Diiron Cluster Research Articles

Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to effect oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate [myo-inositol (MI)] by four electrons to d-glucuronate. Abstraction of hydrogen from C1 by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[2H]6-MI (D6-MI), has now permitted initial characterization of the C-H-cleaving intermediate. The MIOX.1,2,3,4,5,6-[2H]6-MI complex reacts rapidly and reversibly with O2 to form an intermediate, G, with a g = (2.05, 1.98, 1.90) EPR signal. The rhombic g-tensor and observed hyperfine coupling to 57Fe are rationalized in terms of a (superoxo)diiron(III/III) structure with coordination of the superoxide to a single iron. G decays to H, the intermediate previously detected in the reaction with unlabeled substrate. This step is associated with a kinetic isotope effect of > or =5, showing that the superoxide-level complex does indeed cleave a C-H(D) bond of MI.

A Coupled Dinuclear Iron Cluster that Is Perturbed by Substrate Binding in myo-Inositol Oxygenase

myo-Inositol oxygenase (MIOX) uses iron as its cofactor and dioxygen as its cosubstrate to effect the unique, ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate to d-glucuronate. The nature of the iron cofactor and its interaction with the substrate, myo-inositol (MI), have been probed by electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The data demonstrate the formation of an antiferromagnetically coupled, high-spin diiron(III/III) cluster upon treatment of solutions of Fe(II) and MIOX with excess O(2) or H(2)O(2) and the formation of an antiferromagnetically coupled, valence-localized, high-spin diiron(II/III) cluster upon treatment with either limiting O(2) or excess O(2) in the presence of a mild reductant (e.g., ascorbate). Marked changes to the spectra of both redox forms upon addition of MI and analogy to changes induced by binding of phosphate to the diiron(II/III) cluster of the protein phosphatase, uteroferrin, suggest that MI coordinates directly to the diiron cluster, most likely in a bridging mode. The addition of MIOX to the growing family of non-heme diiron oxygenases expands the catalytic range of the family beyond the two-electron oxidation (hydroxylation and dehydrogenation) reactions catalyzed by its more extensively studied members such as methane monooxygenase and stearoyl acyl carrier protein Delta(9)-desaturase.

Oxygen Activation by a Mixed-Valent, Diiron(II/III) Cluster in the Glycol Cleavage Reaction Catalyzed by myo-Inositol Oxygenase

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by Mössbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.

Regulation of Methane Monooxygenase Catalysis Based on Size Exclusion and Quantum Tunneling

The hydroxylase component (MMOH) of the soluble form of methane monooxygenase (sMMO) isolated from Methylosinus trichosporium OB3b catalyzes both the O2 activation and the CH4 oxidation reactions at the oxygen-bridged dinuclear iron cluster present in its buried active site. During the reaction cycle, the diiron cluster forms a bis-mu-oxo-(Fe(IV))2 intermediate termed compound Q (Q) that reacts directly with methane. Many adventitious substrates also react with Q, most at a relatively slow rate. We have proposed that Q reacts preferentially with CH4 because the sMMO regulatory component MMOB induces a size selective pore into the MMOH active site as the two components form a complex. Support for this proposal has come through the observation of a nonlinear Arrhenius plot for the CH4 oxidation, presumably due to a shift in rate-limiting step from substrate binding at low temperature to C-H bond cleavage at high temperature. Reactions of all substrates other than CH4 fail to exhibit a break in the Arrhenius plot because binding is always rate limiting in the temperature range explored. Here we show that it is possible to induce a break in the Arrhenius plot for the ethane reaction with Q by using an MMOB mutant termed DBL2 (S109A/T111A) in which residues at the MMOH-MMOB interface are reduced in size. We hypothesize that this increases the ethane binding rate and shifts the Arrhenius breakpoint into the observable temperature range. As a result of this shift, the kinetic and activation parameters of the C-H bond breaking reaction for both methane and ethane can be observed using the DBL2 mutant. A 2H-KIE is observed for both substrate oxidation reactions when using DBL2, whereas only CH4 oxidation exhibits an effect when using wild type MMOB, consistent with the C-H bond cleaving reaction becoming at least partially rate limiting for ethane. Analysis of the temperature dependence of the 2H-KIE for ethane and methane for reactions using both mutant and wild type forms of MMOB suggests that quantum tunneling plays a significant role in methane oxidation but not ethane oxidation.

