Hydroxylase Component Of Methane Monooxygenase Research Articles

Phenomenological simulation and density functional theory prediction of 57 Fe Mössbauer parameters: application to magnetically coupled diiron proteins

The use of phenomenological spin Hamiltonians and of spin density functional theory for the analysis and interpretation of Mossbauer spectra of antiferromagnetic or ferromagnetic diiron centers is briefly discussed. The spectroscopic parameters of the hydroxylase component of methane monooxygenase (MMOH), an enzyme that catalyzes the conversion of methane to methanol, have been studied. In its reduced diferrous state (MMOHRed) the enzyme displays 57Fe Mossbauer and EPR parameters characteristic of two ferromagnetically coupled high spin ferrous ions. However, Mossbauer spectra recorded for MMOHRed from two different bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, display slightly different electric quadrupole splittings (ΔEQ) in apparent contradiction to their essentially identical active site crystallographic structures and biochemical functions. Herein, the Mossbauer spectral parameters of MMOHRed have been predicted and studied via spin density functional theory. The somewhat different ΔEQ recorded for the two bacteria have been traced to the relative position of an essentially unbound water molecule within their diiron active sites. It is shown that the presence or absence of the unbound water molecule mainly affects the electric field gradient at only one iron ion of the binuclear active sites.

Hydroxylation mechanism of methane and its derivatives over designed methane monooxygenase model with peroxo dizinc core

The peroxo dizinc Zn(2)O(2) complex Q coordinated by imidazole and carboxylate groups for each Zn center has been designed to model the hydroxylase component of methane monooxygenase (MMO) enzyme, on the basis of the experimentally available structure information of enzyme with divalent zinc ion and the MMO with Fe(2)O(2) core. The reaction mechanism for the hydroxylation of methane and its derivatives catalyzed by Q has been investigated at the B3LYP*/cc-pVTZ, Lanl2tz level in protein solution environment. These hydroxylation reactions proceed via a radical-rebound mechanism, with the rate-determining step of the C-H bond cleavage. This radical-rebound reaction mechanism is analogous to the experimentally available MMOs with diamond Fe(2)O(2) core accompanied by a coordinate number of six for the hydroxylation of methane. The rate constants for the hydroxylation of substrates catalyzed by Q increase along CH(4) < CH(3)F < CH(3)CN ≈ CH(3)NO(2) < CH(3)CH(3). Both the activation strain ΔE(≠)(strain) and the stabilizing interaction ΔE(≠)(int) jointly affect the activation energy ΔE(≠). For the C-H cleavage of substrate CH(3)X, with the decrease of steric shielding for the substituted CH(3)X (X = F > H > CH(3) > NO(2) > CN) attacking the O center in Q, the activation strain ΔE(≠)(strain) decreases, whereas the stabilizing interaction ΔE(≠)(int) increases. It is predicted that the MMO with peroxo dizinc Zn(2)O(2) core should be a promising catalyst for the hydroxylation of methane and its derivatives.

TRNA-modifying MiaE protein from Salmonella typhimurium is a nonheme diiron monooxygenase.

MiaE catalyzes the posttranscriptional allylic hydroxylation of 2-methylthio-N-6-isopentenyl adenosine in tRNAs. The Salmonella typhimurium enzyme was heterologously expressed in Escherichia coli. The purified enzyme is a monomer with two iron atoms and displays activity in in vitro assays. The type and properties of the iron center were investigated by using a combination of UV-visible absorption, EPR, HYSCORE, and Mössbauer spectroscopies which demonstrated that the MiaE enzyme contains a nonheme dinuclear iron cluster, similar to that found in the hydroxylase component of methane monooxygenase. This is the first example of an enzyme from this important class of diiron monooxygenases to be involved in the hydroxylation of a biological macromolecule and the second example of a redox metalloenzyme participating in tRNA modification.

