CmlI <i>N</i>‑Oxygenase Catalyzes the Final Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates

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 <b>P</b> 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 <b>P</b> in the absence of substrates (decay <i>t</i>_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 NH₂-CAM and O₂ in either order to form a <b>P</b>-NH₂-CAM intermediate. <b>P</b>-NH₂-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. O₂ binding results in formation of <b>P</b>-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

Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI

The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH₂-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O₂, CmlI forms a long-lived peroxo intermediate, <b>P</b>, which reacts with NH₂-CAM to form CAM. Since <b>P</b> is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether <b>P</b> is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under ¹⁸O₂ show that both oxygen atoms in the nitro function of CAM derive from O₂. However, when the single-turnover reaction between ¹⁸O₂-<b>P</b> and NH₂-CAM is carried out in an ¹⁶O₂ atmosphere, CAM nitro groups contain both ¹⁸O and ¹⁶O, suggesting that <b>P</b> can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH­(OH)-CAM. Accordingly, the aerobic reaction of NH­(OH)-CAM with diferric CmlI yields <b>P</b> and then CAM without an external reductant. A catalytic cycle is proposed in which NH₂-CAM reacts with <b>P</b> to form NH­(OH)-CAM and diferric CmlI. Then the NH­(OH)-CAM rereduces the enzyme diiron cluster, allowing <b>P</b> to reform upon O₂ binding, while itself being oxidized to NO-CAM. Finally, the reformed <b>P</b> oxidizes NO-CAM to CAM with incorporation of a second O₂-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site

An Unusual Peroxo Intermediate of the Arylamine Oxygenase of the Chloramphenicol Biosynthetic Pathway

Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O₂ to the diferrous state of the cluster results in an exceptionally long-lived intermediate (<i>t</i>_1/2 = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an ¹⁸O₂-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed <i>cis</i>-μ-1,2-peroxo (μ-η¹:η¹) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a ν­(O–O) that is at least 60 cm^–1 lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ-η¹:η¹-peroxo ligand; we propose that a μ-η¹:η²-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step

NRVS Studies of the Peroxide Shunt Intermediate in a Rieske Dioxygenase and Its Relation to the Native Fe^II O₂ Reaction

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin Fe^III–(hydro)­peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native Fe^II/O₂ reaction is evaluated. For the native Fe^II/O₂ reaction, an Fe^III–superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of Fe^III–superoxo and Fe^III–hydroperoxo species in nonheme Fe biochemistry