Role of the C-Terminal Region of the B Component of Methylosinus trichosporium OB3b Methane Monooxygenase in the Regulation of Oxygen Activation

The effects of the C-terminal region of the B component (MMOB) of soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b on steady-state turnover, the transient kinetics of the reaction cycle, and the properties of the sMMO hydroxylase (MMOH) active site diiron cluster have been explored. MMOB is known to have many profound effects on the rate and specificity of sMMO. Past studies have revealed specific roles for the well-folded core structure of MMOB as well as the disordered N-terminal region. Here, it is shown that the disordered C-terminal region of MMOB also performs critical roles in the regulation of catalysis. Deletion mutants of MMOB missing 5, 8, and 13 C-terminal residues cause progressive decreases in the maximum steady-state turnover number, as well as lower apparent rate constants for formation of the key reaction cycle intermediate, compound Q. It is shown that this latter effect is actually due to a decrease in the rate constant for formation of an earlier intermediate, probably the hydroperoxo species, compound P. Moreover, the deletions result in substantial uncoupling at or before the P intermediate. It is proposed that this is due to competition between slow H(2)O(2) release from one of the intermediates and the reaction that carries this intermediate on to the next step in the cycle, which is slowed by the mutation. Electron paramagnetic resonance (EPR) studies of the hydroxylase component (MMOH) in the mixed valent state suggest that complexation with the mutant MMOBs alters the electronic properties of the diiron cluster in a manner distinct from that observed when wild-type MMOB is used. Active site structural changes are also suggested by a substantial decrease in the deuterium kinetic isotope effect for the reaction of Q with methane thought to be associated with a decrease in quantum tunneling in the C-H bond breaking reaction. Thus, the surface interactions between MMOH and MMOB that affect substrate oxidation and its regulation appear to require the complete MMOB C-terminal region for proper function.

A dityrosyl-diiron radical cofactor center is essential for human ribonucleotide reductases

Ribonucleotide reductase catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA biosynthesis. A tyrosine residue in the small subunit of class I ribonucleotide reductase harbors a stable radical, which plays a central role in the catalysis process. We have discovered that an additional tyrosine residue, conserved in human small subunits hRRM2 and p53R2, is required for the radical formation and enzyme activity. Mutations of this newly identified tyrosine residue obliterated the stable radical and the enzymatic activity of human ribonucleotide reductases shown by electron paramagnetic resonance spectroscopy and enzyme activity assays. Three-dimensional structural analysis reveals for the first time that these two tyrosines are located at opposite sides of the diiron cluster. We conclude that both tyrosines are necessary in maintaining the diiron cluster of the enzymes, suggesting that the assembly of a dityrosyl-diiron radical cofactor center in human ribonucleotide reductases is essential for enzyme catalytic activity. These results should provide insights to design better ribonucleotide reductase inhibitors for cancer therapy.

Diphenylphosphide-Bridged Diiron Derivatives of [Fe2(η5-C5H5)2(μ-H)(μ-PPh2)(CO)2

The complex trans-[Fe2Cp2(μ-H)(μ-PPh2)(CO)2] is obtained in 91% yield by refluxing toluene solutions of [Fe2Cp2(CO)4] (Cp = η5-C5H5) and the secondary phosphine PPh2H. This compound isomerizes upon irradiation with visible−UV light under a CO atmosphere to yield cis-[Fe2Cp2(μ-H)(μ-PPh2)(CO)2]. The above hydride complexes react under photochemical conditions with 1 equiv of secondary phosphines PR2H (R = Et, Ph) to give the corresponding monocarbonyl compounds [Fe2Cp2(μ-PPh2)(μ-PR2)(μ-CO)] via the hydride intermediates [Fe2Cp2(μ-H)(μ-PPh2)(CO)(PR2H)] (detected and isolated for R = Et). Deprotonation of trans-[Fe2Cp2(μ-H)(μ-PPh2)(CO)2] with LiBu gives the binuclear anion [Fe2Cp2(μ-PPh2)(CO)2]-. This highly nucleophilic carbonylate reacts rapidly with [AuCl(PiPr3)] or MeI to give the corresponding gold diiron cluster [AuFe2Cp2(μ-PPh2)(CO)2(PiPr3)] or methyl derivative [Fe2Cp2(Me)(μ-PPh2)(μ-CO)(CO)2], respectively. Both hydrides cis- and trans-[Fe2Cp2(μ-H)(μ-PPh2)(CO)2] can be reversibly oxidized at low temperature to the corresponding cation radicals cis- and trans-[Fe2Cp2(μ-H)(μ-PPh2)(CO)2]+. At room temperature, however, the trans dicarbonyl cation isomerizes to its cis isomer, which in turn experiences a degradation process involving the reductive elimination of the bridging groups. The structures of the new complexes are analyzed on the basis of the corresponding IR and NMR (1H, 31P and 13C) spectroscopic data. The nature of the new radical cations is analyzed also on the basis of cyclic voltammetry and ESR measurements.

