Dinuclear Iron Center Research Articles

Di-iron—carboxylate proteins

Di-iron centers bridged by carboxylate residues and oxide/hydroxide groups have so far been seen in four classes of proteins involved in dioxygen chemistry or phosphoryl transfer reactions. The dinuclear iron centers in these proteins are coordinated by histidines and additional carboxylate ligands. Recent structural data on some of these enzymes, combined with spectroscopic and kinetic data, can now serve as a base for detailed mechanistic suggestions. The di-iron sites in the major class of hydroxylase-oxidase enzymes, which contains ribonucleotide reductase and methane monooxygenase, show significant flexibility in the geometry of their coordination of three or more carboxylate groups. This flexibility, combined with a relatively low coordination number, and a buried environment suitable for reactive oxygen chemistry, explains their efficient harnessing of the oxidation power of molecular oxygen.

Identification of the ferroxidase centre of Escherichia coli bacterioferritin

The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit [Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett. 333, 197-202]. We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit. The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR. The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase. Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions. Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation. The mechanism of iron (II) oxidation is discussed in the light of this and previous work.

Iron(II) oxidation by H chain ferritin: evidence from site-directed mutagenesis that a transient blue species is formed at the dinuclear iron center.

The iron storage molecule, ferritin, consists of an iron core surrounded by a shell of 24 protein subunits, which, in mammals, are of two types, H and L. Prior to storage of iron as a hydrous ferric oxide within the protein shell, Fe(II) is catalytically oxidized at dinuclear centers within H chains. When 48 Fe(II) atoms/molecule were added to 1 microM recombinant human H chain apoferritin (apo-HuHF), in 0.1 M Mes (pH 6.5), oxidation was 80% complete within about 0.2 s while 99% of the Fe(II) was oxidized within 10 s. A broad visible absorption band (400-800 nm, with a maximum at 650 nm) appeared during the fast phase of Fe(II) oxidation. It reached a plateau at 0.2-0.3 s and then declined while Fe(II) oxidation proceeded to completion and absorbance in the near-UV (300-400 nm) increased. The transient visible species was not observed when Tyr-34 was replaced by phenylalanine or when other conserved amino acids at the ferroxidase centers were substituted by residues which are unable to bind iron or which alter the charge balance. When a second increment of 48 iron atoms was added, 10 min after the first, the visible absorbance was absent and the rate of oxidation slower. Restoration of full oxidative activity took over 24 h. The data indicate that the fast oxidation of Fe(II) by apo-HuHF and the transient visible absorbance associated with it are due to Fe(II) oxidation at the ferroxidase centers.

Electron transfer properties of the R2 protein of ribonucleotide reductase from Escherichia coli.

The enzyme ribonucleotide reductase from Escherichia coli consists of two proteins, R1 and R2. The active R2 protein contains two dinuclear iron centers and the catalytically essential tyrosyl radical. We have explored the redox properties of the tyrosyl radical and estimate an apparent redox potential of +1000 +/- 100 mV (vs SHE) on the basis of the behavior of numerous mediators. The inability of most of these mediators to equilibrate with the tyrosyl radical supports the notion that the radical exists in an extremely protected hydrophobic pocket that prevents most radical scavengers from interacting with the radical, resulting in its unusual stability. The formal midpoint potential of the diiron clusters of the R2 protein was determined to be -115 +/- 2 mV at pH 7.6 and 4 degrees C. This reduction is a two-electron transfer process, making the R2 protein the first of the nonheme diiron proteins not to stabilize a mixed valence intermediate at ambient temperature. The formal midpoint potential of the dinuclear iron centers is pH dependent, exhibiting a 30 mV/pH unit variance, which indicates that one proton is accepted from the solvent per two electrons transferred to the dinuclear iron center upon reduction. The midpoint potential of the site-directed mutant Y122F R2 protein was also investigated under the same conditions, and this midpoint potential was determined to be -178 mV, providing the first direct evidence that the presence of the Y122 residue modulates the redox properties of the diiron clusters. The redox potentials of both the wild type and Y122F proteins experience cathodic shifts when measured in the presence of azide or the R1 protein. For the latter, the midpoint potentials were determined to be -226 mV for the wild type protein and -281 mV for the Y122F mutant protein, representing a negative shift of over 100 mV for both proteins. These results indicate that the presence of the Y122 residue does not influence the effect of R1 binding, that the R1 protein preferentially binds the oxidized form of R2, and that the binding of R1 acts as a regulatory control mechanism to prevent unnecessary turnover of the dinuclear iron centers.

Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states

The hydroxylase component of soluble methane monooxygenase (sMMO) contains a dinuclear iron center responsible for the oxidation of methane to methanol. As isolated, the center is in the oxidized, diiron(III) state. The 2.2 A resolution X-ray structure of the oxidized hydroxylase, Hox, from Methylococcus capsulatus (Bath) was previously determined at 4 degrees C. In this structure the two iron atoms are bridged by a glutamate, a hydroxide ion, and an acetate ion, and additionally coordinated to two His residues, three Glu residues, and a water molecule. The 1.7 A resolution crystal structures of the sMMO hydroxylase from Methylococcus capsulatus (Bath) in both its oxidized diiron(III), Hox, and dithionite-treated, reduced diiron(II), Hred, oxidation states were determined at -160 degrees C. The structure of the diiron center in Hox differs from that previously reported at 2.2 A resolution and 4 degrees C. Although the hydroxide bridge is retained, the bidentate, bridging ligand assigned as acetate is replaced by a weakly coordinating monoatomic water bridge. In the resulting four-membered Fe(OH)Fe(OH2) ring, the Fe ... Fe distance is shortened from 3.4 A to 3.1 A. In protomer A of Hred, the hydroxide bridge is displaced by an oxygen atom of Glu243, which undergoes a carboxylate shift from its terminal monodentate binding mode in Hox to a mode in which the carboxylate is both monoatomic bridging and bidentate chelating. We therefore conclude that the center has been reduced to the diiron(II) oxidation state. Both iron atoms are coordinated to five ligands and weakly to a sixth water molecule in the resulting structure. The diiron center in protomer B of Hred has the same composition as those in Hox. In both the oxidized and reduced structures, the diiron core is connected through hydrogen bonds involving exogenous species to Thr213 in the active site cavity. The diiron center in Hox can change its exogenous ligand coordination and geometry, a property that could be important in the catalytic cycle of sMMO. In Hred, a carboxylate shift occurs, extruding hydroxide ion and opening coordination sites for reaction with O2 to form the diiron(III) peroxo intermediate, Hperoxo. Residue Thr213 may function in catalysis.

Residues Important for Radical Stability in Ribonucleotide Reductase from Escherichia coli

The R2 protein of ribonucleotide reductase contains at the side chain of tyrosine 122 a stable free radical, which is essential for enzyme catalysis. The tyrosyl radical is buried in the protein matrix close to a dinuclear iron center and a cluster of three hydrophobic residues (Phe-208, Phe-212, and Ile-234) conserved throughout the R2 family. A key step in the generation of the tyrosyl radical is the activation of molecular oxygen at the iron center. It has been suggested that the hydrophobic cluster provides an inert binding pocket for molecular oxygen bound to the iron center and that it may play a role in directing the oxidative power of a highly reactive intermediate toward tyrosine 122. We have tested these hypotheses by constructing the following mutant R2 proteins:F208Y, F212Y, F212W, and I234N. The resulting mutant proteins all have the ability to form a tyrosine radical, which indicates that binding of molecular oxygen can occur. In the case of F208Y, the yield of tyrosyl radical is substantially lower than in the wild-type case. A competing reaction resulting in hydroxylation of Tyr-208 implies that the phenylalanine at position 208 may influence the choice of target for electron abstraction. The most prominent result is that all mutant proteins show impaired radical half-life; in three of the four mutants, the half-lives are several orders of magnitude shorter than that of the wild-type radical. This suggests that the major role of the hydrophobic pocket is to stabilize the tyrosyl radical. This hypothesis is corroborated by comparative studies of the environment of other naturally occurring tyrosyl radicals.

A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.

The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the R1 and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S. typhimurium nrdE and nrdF genes (R1E and R2F). They correspond to the R1 and R2 proteins. Each protein is a homodimer. Together they catalyze the reduction of CDP to dCDP, using dithiothreitol or reduced glutaredoxin, but not thioredoxin, as an electron donor. CDP reduction is strongly stimulated by low concentrations of dATP, presumably acting as an allosteric effector. Protein R2F contains an antiferromagnetically coupled dinuclear iron center and a tyrosyl free radical. The E. coli and S. typhimurium chromosome thus have maintained the information for a potentially active additional class I ribonucleotide reductase, whose role in vivo is as yet unknown. The allosteric regulation of this enzyme differs from that of the normally expressed reductase.

Modeling Hydrolysis at Dinuclear Iron Centers

Modeling Hydrolysis at Dinuclear Iron Centers

The conserved serine 211 is essential for reduction of the dinuclear iron center in protein R2 of Escherichia coli ribonucleotide reductase.