DFT calculations of 57Fe Mössbauer isomer shifts and quadrupole splittings for iron complexes in polar dielectric media: Applications to methane monooxygenase and ribonucleotide reductase

To predict the isomer shifts of Fe complexes in different oxidation and spin states more accurately, we have performed linear regression between the measured isomer shifts (delta(exp)) and DFT (PW91 potential with all-electron triple-zeta plus polarization basis sets) calculated electron densities at Fe nuclei [rho(0)] for the Fe(2+,2.5+) and Fe(2.5+,3+,3.5+,4+) complexes separately. The geometries and electronic structures of all complexes in the training sets are optimized within the conductor like screening (COSMO) solvation model. Based on the linear correlation equation delta(exp) = alpha[rho(0) - 11884.0] + C, the best fitting for 17 Fe(2+,2.5+) complexes (totally 31 Fe sites) yields alpha = -0.405 +/- 0.042 and C = 0.735 +/- 0.047 mm s(-1). The correlation coefficient is r = -0.876 with a standard deviation of SD = 0.075 mm s(-1). In contrast, the linear fitting for 19 Fe(2.5+,3+,3.5+,4+) complexes (totally 30 Fe sites) yields alpha = -0.393 +/- 0.030 and C = 0.435 +/- 0.014 mm s(-1), with the correlation coefficient r = -0.929 and a standard deviation SD = 0.077 mm s(-1). We provide a physical rationale for separating the Fe(2+,2.5+) fit from the Fe(2.5+,3+,3.5+,4+) fit, which also is clearly justified on a statistical empirical basis. Quadrupole splittings have also been calculated for these systems. The correlation between the calculated (DeltaE(Q(cal))) and experimental (DeltaE(Q(exp))) quadrupole splittings based on |DeltaE(Q(exp))| = A |DeltaE(Q(cal))| + B yields slope A, which is almost the ideal value 1.0 (A = 1.002 +/- 0.030) and intercept B almost zero (B = 0.033 +/- 0.068 mm s(-1)). Further calculations on the reduced diferrous and oxidized diferric active sites of class-I ribonucleotide reductase (RNR) and the hydroxylase component of methane monooxygenase (MMOH), and on a mixed-valent [(tpb)Fe3+(mu-O)(mu-CH3CO2)Fe4+(Me3[9]aneN3)]2+ (S = 3/2) complex and its corresponding diferric state have been performed. Calculated results are in very good agreement with the experimental data.

Spin states in polynuclear clusters: The [Fe2O2] core of the methane monooxygenase active site

The ability to provide a correct description of different spin states of mono- and polynuclear transition metal complexes is essential for a detailed investigation of reactions that are catalyzed by such complexes. We study the energetics of different total and local spin states of a dinuclear oxygen-bridged iron(IV) model for the intermediate Q of the hydroxylase component of methane monooxygenase by means of spin-unrestricted Kohn-Sham density functional theory. Because it is known that the spin state total energies depend systematically on the density functional, and that this dependence is intimately connected to the exact exchange admixture of present-day hybdrid functionals, we compare total energies, local and total spin values, and Heisenberg coupling constants calculated with the established functionals BP86 and B3LYP as well as with a modified B3LYP version with an exact exchange admixture ranging from 0 to 24%. It is found that exact exchange enhances local spin polarization. As the exact exchange admixture increases, the high-spin state is energetically favored, although the Broken-Symmetry state always is the ground state. Instead of the strict linear variation of the energy splittings observed for mononuclear complexes, a slightly nonlinear dependence is found. The Heisenberg coupling constants J(Fe1Fe2) --evaluated according to three different proposals from the literature -- are found to vary from -129 to -494 cm(-1) accordingly. The experimental finding that intermediate Q has an antiferromagnetic ground state is thus confirmed.

Cover Picture: Angew. Chem. Int. Ed. 7/2002

The cover picture shows Cu2(μ-O)2 and Fe2(μ-O)2 complexes with the M2(μ-O)2 diamond core motif (the core is shown bottom right, M=green and oxygen=red spheres) and a representative example of a non-heme multimetal enzyme (hydroxylase component of methane monooxygenase, in the background). Although quite a familiar feature in high-valent manganese chemistry, the M2(μ-O)2 diamond core motif has only recently been found in synthetic complexes for M=Cu or Fe. Despite differences in electronic structures that have been revealed through experimental and theoretical studies, Cu2(μ-O)2 and Fe2(μ-O)2 cores exhibit analogously covalent metal–oxo bonding, and similar tendencies to abstract hydrogen atoms from substrates. Our understanding of biocatalysis has been enhanced significantly through the isolation and comprehensive characterization of the Cu2(μ-O)2 and Fe2(μ-O)2 complexes. In particular, it has led to the development of new mechanistic notions about how non-heme multimetal enzymes, such as, methane monooxygenase, fatty acid desaturase, and tyrosinase, may function in the activation of dioxygen to catalyze a diverse array of organic transformations. To find out more see the review by L. Que, Jr. and W. B. Tolman on p.1114 ff.

