Structural basis of regiospecificity of a mononuclear iron enzyme in antibiotic fosfomycin biosynthesis.

Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that uses dioxygen to catalyze the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid (S-HPP) in the biosynthesis of the antibiotic fosfomycin. Additionally, the enzyme converts the R-enantiomer of the substrate (R-HPP) to 2-oxo-propylphosphonic acid. To probe the mechanism of HppE regiospecificity, we determined three X-ray structures: R-HPP with inert cobalt-containing enzyme (Co(II)-HppE) at 2.1 Å resolution; R-HPP with active iron-containing enzyme (Fe(II)-HppE) at 3.0 Å resolution; and S-HPP-Fe(II)-HppE in complex with dioxygen mimic NO at 2.9 Å resolution. These structures, along with previously determined structures of S-HPP-HppE, identify the dioxygen binding site on iron and elegantly illustrate how HppE is able to recognize both substrate enantiomers to catalyze two completely distinct reactions.

Formation and function of the Manganese(IV)/Iron(III) cofactor in Chlamydia trachomatis ribonucleotide reductase.

The beta(2) subunit of a class Ia or Ib ribonucleotide reductase (RNR) is activated when its carboxylate-bridged Fe(2)(II/II) cluster reacts with O(2) to oxidize a nearby tyrosine (Y) residue to a stable radical (Y(*)). During turnover, the Y(*) in beta(2) is thought to reversibly oxidize a cysteine (C) in the alpha(2) subunit to a thiyl radical (C(*)) by a long-distance ( approximately 35 A) proton-coupled electron-transfer (PCET) step. The C(*) in alpha(2) then initiates reduction of the 2' position of the ribonucleoside 5'-diphosphate substrate by abstracting the hydrogen atom from C3'. The class I RNR from Chlamydia trachomatis (Ct) is the prototype of a newly recognized subclass (Ic), which is characterized by the presence of a phenylalanine (F) residue at the site of beta(2) where the essential radical-harboring Y is normally found. We recently demonstrated that Ct RNR employs a heterobinuclear Mn(IV)/Fe(III) cluster for radical initiation. In essence, the Mn(IV) ion of the cluster functionally replaces the Y(*) of the conventional class I RNR. The Ct beta(2) protein also autoactivates by reaction of its reduced (Mn(II)/Fe(II)) metal cluster with O(2). In this reaction, an unprecedented Mn(IV)/Fe(IV) intermediate accumulates almost stoichiometrically and decays by one-electron reduction of the Fe(IV) site. This reduction is mediated by the near-surface residue, Y222, a residue with no functional counterpart in the well-studied conventional class I RNRs. In this review, we recount the discovery of the novel Mn/Fe redox cofactor in Ct RNR and summarize our current understanding of how it assembles and initiates nucleotide reduction.

Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse.

Activation of O2 by the diiron(II/II) cluster in protein R2 of class I ribonucleotide reductase generates the enzyme's essential tyrosyl radical. A crucial step in this reaction is the transfer of an electron from solution to a diiron(II/II)-O2 adduct during formation of the radical-generating, diiron(III/IV) intermediate X. In the reaction of R2 from Escherichia coli, this electron injection is initiated by the rapid (>400 s-1 at 5 degrees C), transient oxidation of the near-surface residue, tryptophan 48, to a cation radical and is blocked by substitution of W48 with F, A, G, Y, L, or Q. By contrast, a study of the cognate reaction in protein R2 from mouse suggested that electron injection might be the slowest step in generation of its tyrosyl radical, Y177* [Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472]. The crucial evidence was the observation that Y177* production is slowed by approximately 30-fold upon substitution of W103, the cognate of the electron-shuttling W48 in E. coli R2, with tyrosine. In this work, we have applied stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies to the mouse R2 reaction to evaluate the possibility that an already sluggish electron-transfer step is slowed by 30-fold by substitution of this key residue. The drastically reduced accumulation of cluster X, failure of precursors to the intermediate to accumulate, and, most importantly, first-order dependence of the rate of Y177* formation on the concentration of O2 prove that addition of O2 to the diiron(II/II) cluster, rather than electron injection, is the slowest step in the R2-W103Y reaction. This finding indicates that the basis for the slowing of Y177* formation by the W103Y substitution is an unexpected secondary effect on the structure or dynamics of the protein, its diiron(II/II) cluster, or both rather than the expected chemical effect on the electron injection step.

A manganese(IV)/iron(III) cofactor in Chlamydia trachomatis ribonucleotide reductase.

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.

(Mu-1,2-peroxo)diiron(III/III) complex as a precursor to the diiron(III/IV) intermediate X in the assembly of the iron-radical cofactor of ribonucleotide reductase from mouse.

Stopped-flow absorption and freeze-quench electron paramagnetic resonance (EPR) and Mössbauer spectroscopies have been used to obtain evidence for the intermediacy of a (mu-1,2-peroxo)diiron(III/III) complex on the pathway to the tyrosyl radical and (mu-oxo)diiron(III/III) cluster during assembly of the essential cofactor in the R2 subunit of ribonucleotide reductase from mouse. The complex accumulates to approximately 0.4 equiv in the first few milliseconds of the reaction and decays concomitantly with accumulation of the previously detected diiron(III/IV) cluster, X, which generates the tyrosyl radical and product (mu-oxo)diiron(III/III) cluster. Kinetic complexities in the reaction suggest the existence of an anti-cooperative interaction of the monomers of the R2 homodimer in Fe(II) binding and perhaps O2 activation. The detection of the (mu-1,2-peroxo)diiron(III/III) complex, which has spectroscopic properties similar to those of complexes previously characterized in the reactions of soluble methane monooxygenase, stearoyl acyl carrier protein Delta9 desaturase, and variants of Escherichia coli R2 with the iron ligand substitution, D84E, provides support for the hypothesis that the reactions of the diiron-carboxylate oxidases and oxygenases commence with the formation of this common intermediate.

Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse.

The kinetics and mechanism of formation of the tyrosyl radical and mu-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent mu-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (k(obs) = 13 +/- 3 s(-1) at 5 degrees C and 0.95 mM O(2)) and the decay of the intermediate to give the Y177 radical (k(obs) = 5 +/- 2 s(-1)) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)(aq) (k(obs) = 0.29 +/- 0.03 s(-1)), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.