The binuclear non-heme iron (NHFe) enzyme CmlI performs the six electron oxidation of an aryl-amine to an aryl-nitro group to produce the antibiotic chloramphenicol at ambient conditions. CmlI’s chemistry occurs via a unique oxygen activation pathway, in which the biferrous active site reacts with molecular oxygen to form the novel biferric peroxy level reactive intermediate P’. In contrast, soluble methane monooxygenase, another binuclear NHFe enzyme that activates O₂, passes through a peroxy intermediate to form a high valent Fe(IV)₂O₂ intermediate Q to perform its chemistry. Understanding the utilization of high valent oxo intermediates versus biferric peroxy level intermediates provides fundamental insight into the mechanism of oxygen activation by binuclear NHFe enzymes.
Our group has been characterizing the novel P’ intermediate utilizing ⁵⁷Fe nuclear resonance vibration spectroscopy (NRVS) paired with density functional theory (DFT) computation on the isotope labeled (¹⁶O₂ & ¹⁸O₂) P’ intermediate and Variable Temperature Variable Field (VTVH) Magnetic Circular Dichroism (MCD) on the one-electron cryoreduced P’ intermediate to define the active site structure. Further, we have performed normal coordinate analysis (NCA), density functional theory (DFT) computation and reaction coordinate calculations to finalize assignment of the active site structure of the P’ intermediate, and understand the geometric and electronic contributions of this active site to CmlI’s reactivity. We have further characterized the structure of the biferrous active site utilizing MCD, and investigated its reaction with O₂ to produce the P’ intermediate utilizing DFT. The spectroscopic and computational definition of both the biferrous and P’ intermediate provides insight into the mechanism of oxygen activation and reactivity of this class of binuclear NHFe enzymes.

Esen Alp

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