The R2 protein family of class I ribonucleotide reductases contains a highly conserved serine residue close to the essential tyrosyl radical and the dinuclear iron center. In order to test its physiological importance, we have engineered the Ser-211 of Escherichia coli R2 to an alanine and a cysteine residue. The three-dimensional structure of R2 S211A solved to 2.4-A resolution is virtually identical to the wild-type structure apart from the substituted residue. Both mutant proteins contain oxidized dinuclear iron and tyrosyl radical, and their specific enzyme activity per radical are comparable to that of the wild-type protein. In R2 S211A the stability of the tyrosyl radical is substantially decreased, probably caused by movement of Gln-80 into hydrogen bonding distance of Tyr-122. The major defect in R2 S211A, however, is the inability of its iron center to be reduced by enzymic or chemical means, a characteristic not found in R2 S211C. We propose that Ser-211 is needed as a proton donor/transporter during reduction of the iron center of R2, a reaction which in vivo precedes reconstitution of the tyrosyl radical. This offers a physiological explanation for the high conservation of a serine residue at this position in the R2 family.

Dioxygen is the source of the mu-oxo bridge in iron ribonucleotide reductase.

The formation of the iron-radical cofactor in the R2 subunit of ribonucleotide reductase has been monitored by resonance Raman spectroscopy. The differrous cluster in reduced R2 functions as a tyrosine oxidase; it uses O2 to oxidize Tyr-122 to a stable radical and results in an oxo-bridged diferric cluster. The Phe-122 mutant produces an identical dinuclear iron center and provides a simplified model for O2 activation. Oxidation with 18O2 results in quantitative incorporation of 18O into the diferric cluster as evidenced by the 13-cm-1 downshift in the Fe-O-Fe stretching vibration at 500 cm-1. Thus, O2 must be coordinated to the diiron center during O-O bond cleavage. When the Phe-208 adjacent to the diferous cluster is mutated to Tyr, reaction with O2 results in its oxidation to dihydroxyphenylalanine (DOPA-208) and subsequent coordination to Fe as a catecholate ligand. The Fe-O/(catecholate) stretching modes at 512 and 592 cm-1 shift by -13 and -8 cm-1, respectively, when the oxidation is performed in H(2)18O. These isotope shifts indicate that the second oxygen atom of DOPA-208 originates from H2O rather than O2. Taken together, our results are consistent with a mu-1,1-peroxide and a high valent iron-oxo species as reaction intermediates. A common pathway for oxygen activation by the related iron-oxo enzymes methane monooxygenase and fatty acid desaturase is proposed.

Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii.

Structure and Function of the Escherichia coli Ribonucleotide Reductase Protein R2

The crystal structure of the ribonucleotide reductase free radical protein R2 from Escherichia coli has been determined by multiple isomorphous replacement and twofold molecular averaging. The structure has been refined at 2·2 Å resolution to R = 0·175. The subunit structure of the R2 protein has a novel fold where the basic motif is a bundle of eight long helices. The R2 dimer has two equivalent dinuclear iron centers. Each iron center is well buried in the subunit. The iron atoms have both histidine and carboxyl acid ligands and are bridged by an oxide ion and the carboxylate group of Glu115. One iron atom is octahedrally coordinated with small deviations from ideal values, while the coordination of the other iron ion is more distorted, mainly due to the fact that Asp84 is a bidental ligand to this iron atom. The oxidation of the enzymatically essential tyrosine residue (Tyr122) and the dinuclear iron center by molecular oxygen is suggested to take part in a suitable conserved oxygen-binding pocket between the iron center and the tyrosine η-oxygen 5·3 Å away from the closest iron ion. The tyrosine proton can be abstracted by the dioxygen and the deprotonated tyrosine residue is then more easily oxidized to a radical species. Tyr122 is buried inside the protein about 10 Å from the surface. This has the consequence that the tyrosyl radical cannot participate directly in hydrogen abstraction from the substrate ribose at the active site of the holoenzyme located on the R1 subunit. The radical must then be indirectly involved in the mechanism of the enzyme and an electron transfer reaction between the active site and the tyrosine must take place. Based on the analysis of the available ribonucleotide reductase sequences, the binding surface for the large ribonucleotide reductase protein R1, and a possible route for an electron transport between the buried radical and this surface is described.

Crystallization and preliminary X-ray analysis of the methane monooxygenase hydroxylase protein from Methylococcus capsulatus (Bath)

Methane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of methane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition α 2β 2γ 2 . The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol, lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group P2 12 12 1 , with one dimer of relative molecular mass M r = 252,000 in the asymmetric unit. The unit cell dimensions are a = 62.6 Å, b = 110.1 Å, c = 333.5 Å. The crystals diffract uniformly beyond 2.5 Å resolution. Crystals of the related hydroxylase from Methylosinus trichosporium OB3b have also been obtained.

