Moessbauer and EPR Studies of the Photoactivation of Nitrile Hydratase

• Published in: Biochemistry (Easton) 2001-07, Vol.40 (27), p.7984-7991
• Main Authors: Popescu, V-C, Muenck, E, Fox, B G, Sanakis, Y, Cummings, J G, Turner, I M, Nelson, MJ
• Format: Article
• Language: English
• Subjects: Rhodococcus
• ISSN: 0006-2960
• DOI: 10.1021/bi010198f

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 super(-). 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 NH sub(dark). 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 NH sub(dark) to a low-spin ferric complex, NH sub(light), exhibiting an S = one half EPR signal with g values of 2.27, 2.13, and 1.97. We have characterized both NH sub(dark) and NH sub(light) with Moessbauer spectroscopy. The z-axis of the super(57)Fe magnetic hyperfine tensor, A, of NH sub(light) was found to be rotated by similar to 45 degree relative to the z-axis of the g tensor (g sub(z) = 1.97). Comparison of the A tensor of NH sub(light) 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 Moessbauer spectroscopy. Photolysis at 4.2 K in the Moessbauer spectrometer yielded a five-coordinate low-spin ferric species, NH sub(A), which converted back into NH sub(dark) when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.