General knowledge of dioxygen-activating mononuclear non-heme iron(II) enzymes containing a 2-His-1-carboxylate facial triad has significantly expanded in the last few years, due in large part to the extensive library of crystal structures that is now available. The common structural motif utilized by this enzyme superfamily acts as a platform upon which a wide assortment of substrate transformations are catalyzed. The facial triad binds a divalent metal ion at the active site, which leaves the opposite face of the octahedron available to coordinate a variety of exogenous ligands. The binding of substrate activates the metal center for attack by dioxygen, which is subsequently converted to a high-valent iron intermediate, a formidable oxidizing species. Herein, we summarize crystallographic and mechanistic features of this metalloenzyme superfamily, which has enabled the proposal of a common but flexible pathway for dioxygen activation.

Superoxide reductases (SORs) contain a novel square pyramidal ferrous [Fe(NHis)(4)(SCys)] site that rapidly reduces superoxide to hydrogen peroxide. Here we report extensive pulse radiolysis studies on recombinant two-iron SOR (2Fe-SOR) from Desulfovibrio vulgaris. The results support and elaborate on our originally proposed scheme for reaction of the [Fe(NHis)(4)(SCys)] site with superoxide [Coulter, E. D., Emerson, J. E., Kurtz, D. M., Jr., and Cabelli, D. E. (2000) J. Am. Chem. Soc. 122, 11555-11556]. This scheme consists of second-order diffusion-controlled formation of an intermediate absorbing at approximately 600 nm, formulated as a ferric-(hydro)peroxo species, and its decay to the carboxylate-ligated ferric [Fe(NHis)(4)(SCys)] site with loss of hydrogen peroxide. The second-order rate constant for formation of the 600-nm intermediate is essentially pH-independent (pH 5-9.5), shows no D(2)O solvent isotope effect at pH 7.7, and decreases with increasing ionic strength. These data indicate that formation of the intermediate does not involve a rate-determining protonation, and are consistent with interaction of the incoming superoxide anion with a positive charge at or near the ferrous [Fe(NHis)(4)(SCys)] site. The rate constant for decay of the 600-nm intermediate follows the pH-dependent rate law: k(2)(obs) = k(2)'[H(+)] + k(2)' ' and shows a significant D(2)O solvent isotope effect at pH 7.7. The values of k(2)' and k(2)' ' indicate that the 600-nm intermediate decays via diffusion-controlled protonation at acidic pHs and a first-order process involving either water or a water-exchangeable proton on the protein at basic pHs. The formation and decay rate constants for an E47A variant of 2Fe-SOR are not significantly perturbed from their wild-type values, indicating that the conserved glutamate carboxylate does not directly displace the (hydro)peroxo ligand of the intermediate at basic pHs. The kinetics of a K48A variant are consistent with participation of the lysyl side chain in directing the superoxide toward the active site and in directing the protonation pathway of the ferric-(hydro)peroxo intermediate toward release of hydrogen peroxide.

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bioinorganic chemistry ͉ extradiol dioxygenase ͉ nonheme iron ͉ manganese E xtradiol catecholic dioxygenases catalyze the cleavage of dihydroxybenzene rings with incorporation of both atoms from O 2 to yield muconic semialdehyde products (1-3). As such, these enzymes play key roles in the ability of nature to reclaim the vast quantities of organic carbon sequestered in aromatic compounds in the environment. The metal in these enzymes is coordinated by two His residues and one Glu/Asp residue that occupy one face of a (pseudo)octahedron, representing the first examples of what has become recognized as the ''2-His-1-carboxylate facial triad'' (2H1C triad) motif in many nonheme iron enzymes that activate dioxygen (4, 5). Indeed, a truly remarkable array of oxidative reactions is catalyzed by 2H1C triad enzymes, including C-H hydroxylation, ring expansion, and COC bond cleavage (5).Studies from our laboratories and others have resulted in a mechanistic proposal shown in Fig. 1 for the extradiol dioxygenases that takes advantage of the 2H1C triad motif (3,(6)(7)(8).Crystal structures show that the catecholic substrate binds in a bidentate fashion to the reduced metal center trans from the histidines, displacing two or three water molecules (9-11). This primes the metal center for O 2 binding in the coordination site trans to the carboxylate ligand after substrate is in place. We have proposed that electron density is transferred from the aromatic substrate to bound dioxygen via the iron, thereby giving them both radical character and activating them for reaction with each other (6,12,13