Exploring the molecular nature of alternative oxidase regulation and catalysis

Plant mitochondria contain a non-protonmotive alternative oxidase (AOX) that couples the oxidation of ubiquinol to the complete reduction of oxygen to water. In this paper we review theoretical and experimental studies that have contributed to our current structural and mechanistic understanding of the oxidase and to the clarification of the molecular nature of post-translational regulatory phenomena. Furthermore, we suggest a catalytic cycle for AOX that involves at least one transient protein-derived radical. The model is based on the reviewed information and on recent insights into the mechanisms of cytochrome c oxidase and the hydroxylase component of methane monooxygenase.

Density functional studies of oxidized and reduced methane monooxygenase. Optimized geometries and exchange coupling of active site clusters.

The conflicting protein crystallography data for the oxidized form (MMOH(ox)) of methane monooxygenase present a dilemma regarding the identity of the solvent-derived bridging ligands within the active site: do they comprise a diiron unit bridged by 1H2O and 1OH(-) as postulated for Methylococcus capsulatus or 2OH(-) ligands as suggested for Methylosinus trichosporium? Using models derived explicitly from the M. capsulatus and M. trichosporium protein data, spin-unrestricted density functional methods have been used to study two structurally characterized forms of the hydroxylase component of methane monooxygenase. The active site geometries of the oxidized (MMOH(ox)) and two-electron-reduced (MMOH(red)) states have been geometry optimized using several quantum cluster models which take into account the antiferromagnetic (AF) and ferromagnetic (F) coupling of electron spins. Trends in cluster geometries, energetics, and Heisenberg J values have been evaluated. For the majority of models, calculated geometries are in good agreement with the X-ray analyses and appear relatively insensitive to the F or AF alignment of electron spins on adjacent Fe sites. Discrepancies between calculation and experiment appear in the orientation of the coordinated His and Glu amino acid side chains for both MMOH(ox) and MMOH(red) and also in unexpected intramolecular proton transfer in the MMOH(ox) cluster models. There is additional dispersion between (and among) calculated and experimental Fe(3+)-OH(-) distances with relevance to the correct protonation state of the solvent-derived ligands. In an accompanying paper (Lovell, T.; Li, J.; Noodleman, L. Inorg. Chem. 2001, 40, 5267), a comparison of the related energetics of the active site models examined herein is further evaluated in the full protein and solvent environment.

The Flexibility of Carboxylate Ligands in Methane Monooxygenase and Ribonucleotide Reductase: A Density Functional Study

Available experimental data for the active sites of the hydroxylase component of methane monooxygenase (MMOH) and the R2 subunit of ribonucleotide reductase (R2) indicates high flexibility of the ligand environment of the iron centers in these two metalloproteins, suggesting that carboxylate ligands may play a special role for proper enzymatic functioning. By using quantum chemical methods, here we have investigated (1) the so-called 1,2-carboxylate shift (i.e., shift of a bridging carboxylate ligand from μ-1,1 to μ-1,2 between two metal centers), and (2) the monodentate ↔ bidentate rearrangement of terminal carboxylate ligands (bound to only one metal center), in the reduced forms of MMOH and R2. Our results show that (i) MMOH-like and R2-like structures, with a μ-1,1 and μ-1,2 bridged carboxylate ligand, respectively, are energetically very close; (ii) complexes with lower coordination numbers in the Fe2 center are computed to be slightly more stable than those with higher coordination numbers, and (iii...

Oxygen activation catalyzed by methane monooxygenase hydroxylase component: proton delivery during the O-O bond cleavage steps.