Mediation by Indole Analogues of Electron Transfer during Oxygen Activation in Variants of Escherichia coli Ribonucleotide Reductase R2 Lacking the Electron-Shuttling Tryptophan 48

Activation of dioxygen by the carboxylate-bridged diiron(II) cluster in the R2 subunit of class I ribonucleotide reductase from Escherichia coli results in the one-electron oxidation of tyrosine 122 (Y122) to a stable radical (Y122*). A key step in this reaction is the rapid transfer of a single electron from a near-surface residue, tryptophan 48 (W48), to an adduct between O(2) and diiron(II) cluster to generate a readily reducible cation radical (W48(+)(*)) and the formally Fe(IV)Fe(III) intermediate known as cluster X. Previous work showed that this electron injection step is blocked in the R2 variant with W48 replaced by phenylalanine [Krebs, C., Chen, S., Baldwin, J., Ley, B. A., Patel, U., Edmondson, D. E., Huynh, B. H., and Bollinger, J. M., Jr. (2000) J. Am. Chem. Soc. 122, 12207-12219]. In this study, we show that substitution of W48 with alanine similarly disables the electron transfer (ET) but also permits its chemical mediation by indole compounds. In the presence of an indole mediator, O(2) activation in the R2-W48A variant produces approximately 1 equiv of stable Y122* and more than 1 equiv of the normal (micro-oxo)diiron(III) product. In the absence of a mediator, the variant protein generates primarily altered Fe(III) products and only one-fourth as much stable Y122* because, as previously reported for R2-W48F, most of the Y122* that is produced decays as a consequence of the inability of the protein to mediate reductive quenching of one of the two oxidizing equivalents of the initial diiron(II)-O(2) complex. Mediation of ET is effective in W48A variants containing additional substitutions that also impact the reaction mechanism or outcome. In the reaction of R2-W48A/F208Y, the presence of mediator suppresses formation of the Y208-derived diiron(III)-catecholate product (which is predominant in R2-F208Y in the absence of reductants) in favor of Y122*. In the reaction of R2-W48A/D84E, the presence of mediator affects the outcome of decay of the peroxodiiron(III) intermediate known to accumulate in D84E variants, increasing the yield of Y122* by as much as 2.2-fold to a final value of 0.75 equiv and suppressing formation of a 490 nm absorbing product that results from decay of the two-electron oxidized intermediate in the absence of a functional ET apparatus.

Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase.

A key step in generation of the catalytically essential tyrosyl radical (Y122(*)) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe(2)O(2))(4+) complex formed by addition of O(2) to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe(2)O(2))(4+) complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48(+)(*)). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe(2)O(2))(4+) state that is probably the precursor to the X-W48(+)(*) diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O(2) results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent Mössbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mössbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or Mössbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively mu-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H(peroxo)) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe(2)O(2))(4+) state in R2-W48A/Y122F. It is postulated that the new (Fe(2)O(2))(4+) state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (mu-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.

Redox Studies of Subunit Interactivity in Aerobic Ribonucleotide Reductase from Escherichia coli

Ribonucleotide reductase is a heterodimeric (alpha(2)beta(2)) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (-163 +/- 3 mV) different from that of R2(Met) produced by hydroxyurea (-115 +/- 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming -223 +/- 6 and -226 +/- 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to -192 +/- 2 mV for dGDP/dTTP and to -203 +/- 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mm) and dATP (0.3-0.6 mm) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.

