In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y*). The Y* reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C*), which abstracts the 3'-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y*, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C* in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.

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Recent studies on taurine:alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli have provided evidence for a three-step, minimal kinetic mechanism involving the quaternary TauD.Fe(II).alpha-ketoglutarate.taurine complex, the taurine-hydroxylating Fe(IV)-oxo intermediate (J) that forms upon reaction of the quaternary complex with O(2), and a poorly defined, Fe(II)-containing intermediate state that converts in the rate-limiting step back to the quaternary complex [Price, J. C., Barr, E. W., Tirupati, B., Bollinger, J. M., Jr., and Krebs, C. (2003) Biochemistry 42, 7497-7508]. The mapping of this kinetic mechanism onto the consensus chemical mechanism for the Fe(II)- and alpha-ketoglutarate-dependent engendered several predictions and additional questions that have been experimentally addressed in the present study. The results demonstrate (1) that postulated intermediates between the quaternary complex and J accumulate very little or not at all; (2) that decarboxylation of alpha-ketoglutarate occurs prior to or concomitantly with formation of J; (3) that the second intermediate state comprises one or more product complex with Mossbauer features that are partially resolved from those of the binary TauD.Fe(II), ternary TauD.Fe(II).alpha-ketoglutarate, and quaternary TauD.Fe(II).alpha-ketoglutarate.taurine complexes; and (4) that the rate-determining step in the catalytic cycle is release of product(s) prior to the rapid, ordered binding of alpha-ketoglutarate and then taurine to regenerate the O(2)-reactive quaternary complex. The results thus integrate the previously proposed kinetic and chemical mechanisms and indicate which of the postulated intermediates in the latter will be detectable only upon perturbation of the kinetics by changes in reaction conditions (e.g., temperature), protein mutagenesis, the use of substrate analogues, or some combination of these.

. Recent studies have suggested that the enzyme, which was shown nearly 50 years ago to require iron (1, 2), contains a coupled dinuclear nonheme iron cluster (5), making MIOX the most recent addition to the nonheme diiron oxygenase͞oxidase family that also includes bacterial hydrocarbon hydroxylases (e.g., soluble methane monooxygenase), plant fatty acyl desaturases (e.g., stearoyl acyl carrier protein ⌬ 9 desaturase), and protein R2 of class I ribonucleotide reductase (R2) (6-10). Mössbauer and EPR spectra showed that treatment of recombinant Mus musculus MIOX isolated in its iron-free form from Escherichia coli with Fe(II) and O 2 leads to formation of an antiferromagnetically coupled diiron cluster in either the II͞III or III͞III oxidation state, depending on the O 2 ͞MIOX ratio and the presence or absence of a reductant (ascorbate or cysteine). Binding of MI was shown to perturb the spectra of both oxidation states in a manner consistent with direct coordination of the substrate to the cluster (5).All nonheme diiron oxygenases and oxidases characterized before MIOX activate O 2 with the II͞II oxidation state of the cofactor (11,12). For several of the reactions, (peroxo)diiron(III͞III) intermediates have been demonstrated. These complexes are generally proposed to undergo O-O-bond cleavage to generate high-valent iron complexes that cleave strong C-H or O-H bonds of their oxidation targets (8,(11)(12)(13)(14). Indeed, the diiron(III͞IV) cluster, X (15, 16), and the diiron(IV͞IV) cluster, Q (8, 13, 17), have been directly characterized in the R2 and soluble methane monooxygenase reactions, respectively. In each of the previously characterized diiron-oxygenase͞oxidase reactions, a diiron(III͞III) ''product'' state of the cluster is generated at the end of the oxidation sequence. Subsequent events require reduction of the cluster back to the diiron(II͞II) state by additional proteins, with electrons provided ultimately by NAD(P)H. This redox cycling of the cofactor and provision of two electrons by the nicotinamide ''cosubstrate'' ensure that at most two electrons can be extracted from the substrate. The MIOX reaction, a four-electron oxidation, would seem to require a different mechanism.Indeed, a recent study concluded that the mixed-valent, II͞III state of the cofactor, rather than the conventional II͞II state, activates O 2 for DG production in the MIOX reaction (4). Single-turnover experiments showed that the fully reduced enzyme (MIOX II/II ) reacts with limiting O 2 in the presence of saturating MI to generate the mixed-valent enzyme as a stable product with unit stoichiometry and with only a low yield of DG. By contrast, the

The iron(II)-and α-ketoglutarate-dependent dioxygenases comprise enzymes that catalyze a variety of important reactions in biology, including steps in the biosynthesis of collagen and antibiotics, the degradation of xenobiotics, the repair of alkylated DNA, and the sensing of oxygen and response to hypoxia. In these reactions, the reductive activation of oxygen is coupled to hydroxylation of the substrate and decarboxylation of the co-substrate, α-ketoglutarate. It is believed that a single, conserved mechanistic pathway for formation of a high-valent iron intermediate that attacks the substrate is operant in all members of this family. Application 4245 of a combination of rapid kinetic and spectroscopic techniques to the reaction of taurine/α-ketoglutarate dioxygenase (TauD), one member of this large enzyme family, has led to the detection of two reaction intermediates. The first intermediate, which is termed J, is a high-spin Fe IV -oxo complex. Decay of J exhibits a large, normal C1 deuterium kinetic isotope effect, demonstrating that it is the species activating the C-H bond for hydroxylation. The second intermediate is an Fe II -containing product(s) complex.