Mossbauer and EPR studies of the photoactivation of nitrile hydratase

The alpha beta dimer of active nitrile hydratase from Rhodococcus sp. R312 contains one low-spin ferric ion that is coordinated by three Cys residues, two N-amide groups from the protein backbone, and one OH-. The enzyme isol ated from bacteria grown in the dark is inactive and contains the iron site as a six-coordinate diamagnetic Fe-nitrosyl complex, called NHdark The act ive state can be obtained from the dark state by photolysis of the Fe-NO bo nd at room temperature. Activation is accompanied by the conversion of NHda rk to a low-spin ferric complex, NHlight exhibiting an S = 1/2 EPR signal w ith g values of 2.27, 2.13, and 1.97. We have characterized both NHdark and NHlight With Mossbauer spectroscopy. The z-axis of the Fe-57 magnetic hype rfine tenser, A, of NHlight was found to be rotated by similar to 45 degree s relative to the z-axis of the g tenser (g(z) = 1.97). Comparison of the A tenser of NHlight With the A tensors of low-spin ferric hemes indicates a substantially larger degree of covalency for nitrile hydratase. We have als o performed photolysis experiments between 2 and 20 K and characterized the photolyzed products by EPR and Mossbauer spectroscopy. Photolysis at 4.2 K in the Mossbauer spectrometer yielded a five-coordinate low-spin ferric sp ecies, NHA which converted back into NHdark when the sample was briefly war med to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.