NRVS Studies of the Peroxide Shunt Intermediate in a Rieske Dioxygenase and Its Relation to the Native FeII 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 FeIII-(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 FeII/O2 reaction is evaluated. For the native FeII/O2 reaction, an FeIII-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 FeIII-superoxo and FeIII-hydroperoxo species in nonheme Fe biochemistry.

CmlI N-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 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.

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, P, which reacts with NH2-CAM to form CAM. Since P is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether P is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under (18)O2 show that both oxygen atoms in the nitro function of CAM derive from O2. However, when the single-turnover reaction between (18)O2-P and NH2-CAM is carried out in an (16)O2 atmosphere, CAM nitro groups contain both (18)O and (16)O, suggesting that P 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 P and then CAM without an external reductant. A catalytic cycle is proposed in which NH2-CAM reacts with P to form NH(OH)-CAM and diferric CmlI. Then the NH(OH)-CAM rereduces the enzyme diiron cluster, allowing P to reform upon O2 binding, while itself being oxidized to NO-CAM. Finally, the reformed P 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.

Rate-Determining Attack on Substrate Precedes Rieske Cluster Oxidation during Cis-Dihydroxylation by Benzoate Dioxygenase.

Rieske dearomatizing dioxygenases utilize a Rieske iron-sulfur cluster and a mononuclear Fe(II) located 15 Šacross a subunit boundary to catalyze O2-dependent formation of cis-dihydrodiol products from aromatic substrates. During catalysis, O2 binds to the Fe(II) while the substrate binds nearby. Single-turnover reactions have shown that one electron from each metal center is required for catalysis. This finding suggested that the reactive intermediate is Fe(III)-(H)peroxo or HO-Fe(V)═O formed by O-O bond scission. Surprisingly, several kinetic phases were observed during the single-turnover Rieske cluster oxidation. Here, the Rieske cluster oxidation and product formation steps of a single turnover of benzoate 1,2-dioxygenase are investigated using benzoate and three fluorinated analogues. It is shown that the rate constant for product formation correlates with the reciprocal relaxation time of only the fastest kinetic phase (RRT-1) for each substrate, suggesting that the slower phases are not mechanistically relevant. RRT-1 is strongly dependent on substrate type, suggesting a role for substrate in electron transfer from the Rieske cluster to the mononuclear iron site. This insight, together with the substrate and O2 concentration dependencies of RRT-1, indicates that a reactive species is formed after substrate and O2 binding but before electron transfer from the Rieske cluster. Computational studies show that RRT-1 is correlated with the electron density at the substrate carbon closest to the Fe(II), consistent with initial electrophilic attack by an Fe(III)-superoxo intermediate. The resulting Fe(III)-peroxo-aryl radical species would then readily accept an electron from the Rieske cluster to complete the cis-dihydroxylation reaction.

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 O(2) to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t(1/2) = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an (18)O(2)-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-µ-1,2-peroxo (µ-η(1):η(1)) 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-η(1):η(1)-peroxo ligand; we propose that a µ-η(1):η(2)-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.