Enzymatic Mechanism of Fe-Only Hydrogenase: Density Functional Study on H−H Making/Breaking at the Diiron Cluster with Concerted Proton and Electron Transfers

The mechanism of the enzymatic hydrogen bond forming/breaking (2H(+) + 2e<==>H(2)) and the plausible charge and spin states of the catalytic diiron subcluster [FeFe](H) of the H cluster in Fe-only hydrogenases are probed computationally by the density functional theory. It is found that the active center [FeFe](H) can be rationally simulated as [[H](CH(3)S)(CO)(CN(-))Fe(p)(CO(b))(mu-SRS)Fe(d)(CO)(CN(-))L], where the monovalence [H] stands for the [4Fe4S](H)(2+) subcluster bridged to the [FeFe](H) moiety, (CH(3)S) represents a Cys-S, and (CO(b)) represents a bridging CO. L could be a CO, H(2)O, H(-), H(2), or a vacant coordination site on Fe(d). Model structures of possible redox states are optimized and compared with the X-ray crystallographic structures and FTIR experimental data. On the basis of the optimal structures, we study the most favorable path of concerted proton transfer and electron transfer in H(2)-forming/breaking reactions at [FeFe](H). Previous mechanisms derived from quantum chemical computations of Fe-only hydrogenases (Cao, Z.; Hall, M. B. J. Am. Chem. Soc. 2001, 123, 3734; Fan, H.; Hall, M. B. J. Am. Chem. Soc. 2001, 123, 3828) involved an unidentified bridging residue (mu-SRS), which is either a propanedithiolate or dithiomethylamine. Our proposed mechanism, however, does not require such a ligand but makes use of a shuttle of oxidation states of the iron atoms and a reaction site between the two iron atoms. Therefore, the hydride H(b)(-) (bridged to Fe(p) and Fe(d)) and eta(2)-H(2) at Fe(p) or Fe(d) most possibly play key roles in the dihydrogen reversible oxidation at the [FeFe](H) active center. This suggested way of H(2) formation/splitting is reminiscent of the mechanism of [NiFe] hydrogenases and therefore would unify the mechanisms of the two related enzymes.

Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli.

The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by Mössbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster.

Mechanistic implications for the formation of the Diiron cluster in ribonucleotide reductase from quantitative EPR spectroscopy

Mechanistic implications for the formation of the Diiron cluster in ribonucleotide reductase from quantitative EPR spectroscopy

Mechanistic implications for the formation of the diiron cluster in ribonucleotide reductase provided by quantitative EPR spectroscopy.