Deoxyhypusine hydroxylase is the key enzyme in the biosynthesis of hypusine containing eukaryotic translation initiation factor 5A (eIF5A), which plays an essential role in the regulation of cell proliferation. Recombinant human deoxyhypusine hydroxylase (hDOHH) has been reported to have oxygen-and iron-dependent activity, an estimated iron/holoprotein stoichiometry of 2, and a visible band at 630 nm responsible for the blue color of the as-isolated protein. EPR, Mö ssbauer, and XAS spectroscopic results presented herein provide direct spectroscopic evidence that hDOHH has an antiferromagnetically coupled diiron center with histidines and carboxylates as likely ligands, as suggested by mutagenesis experiments. Resonance Raman experiments show that its blue chromophore arises from a ( -1,2-peroxo)diiron(III) center that forms in the reaction of the reduced enzyme with O 2, so the peroxo form of hDOHH is unusually stable. Nevertheless we demonstrate that it can carry out the hydroxylation of the deoxyhypusine residue present in the elF5A substrate. Despite a lack of sequence similarity, hDOHH has a nonheme diiron active site that resembles both in structure and function those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein ⌬ 9 -desaturase from plants, suggesting that the oxygen-activating diiron motif is a solution arrived at by convergent evolution. Notably, hDOHH is the only example thus far of a human hydroxylase with such a diiron active site.H ypusine is an unusual, but highly conserved, amino acid that is found only in the eukaryotic translational initiation factor 5A (eIF5A), a protein that regulates cell proliferation (1, 2). The biosynthesis of eIF5A involves a posttranslational modification of the eIF5A precursor, where a lysine residue is first modified to deoxyhypusine (Dhp) by deoxyhypusine synthase (DHS) and then the nascent Dhp is hydroxylated by deoxyhypusine hydroxylase (DOHH) to form hypusine (Hpu) (Scheme 1) (1, 2). The importance of hypusine and these 2 enzymes has been shown by several studies where depletion of spermidine (3) or inhibition of either DHS or DOHH (4, 5) leads to a decrease of hypusinecontaining eIF5A [eIF5A(Hpu)] and inhibition of eukaryotic cell growth. Consequently, these results suggest that eIF5A and DOHH could be promising targets for antitumor (6) and anti-HIV-1 therapies (7).The hydroxylase activity of recombinant human DOHH (hDOHH) has been shown to depend on Fe(II) and not on any other physiologically relevant divalent metal ion. An estimated iron-to-holoprotein stoichiometry of 2 is observed (8). Sequence examination, homology modeling, and mutagenesis experiments suggest 2 possible iron binding sites consisting of histidine and carboxylate ligands (8,9). Thus at first glance, hDOHH appears to resemble members of the superfamily of bacterial diiron multicomponent monooxygenases, like methane or toluene monooxygenase, that use nonheme diiron centers to activate dioxygen for the hydroxylat...

The electronic and vibrational properties of the [Fe(His)(4)(Cys)] site (Center II) responsible for catalysis of superoxide reduction in the two-iron superoxide reductase (2Fe-SOR) from Desulfovibrio vulgaris have been investigated using the combination of EPR, resonance Raman, UV/visible/near-IR absorption, CD, and VTMCD spectroscopies. Deconvolution of the spectral contributions of Center II from those of the [Fe(Cys)(4)] site (Center I) has been achieved by parallel investigations of the C13S variant, which does not contain Center I. The resonance Raman spectrum of ferric Center II has been assigned based on isotope shifts for (34)S and (15)N globally labeled proteins. As for the [Fe(His)(4)(Cys)] active site in 1Fe-SOR from Pyrococcus furiosus, the spectroscopic properties of ferric and ferrous Center II in D. vulgaris 2Fe-SOR are indicative of distorted octahedral and square-pyramidal coordination geometries, respectively. Differences in the properties of the ferric [Fe(His)(4)(Cys)] sites in 1Fe- and 2Fe-SORs are apparent in the rhombicity of the S=5/2 ground state ( E/ D=0.06 and 0.28 in 1Fe- and 2Fe-SORs, respectively), the energy of the CysS(-)(p(pi))-->Fe(3+)(d(pi)) CT transition (15150+/-150 cm(-1) and 15600+/-150 cm(-1) in 1Fe- and 2Fe-SORs, respectively) and in changes in the Fe-S stretching region of the resonance Raman spectrum indicative of a weaker Fe-S(Cys) bond in 2Fe-SORs. These differences are interpreted in terms of small structural perturbations in the Fe coordination sphere with changes in the Fe-S(Cys) bond strength resulting from differences in the peptide N-H.S(Cys) hydrogen bonding within a tetrapeptide bidentate "chelate". Observation of the characteristic intervalence charge transfer transition of a cyano-bridged [Fe(III)-NC-Fe(II)(CN)(5)] unit in the near-IR VTMCD spectra of ferricyanide-oxidized samples of both P. furiosus 1Fe-SOR and D. vulgaris 2Fe-SOR has confirmed the existence of novel ferrocyanide adducts of the ferric [Fe(His)(4)(Cys)] sites in both 1Fe- and 2Fe-SORs.

