CmlI catalyzes the six-electron oxidation of an aryl-amine precursor (NH2-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 t1/2 = 3 h at 4 °C) and the visible chromophore of the diiron cluster. It is found that diferrous CmlI (CmlIred) can react with NH2-CAM and O2 in either order to form a P-NH2-CAM intermediate. P-NH2-CAM undergoes rapid oxygen transfer to form a diferric CmlI (CmlIox) complex with the aryl-hydroxylamine [NH(OH)-CAM] pathway intermediate. CmlIox-NH(OH)-CAM undergoes a rapid internal redox reaction to form a CmlIred-nitroso-CAM (NO-CAM) complex. O2 binding results in formation of P-NO-CAM that converts to CmlIox-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.
Bibliographical noteFunding Information:
*E-mail: firstname.lastname@example.org. ORCID Anna J. Komor: 0000-0003-4806-4233 Yisong Guo: 0000-0002-4132-3565 Lawrence Que Jr.: 0000-0002-0989-2813 John D. Lipscomb: 0000-0002-8158-5594 Present Address ⊥A.J.K.: Leibniz Institute for Natural Product Research and Infection Biology, Jena 07745, Germany. Funding The authors acknowledge the financial support of this work by National Institutes of Health (NIH) Grant GM118030 (to J.D.L.), NIH Grant GM38767 (to L.Q.), National Science Foundation Grant CHE-1654060 (to Y.G.), and NIH Graduate Traineeship GM08700 (to A.J.K.). Y.G. also acknowledges financial support from Carnegie Mellon University. Notes The authors declare no competing financial interest.
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