Mössbauer and EPR studies of the photoactivation of nitrile hydratase.
The alphabeta 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 NH(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(dark) to a low-spin ferric complex, NH(light), exhibiting an S = (1)/(2) EPR signal with g values of 2.27, 2.13, and 1.97. We have characterized both NH(dark) and NH(light) with Mössbauer spectroscopy. The z-axis of the 57Fe magnetic hyperfine tensor, A, of NH(light) was found to be rotated by approximately 45 degrees relative to the z-axis of the g tensor (g(z) = 1.97). Comparison of the A tensor of NH(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 Mössbauer spectroscopy. Photolysis at 4.2 K in the Mössbauer spectrometer yielded a five-coordinate low-spin ferric species, NH(A), which converted back into NH(dark) when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.
Popescu
V C
VC
Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Münck
E
E
Fox
B G
BG
Sanakis
Y
Y
Cummings
J G
JG
Turner
I M
IM
Nelson
M J
MJ
D003624:Darkness; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004789:Enzyme Activation / Q000528:radiation effects; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D005615:Freezing; D006836:Hydro-Lyases / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D008027:Light; D010782:Photolysis; D012240:Rhodococcus / Q000201:enzymology; D015204:Spectroscopy, Mossbauer / Q000379:methods
D005290:Ferric Compounds; D006836:Hydro-Lyases; C048831:nitrile hydratase