The small subunit of Escherichia coli ribonucleotide reductase (R2) is a homodimeric (betabeta) protein, in which each beta-peptide contains a diiron cluster composed of two inequivalent iron sites. R2 is capable of reductively activating O(2) to produce a stable tyrosine radical (Y122*), which is essential for production of deoxyribonucleotides on the larger R1 subunit. In this work, the paramagnetic Mn(II) ion is used as a spectroscopic probe to characterize the assembly of the R2 site with EPR spectroscopy. Upon titration of Mn(II) into samples of apoR2, we have been able to quantitatively follow three species (aquaMn(II), mononuclear Mn(II)R2, and dinuclear Mn(2)(II)R2) and fit each to a sequential two binding site model. As previously observed for Fe(II) binding within apoR2, one of the sites has a greater binding affinity relative to the other, K(1) = (5.5 +/- 1.1) x 10(5) M(-)(1) and K(2) = (3.9 +/- 0.6) x 10(4) M(-)(1), which are assigned to the B and A sites, respectively. In multiple titrations, only one dinuclear Mn(2)(II)R2 site was created per homodimer of R2, indicating that only one of the two beta-peptides of R2 is capable of binding Mn(II) following addition of Mn(II) to apoR2. Under anaerobic conditions, addition of only 2 equiv of Fe(II) to R2 (Fe(2)(II)R2) completely prevented the formation of any bound MnR2 species. Upon reaction of this sample with O(2) in the presence of Mn(II), both Y122* and Mn(2)(II)R2 were produced in equal amounts. Previous stopped-flow absorption spectroscopy studies have indicated that apoR2 undergoes a protein conformational change upon binding of metal (Tong et al. J. Am. Chem. Soc. 1996, 118, 2107-2108). On the basis of these observations, we propose a model for R2 metal incorporation that invokes an allosteric interaction between the two beta-peptides of R2. Upon binding the first equiv of metal to a beta-peptide (beta(I)), the aforementioned protein conformational change prevents metal binding in the adjacent beta-peptide (beta(II)) approximately 25 A away. Furthermore, we show that metal incorporation into beta(II) occurs only during the O(2) activation chemistry of the beta(I)-peptide. This is the first direct evidence of an allosteric interaction between the two beta-peptides of R2. Furthermore, this model can explain the generally observed low Fe occupancy of R2. We also demonstrate that metal uptake and this newly observed allosteric effect are buffer dependent. Higher levels of glycerol cause loss of the allosteric effect. Reductive cycling of samples in the presence of Mn(II) produced a novel mixed metal Fe(III)Mn(III)R2 species within the active site of R2. The magnitude of the exchange coupling (J) determined for both the Mn(2)(II)R2 and Fe(III)Mn(III)R2 species was determined to be -1.8 +/- 0.3 and -18 +/- 3 cm(-)(1), respectively. Quantitative spectral simulations for the Fe(III)Mn(III)R2 and mononuclear Mn(II)R2 species are provided. This work represents the first instance where both X- and Q-band simulations of perpendicular and parallel mode spectra were used to quantitatively predict the concentration of a protein bound mononuclear Mn(II) species.

Methane monooxygenase and compound Q: lessons in oxygen activation

Methane monooxygenase (MMO) catalyzes the NADH and O 2 coupled oxidation of CH 4 to yield CH 3OH. The enzyme consists of three components: MMOH (hydroxylase), MMOB (effector), and MMOR (reductase). We have structurally characterized MMOH and MMOB. The active site is located in MMOH and contains a bis-μ-hydroxo-bridged diiron cluster that is responsible for O 2 activation and substrate oxidation. Transient kinetic studies have revealed many reaction cycle intermediates and allowed some of them to be characterized. The unique intermediate Q contains a bis-μ-oxo-Fe(IV)Fe(IV) cluster and can directly oxidize CH 4. Large deuterium isotope effects are observed for the reaction of Q with methane but, remarkably, not ethane. Arrhenius plots are nonlinear for the CH 4 reaction with Q but not for other substrates. We propose that the Q reaction with substrates occurs in two steps, substrate binding and CH bond cleavage, with the latter rate limiting only for CH 4. Mutagenesis of MMOB shows that it affects the kinetics throughout the reaction cycle. One mutant dramatically alters the rates of Q decay and product release according to substrate size. It is proposed that MMOB serves to gate substrates into the active site of MMOH, thereby allowing it to select methane from more readily oxidized substrates.

Biochemical, Mössbauer, and EPR studies of the diiron cluster of phenol hydroxylase from Pseudomonas sp. strain CF 600.

Phenol hydroxylase of Pseudomonas sp. strain CF600 comprises three components: DmpP is an FAD- and [2Fe-2S]-containing reductase; DmpM is a cofactorless activator protein; and DmpLNO is the oxygenase. Single turnover experiments established that DmpLNO contains the active site, but requires DmpM for efficient turnover: the steady-state turnover rate reaches a maximum at 1.5 DmpM:1 DmpLNO. Chemical cross-linking experiments showed that DmpM interacts with the large subunit of the DmpLNO oxygenase complex. Mössbauer studies revealed that the active site of the oxygenase can accommodate two types of diiron clusters, each of these cluster types having two equivalent sites. Cluster form I, representing typically around 85% of total Fe, has DeltaE(Q) = 1.73 mm/s and delta = 0.54 mm/s, while cluster II exhibits DeltaE(Q) = 0.79 mm/s and delta = 0.48 mm/s. Studies in strong applied magnetic fields suggest that the two iron sites of cluster I are bridged by an oxo group while sites in cluster II appear to be hydroxo-bridged. Reduction of the samples with dithionite yields the diferrous forms of the clusters. Air oxidation of the reduced samples leads to an increase of the cluster II fraction, accompanied by a corresponding decrease in catalytic activity. The reduced oxygenase samples exhibit at X-band an integer spin EPR signal centered, in parallel mode, at g = 16.6. Quantitative analysis showed that 19% of the clusters contribute to the EPR signal, suggesting that cluster II is the EPR-active species. Incubation with dithiothreitol (DTT) inactivated the oxygenase by a mechanism apparently involving H(2)O(2) generation. In addition, Mössbauer studies of DTT-inactivated enzyme showed that all ferric iron belonged to one diamagnetic diferric cluster with parameters that indicate that DTT coordinates to the cluster.

