| Abstract
| - The binding of ethanol and 1,1,1-trifluoroethanol (TFE) to both the Hmv and Hox forms of solublemethane monooxygenase (sMMO) in solution has been studied by Q-band (35 GHz) CW and pulsed ENDORspectroscopy of 1H, 2H and 19F nuclei of exogenous ligands. As part of this investigation we introduce 19F,in this case from bound TFE, as a new probe for the binding of small molecules to a metalloenzyme activesite. The Hmv form was prepared in solution by chemical reduction of Hox. For study of Hox itself, frozensolutions were subjected to γ-irradiation in the frozen solution state at 77 K, which affords an EPR-visiblemixed-valent diiron center, denoted (Hox)mv, held in the geometry of the diiron(III) state. The 19F and 2HENDOR spectra of bound TFE together with 1,2H ENDOR spectra of bound ethanol indicate that the alcoholsbind close to the Fe(II) ion of the mixed-valence cluster in Hmv and in a bridging or semi-bridging fashionto Hox. DMSO does not affect the binding of either of the ethanols or of methanol to Hox, nor of ethanol ormethanol to Hmv. It does, however, displace TFE from the diiron site in Hmv. These results provide the firstevidence that crystal structures of sMMO hydroxylase into which product alcohols were introduced bydiffusion represent the structures in solution.
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