Haverford College, Pennsylvania 19041-1392, USA.
authors: R C
authors: RC
authors: Scarrow
authors: B A
authors: BA
authors: Brennan
authors: J G
authors: JG
authors: Cummings
authors: H
authors: H
authors: Jin
authors: D J
authors: DJ
authors: Duong
authors: J T
authors: JT
authors: Kindt
authors: M J
authors: MJ
authors: Nelson
mesh_terms: D015502:Absorptiometry, Photon / Q000379:methods; D001951:Brevibacterium / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D006836:Hydro-Lyases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D011487:Protein Conformation; D012240:Rhodococcus / Q000201:enzymology*; D013455:Sulfur
chemical_list: D007506:Iron-Sulfur Proteins; D008024:Ligands; D013455:Sulfur; D006836:Hydro-Lyases; C048831:nitrile hydratase