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CmlI catalyzes the six-electron oxidation of an aryl-amine precursor (NH 2 -CAM) to the aryl-nitro group of chloramphenicol (CAM). The active site of CmlI contains a (hydr)oxo- and carboxylate-bridged dinuclear iron cluster. During catalysis, a novel diferric-peroxo intermediate P is formed and is thought to directly effect oxygenase chemistry. Peroxo intermediates can facilitate at most two-electron oxidations, so the biosynthetic pathway of CmlI must involve at least three steps. Here, kinetic techniques are used to characterize the rate and/or dissociation constants for each step by taking advantage of the remarkable stability of P in the absence of substrates (decay t 1/2 = 3 h at 4 °C) and the visible chromophore of the diiron cluster. It is found that diferrous CmlI (CmlI red ) can react with NH 2 -CAM and O 2 in either order to form a P-NH 2 -CAM intermediate. P-NH 2 -CAM undergoes rapid oxygen transfer to form a diferric CmlI (CmlI ox ) complex with the aryl-hydroxylamine [NH(OH)-CAM] pathway intermediate. CmlI ox -NH(OH)-CAM undergoes a rapid internal redox reaction to form a CmlI red -nitroso-CAM (NO-CAM) complex. O 2 binding results in formation of P-NO-CAM that converts to CmlI ox -CAM by enzyme-mediated oxygen atom transfer. The kinetic analysis indicates that there is little dissociation of pathway intermediates as the reaction progresses. Reactions initiated by adding pathway intermediates from solution occur much more slowly than those in which the intermediate is generated in the active site as part of the catalytic process. Thus, CmlI is able to preserve efficiency and specificity while avoiding adventitious chemistry by performing the entire six-electron oxidation in one active site.

CmlI N-Oxygenase Catalyzes the Final Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates