Geometric and electronic structure studies of the binuclear nonheme ferrous active site of toluene-4-monooxygenase: parallels with methane monooxygenase and insight into the role of the effector proteins in O2 activation.

Multicomponent monooxygenases, which carry out a variety of highly specific hydroxylation reactions, are of great interest as potential biocatalysts in a number of applications. These proteins share many similarities in structure and show a marked increase in O2 reactivity upon addition of an effector component. In this study, circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD have been used to gain spectroscopic insight into the Fe(II)Fe(II) active site in the hydroxylase component of Toluene-4 monoxygenase (T4moH) and the complex of T4moH bound by its effector protein, T4moD. These results have been correlated to spectroscopic data and density functional theory (DFT) calculations on MmoH and its interaction with MmoB. Together, these data provide further insight into the geometric and electronic structure of these biferrous active sites and, in particular, the perturbation associated with component B/D binding. It is found that binding of the effector protein changes the geometry of one iron center and orientation of its redox active orbital to accommodate the binding of O2 in a bridged structure for efficient 2-electron transfer that can form a peroxo intermediate.