The steady state kinetics of a Desulfovibrio (D.) vulgaris superoxide reductase (SOR) turnover cycle, in which superoxide is catalytically reduced to hydrogen peroxide at a [Fe(His) 4 (Cys)] active site, are reported. A proximal electron donor, rubredoxin, was used to supply reducing equivalents from NADPH via ferredoxin: NADP ؉ oxidoreductase, and xanthine/xanthine oxidase was used to provide a calibrated flux of superoxide. SOR turnover in this system was well coupled, i.e. ؊10 M, during which SOR turns over about once every 6 s, (ii) the diffusion-controlled reaction of reduced SOR with superoxide is the slowest process during turnover, and (iii) neither ligation nor deligation of the active site carboxylate of SOR limits the turnover rate. An intracellular SOR concentration on the order of 10 M is estimated to be the minimum required for lowering superoxide to sublethal levels in aerobically growing SOD knockout mutants of Escherichia coli. SORs from Desulfovibrio gigas and Treponema pallidum showed similar turnover rates when substituted for the D. vulgaris SOR, whereas superoxide dismutases showed no SOR activity in our assay. These results provide quantitative support for previous suggestions that, in times of oxidative stress, SORs efficiently divert intracellular reducing equivalents to superoxide.An emerging paradigm for protecting air-sensitive bacteria and Archaea from the toxic reduction products of dioxygen involves reduction of superoxide and hydrogen peroxide, rather than the classical disproportionation route for their removal characteristic of aerobic microoorganisms (1-9). The reduction of superoxide via Reaction 1 is catalyzed by a novel class of non-heme iron enzymes called superoxide reductases (SORs).

Extradiol catecholic dioxygenases catalyze the cleavage of the aromatic ring of the substrate with incorporation of both oxygen atoms from O 2 . 1 This reaction is a key step in the ability of Nature to reclaim large quantities of carbon sequestered in aromatic compounds. The active sites of these enzymes contain either Mn or Fe, coordinated by a 2-His-1-carboxylate facial triad, which is a common motif of nonheme Fe(II)-containing enzymes that activate dioxygen. 2 Homoprotocatechuate 2,3-dioxygenase from either Brevi-bacterium fuscum or Arthrobacter globiformis catalyzes the ring opening of homoprotocatechuate (HPCA) and contains Fe(II) (FeHPCD) or Mn(II) (MnMndD), respectively, as the native metals. Structures of FeHPCD from crystals that had been incubated with the substrate analogue 4-nitrocatechol and then exposed to low concentrations of O 2 identified three intermediate complexes in the catalytic cycle: semiquinone substrate radical-Fe superoxo (E-SQ), Fe-alkylperoxo (E-AP), and the Fesemialdehyde ring-opened product (EP). 3 Bond length analysis suggests that the iron remains ferrous in each species. A mechanistic proposal developed from these intermediates and earlier studies suggests that electron transfer from the substrate to O 2 , via the metal to form the E-SQ intermediate, results in simultaneous activation of both substrate and oxygen. Recent work has shown that FeHPCD and MnMndD can each be prepared with the nonphysiological metal in the active site (MnHPCD and FeMndD) and that all four forms have approximately the same catalytic parameters. 4 The fact that FeHPCD and MnHPCD also have superimposable structures suggests that the bound metals retain their inherent ~0.7 V difference in redox potential, leading to the proposal that the metal does not undergo a redox state change in the reaction cycle.Here we explore this question through direct electron paramagnetic resonance (EPR) detection and quantitative analysis of the metal oxidation states of MnHPCD as it turns over the natural substrate, HPCA. MnHPCD was chosen for study because it is available in a pure state, it is EPR active, and its full length form is structurally characterized. Four distinct Mn species are observed, two of which are short-lived intermediates. Our advances in EPR simulation software 5, 6 allow characterization of the electronic environment of all Mn species and the determination of concentrations of all species during turnover. Based on this analysis, a lowconcentration intermediate appearing immediately after O 2 addition is found to contain Mn (III) coupled to a radical. Thus, it is possible that the rapid electron transfer to form the reactive Figure 1. The substrate-free enzyme (E, Figure 1A) shows a six-line hyperfine pattern centered at g = 2.00, with splitting of a = 89 G, and a broad feature at g = 2.52. The hyperfine constant is indicative of a Mn(II) species, and the simulation of this spectrum (spectrum A) is calculated for a single protein-bound Mn(II) species using the parameters given in the Figure 1 cap...