Resonance Raman enhancement of the oxo‐bridged dinuclear iron center: Vibrational modes in iron(III) physiological‐type porphyrin complexes

AbstractOxo‐bridged dinuclear iron(III) complexes of two physiological‐type prophyrins, (DPDEFe)2O and (PPDEFe)2O (where DPDE = deuteroporphyrin dimethyl ester and PPDE = protoporphyrin dimethyl ester), were investigated using resonance Raman and infrared spectroscopy. The assignment of the porphyrin vibrational modes was performed using polarization measurements and previous studies concerning the corresponding mononuclear iron(III) complexes, (DPDEFe)Cl, (PPDEFe)Cl, and related compounds. The assignment of the FeOFe axial bridge vibrational modes of the two μ‐oxo complexes investigated and the synthetic analogue (TPPFe)2O (where TPP = tetraphenylporphyrin) was made with the help of 18O substitution. The dependence of the frequencies vs and va on the FeOFe angle for (TPPFe)2O is in good agreement with the angle value previously determined by x‐ray diffraction data. The values of the FeOFe angle in (DPDEFe)2O and (PPDEFe)2O were estimated to be close to 160° and 170°, respectively. The excitation profiles (351.1–647.1 nm) of the porphyrin core marker bands for all the μ‐oxo complexes studied are in good agreement with the expected general trends of the resonance enhancement with moderate vibronic coupling. However, for the μ‐oxo physiological‐type porphyrin complexes, the v4 marker band exhibits and unexpected strong enhancement maximum near the maximum found in the excitation profile of the va and vs (FeOFe) modes. These enhancement maxima tend to correspond to a minor oxo → Fe(III) charge‐transfer band in the electronic absorption spectra. A mixing of several electronic levels under low symmetry may be responsible for this enhancement phenomenon.

Engineering of the iron site in ribonucleotide reductase to a self-hydroxylating monooxygenase.

Protein R2 of ribonucleotide reductase contains a dinuclear ferric iron center adjacent to a tyrosyl radical in the interior of the protein matrix. A patch of hydrophobic residues surrounds the iron-radical cofactor. Its importance during the oxidative generation of the iron-radical cofactor was investigated by site-directed mutagenesis of Phe-208 to tyrosine. The mutant protein R2 F208Y has prominent absorption bands at 460 and 720 nm reminiscent of those in ferric-catecholate complexes. Resonance Raman spectroscopy shows that the iron center of R2 F208Y contains a bidentate catechol ligand. The mechanism for generation of this protein-derived dihydroxyphenylalanine may be similar to the catalytic cycle of methane monooxygenase.

X-ray absorption, Moessbauer, and EPR studies of the dinuclear iron center in the hydroxylase component of methane monooxygenase

X-ray absorption, Moessbauer, and EPR studies of the dinuclear iron center in the hydroxylase component of methane monooxygenase

Dinuclear non-heme iron centers: structure and function

Proteins containing dinuclear non-heme iron centers have been characterized that catalyse reversible binding of O 2, hydroxylation of alkanes, and oxidation of tyrosine to a tyrosyl radical. Physical biochemical methods have revealed novel aspects of both the structure and function of these proteins. Characterization of these centers has been greatly assisted by structural and functional bioinorganic models.

Proteins containing oxo-bridged dinuclear iron centers: a bioinorganic perspective

Proteins containing oxo-bridged dinuclear iron centers: a bioinorganic perspective

Carbon-carbon bond formation at a dinuclear iron center. 4. Synthesis of [Fe2(CO)6(.mu.-C(OR)H)(.mu.-CR'=CR'H)]- anions (R = Me, Et; R' = Ph, H) and the subsequent formation by protonation of the bridging vinylcarbene ligands >CHCR'=CR'H or >CR'CH=CR'H, depending on the reaction conditions

Carbon-carbon bond formation at a dinuclear iron center. 4. Synthesis of [Fe2(CO)6(.mu.-C(OR)H)(.mu.-CR'=CR'H)]- anions (R = Me, Et; R' = Ph, H) and the subsequent formation by protonation of the bridging vinylcarbene ligands >CHCR'=CR'H or >CR'CH=CR'H, depending on the reaction conditions

Electronic and Raman spectroscopic properties of oxo-bridged dinuclear iron centers in proteins and model compounds

Electronic and Raman spectroscopic properties of oxo-bridged dinuclear iron centers in proteins and model compounds