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 H_mv and H_ox 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 H_mv form was prepared in solution by chemical reduction of H_ox. For study of H_ox 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 (H_ox)_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 H_mv and in a bridging or semi-bridging fashion to H_ox. DMSO does not affect the binding of either of the ethanols or of methanol to H_ox, nor of ethanol or methanol to H_mv. It does, however, displace TFE from the diiron site in H_mv. These results provide the first evidence that crystal structures of sMMO hydroxylase into which product alcohols were introduced by diffusion represent the structures in solution.