X-RAY SPECTROSCOPY OF NITRIC-OXIDE BINDING TO IRON IN INACTIVE NITRILE HYDRATASE AND A SYNTHETIC MODEL-COMPOUND

Analysis of the iron K-edge X-ray absorption spectrum (XAS) of the ''d ark'', inactive form of nitrile hydratase (NHdk) from Rhodococcus sp. R312 confirms a 1:1 stoichiometry of nitric oxide bound to low-spin ir on(III). We also report XAS analyses of four iron complexes of the pen tadentate ligands 4-tetramethyl-4,8,12-triaza-3,12-pentadecadiene-2, 1 4-dithiolate (L2-, also denoted as (S2N3)-N-Me2(Pr,Pr)(2-)) and yl-3,7 ,11-triaza-2,11-tridecadiene-1,13-dithiolate (L'(2-), also denoted as S2N3(Pr,Pr)(2-)): five-coordinate (FeL)-L-II' and FeL+ and low-spin si x-coordinate FeL(N-3) and FeL(NO)(+) (cationic species an PF6- salts). The XAS of FeL(N-3) and FeL(NO)(+) closely mimic the spectra of butyr ate-stabilized active nitrile hydratase (NHlt) and NHdk, respectively. The 1s --> 3d pre-edge peak is about twice as intense in five-coordin ate FeL+ than for the remaining samples, suggesting that the iron in b oth 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 o f the EXAFS (including multiple scattering effects) for NHdk and FeL(N O)+ gives the following identical results: a single NO bound per iron with r(FeN) = 1.68 +/- 0.03 Angstrom and angle FeNO approximate to 165 degrees. In NHdk, the presence of the NO Ligand lengthens at least on e of the Fe-S bonds relative to those in NHlt. These data show that sy nthetic inorganic complexes can be designed to assume iron coordinatio n geometries very similar to those of the iron center in nitrile hydra tase and confirm results from EPR spin-trapping experiments (Odaka, M. ; Fujii, K.; Hoshino, M.; Noguchi, T.; Tsujimura, M.; Nagashima, S.; Y ohda, M.; Nagamune, T.; Inoue, Y.; Endo, I. J. Am. Chem. Sec. 1997, 11 9, 3785-3791) that photoactivation of nitrile hydratase includes loss of a single NO ligand from the iron.