The effects of solvent pH and deuteration on the transient kinetics of the key intermediates of the dioxygen activation process catalyzed by the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been studied. MMO consists of hydroxylase (MMOH), reductase, and "B" (MMOB) components. MMOH contains a carboxylate- and oxygen-bridged binuclear iron cluster that catalyzes O2 activation and insertion chemistry. The diferrous MMOH-MMOB complex reacts with O2 to form a diferrous intermediate compound O (O) and subsequently a diferric intermediate compound P (P), presumed to be a peroxy adduct. The O decay reaction was found to be pH-independent within error at 4 degrees C (kobs = 22 +/- 2 s-1 at pH 7.7; kobs = 26 +/- 2 s-1 at pH 7.0). In contrast, the P formation rate was found to decrease sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kP = 9.1 +/- 0.9 s-1 achieved near pH 6.5. The formation of P was slower than the disappearance of O, indicating that at least one other undetected intermediate (P) must form in between. P decays spontaneously to the highly chromophoric intermediate, compound Q (Q). The decay rate of P matched the formation rate of Q, and both rates decreased sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kQ = 2.6 +/- 0.1 s-1 achieved near pH 6.5. No pH dependence was observed for the decay of Q. The formation and decay rates of P and the formation rate of Q decreased linearly with mole fraction of D2O in the reaction mixture. Kinetic solvent isotope effect values of kH/kD = 1.3 +/- 0.1 (P formation) and kH/kD = 1.4 +/- 0.1 (P decay and Q formation) were observed at 5 degrees C. The linearity of the proton inventory plots suggests that only a single proton is transferred in the transition state of the formation reaction for each intermediate. If these protons are transferred to the bound oxygen molecule, as formally required by the reaction stoichiometry, the data are consistent with a model in which water is formed concurrently with the formation of the reactive bis mu-oxo-binuclear Fe(IV) species, Q.

Mössbauer Evidence for Antisymmetric Exchange in a Diferric Synthetic Complex and Diferric Methane Monooxygenase

The dinuclear iron cluster of the oxidized hydroxylase component of methane monooxygenase (MMOH) contains two antiferromagnetically coupled high-spin ferric ions (ℋ = JSA·SB, SA = SB = 5/2, J = 15 ...

Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.

Ligand Field Circular Dichroism and Magnetic Circular Dichroism Studies of Component B and Substrate Binding to the Hydroxylase Component of Methane Monooxygenase

The soluble methane monooxygenase system (MMO), consisting of a hydroxylase (MMOH), a reductase, and component B (MMOB), catalyzes the NADH and O2 dependent hydroxylation of methane and many other hydrocarbons. The binuclear non-heme ferrous active site cluster of the hydroxylase−component B (MMOH−MMOB) complex in the presence of substrate and small molecules has been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. CD studies reveal that addition of the alternative substrate, trans-1,2-dichloroethylene, or inhibitor, tetrachloroethylene, induces a conformational change in the active site pocket only in the presence of MMOB. Complementary MCD data indicate that this conformational change does not result in a direct change in the ligation of the iron atoms. Comparison of the CD/MCD data with the crystal structure of MMOH allows a tentative correlation between the perturbations observed and the iron atoms affected. The binding of MMOB to MMOH distorts the ligand fi...

Carboxylate-Bridged Diiron(II) Complexes: Synthesis, Characterization, and O2-Reactivity of Models for the Reduced Diiron Centers in Methane Monooxygenase and Ribonucleotide Reductase

Diiron(II) complexes with an oxygen-rich coordination environment were assembled with the dinucleating dicarboxylate ligands m-xylylenediamine bis(Kemp's triacid)imide (H2XDK) and the more soluble analogue m-xylylenediamine bis(propyl Kemp's triacid)imide (H2PXDK). X-ray crystallographic analysis revealed that, in most of the complexes, only one monodentate N-donor ligand is bound to each iron(II) ion. In addition to XDK and PXDK, a variety of other ligands bridge the dimetallic core including chloride, fluoride, triflate, or carboxylate. The tris(carboxylate-bridged) complexes [Fe2(μ-XDK)(μ-O2CPh)(ImH)2(O2CPh)(MeOH)] (3) and [Fe2(μ-O2CC(CH3)3)(μ-PXDK)(N-MeIm)2(O2CC(CH3)3)] (4) have the same ligand composition as the diiron(II) cores in the hydroxylase component of methane monooxygenase (MMO) and in the R2 protein of ribonucleotide reductase (RNR). The coordination environments of the two iron centers in 3 and 4 are inequivalent, with one iron being 6-coordinate and the other being 4-coordinate. The bridging benzoate and pivalate ligands have an unusual coordination mode with a Fe−O−C bond angle close to 180°. Fits of magnetic susceptibility data collected for several of the complexes between 300 and 4 K indicated that the two iron(II) centers are weakly antiferromagnetically coupled with a coupling constant which does not depend on the nature of the bridging ligands. Mössbauer spectra of polycrystalline and frozen THF solution samples of 4 exhibited two overlapping doublets of equal intensity, reflecting the different coordination environments of the two iron(II) ions. The nearly identical Mössbauer parameters for the solid and frozen solution samples indicate that the dinuclear core remains intact upon dissolution. Stopped-flow studies of the reaction of 3 and 4 with O2 in THF showed the rapid formation (kψ ≈ 74 s-1 for 3 at 202.5 K and kψ ≈ 300 s-1 for 4 at 197 K) of colored intermediates with broad absorption maxima near 660 and 670 nm, respectively. These values are characteristic of peroxo-to-iron charge-transfer bands and similar to that observed for the (μ-peroxo)diiron(III) intermediate (Hperoxo) in the MMO reaction cycle.

