title: Multi-frequency EPR and Mössbauer spectroscopic studies on freeze-quenched reaction intermediates of nitric oxide synthase.
abstract: 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-pi -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 Mössbauer 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). Mössbauer spectroscopy of (57)Fe-labeled unreacted nNOSox shows a ferric low-spin heme-iron (delta = 0.38 mms(-1), deltaE(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 delta = 0.27 mms(-1) and deltaE(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).
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Jung
authors.forename: F
authors.initials: F
authors.lastname: Lendzian
authors.forename: V
authors.initials: V
authors.lastname: Schünemann
authors.forename: M
authors.initials: M
authors.lastname: Richter
authors.forename: L H
authors.initials: LH
authors.lastname: Böttger
authors.forename: A X
authors.initials: AX
authors.lastname: Trautwein
authors.forename: J
authors.initials: J
authors.lastname: Contzen
authors.forename: M
authors.initials: M
authors.lastname: Galander
authors.forename: D K
authors.initials: DK
authors.lastname: Ghosh
authors.forename: A-L
authors.initials: AL
authors.lastname: Barra
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005615:Freezing; D015394:Molecular Structure; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer
chemical_list: D019001:Nitric Oxide Synthase