Structural and spectroscopic studies of valence-delocalized diiron(II,III) complexes dupported by carboxylate-only bridging ligands.

The synthesis, molecular structures, and spectroscopic properties of a series of valence-delocalized diiron(II,III) complexes are described. One-electron oxidation of diiron(II) tetracarboxylate complexes afforded the compounds [Fe(2)(mu-O(2)CAr(Tol))(4)L(2)]X, where L = 4-(t)BuC(5)H(4)N (1b), C(5)H(5)N (2b), and THF (3b); X = PF(6)(-) (1b and 3b) and OTf(-) (2b). In 1b-3b, four mu-1,3 carboxylate ligands span relatively short Fe...Fe distances of 2.6633(11)-2.713(3) A. Intense (epsilon = 2700-3200 M(-1) cm(-1)) intervalence charge transfer bands were observed at 620-670 nm. EPR spectroscopy confirmed the S = (9)/(2) ground spin state of 1b-3b, the valence-delocalized nature of which was probed by X-ray absorption spectroscopy. The electron delocalization between paramagnetic metal centers is described by double exchange, which, for the first time, is observed in diiron clusters having no single-atom bridging ligand(s).

Exchange coupling constant J of peroxodiferric reaction intermediates determined by Mössbauer spectroscopy.

Oxygen activation at a carboxylate-bridged diiron cluster is employed by a number of enzymes for diverse biological functions. The mechanisms by which O(2) is activated at the diferrous clusters have been studied in detail and peroxodiferric reaction intermediates have been observed in several of these diiron proteins. To understand further the magnetic properties of this common reaction intermediate, we have used Mössbauer spectroscopy to determine the magnitude and sign of the exchange coupling constant J (in the exchange Hamiltonian J S(1) x S(2)) of the peroxodiferric intermediates generated during the reactions of O(2) with two different proteins, the recombinant M ferritin from frog and the site-directed variant W48F/D84E of the R2 subunit of ribonucleotide reductase from Escherichia coli. Both intermediates are antiferromagnetically coupled with a moderate coupling constant J of 50+/-10 cm(-1) for R2-W48F/D84E and 75+/-10 cm(-1) for M ferritin. This work demonstrates the capability of Mössbauer spectroscopy to determine exchange coupling constants of diiron complexes, including reaction intermediates. The approach and its limitations are described.

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.

Desaturation reactions catalyzed by soluble methane monooxygenase.

Soluble methane monooxygenase (MMO) is shown to be capable of catalyzing desaturation reactions in addition to the usual hydroxylation and epoxidation reactions. Dehydrogenated products are generated from MMO-catalyzed oxidation of certain substrates including ethylbenzene and cyclohexadienes. In the reaction of ethylbenzene, desaturation of ethyl C-H occurred along with the conventional hydroxvlations of ethyl and phenyl C-Hs. As a result, styrene is formed together with ethylphenols and phenylethanols. Similarly, when 1,3- and 1,4-cyclohexadienes were used as substrates, benzene was detected as a product in addition to the corresponding alcohols and epoxides. In all cases, reaction conditions were found to significantly affect the distribution among the different products. This new activity of MMO is postulated to be associated with the chemical properties of the substrates rather than fundamental changes in the nature of the oxygen and C-H activation chemistries. The formation of the desaturated products is rationalized by formation of a substrate cationic intermediate, possibly via a radical precursor. The cationic species is then proposed to partition between recombination (alcohol formation) and elimination (alkene production) pathways. This novel function of MMO indicates close mechanistic kinship between the hydroxylation and desaturation reactions catalyzed by the nonheme diiron clusters.