X-ray absorption spectroscopic studies of the methane monooxygenase hydroxylase component from Methylosinus trichosporium OB3b

The conversion from methane to methanol is catalyzed by methane monooxygenase (MMO) in methanotrophic bacteria. Earlier work on the crystal structures of the MMO hydroxylase component (MMOH) from Methylococcus capsulatus (Bath) at 4 °C and –160 °C has revealed two different core arrangements for the diiron active site. To ascertain the generality of these results, we have now carried out the first structural characterization on MMOH from Methylosinustrichosporium OB3b. Our X-ray absorption spectroscopic (XAS) analysis suggests the presence of two Fe-Fe distances of about 3 A and 3.4 A, which are proposed to reflect two populations of MMOH molecules with either a bis(μ-hydroxo)(μ-carboxylato)- or a (μ-hydroxo)(μ-carboxylato)diiron(III) core structure, respectively. The observation of these two different core structures, together with the crystallographic results of the MMOH from Methylococcus capsulatus (Bath), suggests the presence of an equilibrium that may reflect a core flexibility that is required to accommodate the various intermediates in the catalytic cycle of the enzyme. XAS studies on the binding of component B (MMOB) to the hydroxylase component show that MMOB does not perturb either this equilibrium or the gross structure of the oxidized diiron site in MMOH.

Gating Effects of Component B on Oxygen Activation by the Methane Monooxygenase Hydroxylase Component

Component B (MMOB) of the soluble methane monooxygenase (MMO) system accelerates the initial velocity of methane oxidation by up to 150-fold by an unknown mechanism. The active site of MMO contains a diferric, hydroxo-bridged diiron cluster located on the hydroxylase component (MMOH). This cluster is reduced by the NAD(P)H-coupled reductase component to the diferrous state, which then reacts with O2 to yield two reaction cycle intermediates sequentially termed compounds P and Q. The rate of compound P formation is shown here to be independent of O2 concentration, suggesting that an MMOH-O2 complex (compound O) is (congruent to irreversibly) formed before compound P. Compound Q is capable of reacting with hydrocarbons to yield the MMOH-product complex, compound T. It is shown here that MMOB accelerates catalysis by increasing congruent to 1000-fold the rate of O2 association and reaction with diferrous MMOH leading to compound P. Modeling of the single turnover reaction in the presence of substoichiometric MMOB suggests that MMOB also accelerates the compound P to Q conversion by congruent to 40-fold. Due to this O2-gating effect of MMOB, either compound Q or T becomes the dominant species during turnover, depending upon the substrate concentration and type. Because these are the species that either react with substrate (Q) or release product (T), their buildup maximizes the turnover rate. This is the first direct role in catalysis to be recognized for MMOB and represents a novel method for oxygenase regulation.

