Multi-frequency EPR and Mössbauer spectroscopic studies on freeze-quenched reaction intermediates of nitric oxide synthase

✍ Scribed by C. Jung; F. Lendzian; V. Schünemann; M. Richter; L. H. Böttger; A. X. Trautwein; J. Contzen; M. Galander; D. K. Ghosh; A.-L. Barra

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 352 KB
• Volume: 43
• Category: Article
• ISSN: 0749-1581
• DOI: 10.1002/mrc.1694

✦ Synopsis

It is believed by analogy to chloroperoxidase (CPO) from

Caldariomyces fumago that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of nitric oxide synthase (NOS) corresponds to an iron(IV) porphyrin-p -cation radical. Such species can also be produced by the reaction of ferric NOS with external oxidants within the shunt pathway. We present multi-frequency EPR (9.6, 94, 285 GHz) and M össbauer spectroscopic studies on freeze-quenched intermediates of the oxygenase domain of nitric oxide synthase which has reacted with peroxy acetic acid within 8-200 ms. The intermediates of the oxygenase domain of both the cytokine inducible NOS (iNOSox) and the neuronal NOS (nNOSox) show an organic radical signal in the 9.6-GHz spectrum overlapping with the spectrum of an unknown species with g-values of 2.24, 2.23 and 1.96. Using 94-and 285-GHz EPR the organic radical signal is assigned to a tyrosine radical on the basis of g-values (i.e. Tyr*562 in nNOSox and Tyr*341 in iNOSox). M össbauer spectroscopy of 57 Fe-labeled unreacted nNOSox shows a ferric low-spin heme-iron (d = 0.38 mms -1 , 1E Q = 2.58 mms -1 ). The reaction of nNOSox with peroxy acetic acid for 8 ms leads to the disappearance of the magnetic background characteristic for native nNOSox and a new species with d = 0.27 mms -1 and 1E Q = 2.41 mms -1 is detected at 4.2 K which does not resemble the parameters typical for a Fe(IV) center. It is proposed that this intermediate species corresponds to a ferric low-spin species which magnetically couples to an amino acid radical (presumably Trp*409).