Product binding to the diiron(III) and mixed-valence diiron centers of methane monooxygenase hydroxylase studied by 1,2H and 19F ENDOR spectroscopy

The binding of ethanol and 1,1,1-trifluoroethanol (TFE) to both the Hmv and Hox forms of soluble methane monooxygenase (sMMO) in solution has been studied by Q-band (35 GHz) CW and pulsed ENDOR spectroscopy 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 active site. The Hmv form was prepared in solution by chemical reduction of Hox. For study of Hox itself, frozen solutions were subjected to γ-irradiation in the frozen solution state at 77 K, which affords an EPR-visible mixed-valent diiron center, denoted (Hox)mv, held in the geometry of the diiron(III) state. The 19F and 2H ENDOR spectra of bound TFE together with 1,2H ENDOR spectra of bound ethanol indicate that the alcohols bind close to the Fe(II) ion of the mixed-valence cluster in Hmv and in a bridging or semi-bridging fashion to Hox. DMSO does not affect the binding of either of the ethanols or of methan...