X-ray Absorption Spectroscopic Studies of the Diiron Center in Methane Monooxygenase in the Presence of Substrate and the Coupling Protein of the Enzyme System

The interaction among the hydroxylase component of methane monooxygenase (MMO) from Methylococcus capsulatus (Bath), the coupling protein of the MMO enzyme system (component B), and substrate has been investigated by using Fe K-edge X-ray absorption spectroscopy (XAS). Fe K-edge extended X-ray absorption fine structure (EXAFS) studies of the semimet form of the hydroxylase in the presence of the coupling protein, 1-bromo-1-propene, and both the coupling protein and 1-bromo-1-propene revealed small differences in the appearance of the EXAFS above k = 8 {Angstrom}{sup {minus}1} as compared to the noncomplexed hydroxylase. No dramatic change in the Fe coordination was seen in fits to the data. The average first shell Fe-O/N distance for the complexed forms of the semimet hydroxylase ranged between 2.06 and 2.08 {Angstrom}, which is comparable to the distance found for the noncomplexed form, 2.06-2.09 {Angstrom}. Although the average first shell coordination was similar for all samples, a difference was seen in the distribution of long vs short distance contributions to the first shell coordination sphere for samples with component B present. This difference was accompanied by a small but consistent decrease in the Fe-Fe distance of the B-complexed hydroxylase samples, from 3.42 to 3.39 {angstrom}.

Spectroscopic studies of the coupled binuclear non-heme iron active site in the fully reduced hydroxylase component of methane monooxygenase: comparison to deoxy and deoxy-azide hemerythrin. [Erratum to document cited in CA120:3498

Spectroscopic studies of the coupled binuclear non-heme iron active site in the fully reduced hydroxylase component of methane monooxygenase: comparison to deoxy and deoxy-azide hemerythrin. [Erratum to document cited in CA120:3498

Oxidation-reduction potentials of the methane monooxygenase hydroxylase component from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB) protein components. MMOH contains two oxygen-bridged dinuclear iron clusters that are the sites of O2 activation and hydrocarbon oxidation. Each cluster can be stabilized in diferric [Fe(III).Fc(III)], mixed-valence [Fe(II).Fe(III)], and diferrous [Fe(II).Fe(II)] redox states. We have correlated the EPR spin quantitation of the S = 1/2 mixed-valence state with the system electrode potential to determine both formal redox potential values for MMOH at 4 degrees C: E1 degrees' = +76 +/- 15 mV and E2 degrees' = +21 +/- 15 mV (Em = +48 mV, 61% maximum mixed-valence state). Complementary Mössbauer studies of 57Fe-enriched MMOH allowed all three redox states to be quantitated simultaneously in individual samples and revealed that the distribution of redox states was in accord with the measured potential values. EPR spectra of partially reduced MMOH showed that the apparent midpoint potential values of MMOH-MMOR, MMOH-MMOR-MMOB, and MMOH-MMOR-MMOB-substrate complexes were slightly more positive than that of MMOH alone. In contrast, the MMOH-MMOB complex appeared to have a substantially more negative redox potential. The formal redox potential values of the latter complex were determined to be E1 degrees' = -52 +/- 15 mV and E2 degrees' = -115 +/- 15 mV, respectively, at 4 degrees C (Em = -84 mV, 65% maximum mixed-valence state). This negative 132-mV shift in the midpoint potential of MMOH coupled to MMOB binding suggests that MMOB binds approximately 10(4) more strongly to the diferric state of MMOH than to the diferrous state. Since the potential shift is strongly negative, and since a nearly constant separation between the two formal potential values of MMOH is maintained when MMOB binds, the role of the MMOB-MMOH complex must not be to thermodynamically stabilize the formation of the diferrous cluster which is the form that reacts with O2 during catalysis. However, MMOB binding may provide kinetic stabilization of the diferrous state and/or modulation of the interaction of MMOH with O2 and hydrocarbon substrates. Such roles may be effected through cyclic association and dissociation of the MMOB-MMOH complex as MMOH oscillates between redox states during catalysis, thereby dynamically altering the affinity of this complex.

Spectroscopic studies of the coupled binuclear non-heme iron active site in the fully reduced hydroxylase component of methane monooxygenase: comparison to deoxy and deoxy-azide hemerythrin

A combination of circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies has been used to probe the geometric and electronic structure of the binuclear Fe(II) active site of the reduced hydroxylase component of methane monooxygenase (MMOH). Excited-state data provide the numbers and energies of d→d transitions which are interpreted in terms of ligand field calculations to estimate the geometry of each iron. Variable-temperature variable-field (VTVH) MCD data are analyzed by using a non-Kramers doublet model to obtain the zero field splitting (ZFS) and g∥ value of the ground state and the excited sublevel energies. These results are further interpreted in terms of a spin Hamiltonian which includes the ZFS of each Fe 2+ combined with the exchange coupling between iron centers