X-ray spectroscopy of nitrile hydratase at pH 7 and 9.
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].
Scarrow
R C
RC
Department of Chemistry, Haverford College, Pennsylvania 19041-1392, USA.
Brennan
B A
BA
Cummings
J G
JG
Jin
H
H
Duong
D J
DJ
Kindt
J T
JT
Nelson
M J
MJ
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
D007506:Iron-Sulfur Proteins; D008024:Ligands; D013455:Sulfur; D006836:Hydro-Lyases; C048831:nitrile hydratase