Radiolytic reduction of methane monooxygenase dinuclear iron cluster at 77 K. EPR evidence for conformational change upon reduction or binding of component B to the diferric state

The soluble form of methane monooxygenase (MMO) consists of three components: reductase, hydroxylase (MMOH), and 'B' (MMOB). Resting MMOH contains a diferric bis-μ-hydroxodinuclear iron 'diamond core' cluster which is the site of oxygen activation chemistry after reduction. Here it is shown that γ-irradiation of MMOH at 77 K results in reduction of the diiron cluster to an EPR active Fe(II)·Fe(III) mixed valence state. At this temperature, the conformation of the enzyme remains essentially unchanged during reduction, so the EPR-spectrum becomes a probe of the conformation of the diferric state. The γ-irradiated MMOH exhibits EPR spectra that differ in lineshape and saturation properties from those of the mixed valence MMOH generated by chemical reduction in solution; annealing the γ-irradiated sample at 230 K yields the spectra of the chemically reduced sample. This demonstrates that the conformation of diferric MMOH in the vicinity of the diiron cluster changes during reduction to the mixed valence state. The analogous experiment for the MMOB·MMOH complex gives a new mixed valence species following γ-irradiation that differs from all previously reported mixed valence species. Thus, binding of MMOB also causes a change in the conformation of diferric MMOH. It is hypothesized that the structural changes observed for the first time here may involve conversion of the dihydroxo- bridged diamond core structure to one with more readily dissociable bridging oxygen ligands to facilitate reaction with O₂ following cluster reduction.