Mössbauer 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 isolated from bacteria grown in the dark is inactive and contains the iron site as a six-coordinate diamagnetic Fe-nitrosyl complex, called NHdark The active state can be obtained from the dark state by photolysis of the Fe-NO bond at room temperature. Activation is accompanied by the conversion of NHdark to a low-spin ferric complex, NHlight exhibiting an S = 1/2 EPR signal with 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 hyperfine tenser, A, of NHlight was found to be rotated by similar to 45 degrees 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 also 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 species, NHA which converted back into NHdark when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.