Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction-EPR/ENDOR spectroscopy

EPR/ENDOR studies have been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by gamma-irradiation at 77 K in the absence of substrate and in the presence of a variety of substrates including its native hydroxylation substrate, camphor (a), and the alternate substrates, 5-methylenyl-camphor (b), 5,5-difluorocamphor (c), norcamphor (d), and adamantanone (e); the equivalent experiments have been performed on the T252A mutant complexed with a and b. The present study shows that the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate. This includes alterations in the properties of the heme center itself (g tensor; (14)N, (1)H, hyperfine couplings). It also includes dramatic changes in reactivity: the presence of any substrate increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold. Among the substrates, b stands out as having an exceptionally strong influence on the properties and reactivity of the P450cam intermediates, especially in the T252A mutant. The intermediate, 2(T252A)-b, does not lose H(2)O(2), as occurs with 2(T252A)-a, but decays with formation of the epoxide of b. Thus, these observations show that substrate can modulate the properties of both the monoxygenase active-oxygen intermediates and the proton-delivery network that encompasses them.     

On the functional role of a water molecule in clade 3 catalases: a proposal for the mechanism by which NADPH prevents the formation of compound II

X-ray structures of the 13 different monofunctional heme catalases published to date were scrutinized in order to gain insight in the mechanism by which NADPH in Clade 3 catalases may protect the reactive ferryloxo intermediate Compound I (Cpd I; por (*+)Fe (IV)O) against deactivation to the catalytically inactive intermediate Compound II (Cpd II; porFe (IV)O). Striking similarities in the molecular network of the protein subunits encompassing the heme center and the surface-bound NADPH were found for all of the Clade 3 catalases. Unique features in this region are the presence of a water molecule (W1) adjacent to the 4-vinyl group of heme and a serine residue or a second water molecule hydrogen-bonded to both W1 and the carbonyl group of a threonine-proline linkage, with the proline in van der Waals contact with the dihydronicotinamide group of NADPH. A mechanism is proposed in which a hydroxyl anion released from W1 undergoes reversible nucleophilic addition to the terminal carbon of the 4-vinyl group of Cpd I, thereby producing a neutral porphyrin pi-radical ferryloxo (HO-por (*)Fe (IV)O) species of reduced reactivity. This structure is suggested to be the elusive Cpd II' intermediate proposed in previous studies. An accompanying proton-shifting process along the hydrogen-bonded network is believed to facilitate the NADPH-mediated reduction of Cpd I to ferricatalase and to serve as a funnel for electron transfer from NADPH to the heme center to restore the catalase Fe (III) resting state. The proposed reaction paths were fully supported as chemically reasonable and energetically feasible by means of density functional theory calculations at the (U)B3LYP/6-31G* level. A particularly attractive feature of the present mechanism is that the previously discussed formation of protein-derived radicals is avoided.     

Substrate-triggered activation of a synthetic [Fe2(μ-O)2] diamond core for C-H bond cleavage

An [Fe(IV)(2)(μ-O)(2)] diamond core structure has been postulated for intermediate Q of soluble methane monooxygenase (sMMO-Q), the oxidant responsible for cleaving the strong C-H bond of methane and its hydroxylation. By extension, analogous species may be involved in the mechanisms of related diiron hydroxylases and desaturases. Because of the paucity of well-defined synthetic examples, there are few, if any, mechanistic studies on the oxidation of hydrocarbon substrates by complexes with high-valent [Fe(2)(μ-O)(2)] cores. We report here that water or alcohol substrates can activate synthetic [Fe(III)Fe(IV)(μ-O)(2)] complexes supported by tetradentate tris(pyridyl-2-methyl)amine ligands (1 and 2) by several orders of magnitude for C-H bond oxidation. On the basis of detailed kinetic studies, it is postulated that the activation results from Lewis base attack on the [Fe(III)Fe(IV)(μ-O)(2)] core, resulting in the formation of a more reactive species with a [X-Fe(III)-O-Fe(IV)═O] ring-opened structure (1-X, 2-X, X = OH(-) or OR(-)). Treatment of 2 with methoxide at -80 °C forms the 2-methoxide adduct in high yield, which is characterized by an S = 1/2 EPR signal indicative of an antiferromagnetically coupled [S = 5/2 Fe(III)/S = 2 Fe(IV)] pair. Even at this low temperature, the complex undergoes facile intramolecular C-H bond cleavage to generate formaldehyde, showing that the terminal high-spin Fe(IV)═O unit is capable of oxidizing a C-H bond as strong as 96 kcal mol(-1). This intramolecular oxidation of the methoxide ligand can in fact be competitive with intermolecular oxidation of triphenylmethane, which has a much weaker C-H bond (D(C-H) 81 kcal mol(-1)). The activation of the [Fe(III)Fe(IV)(μ-O)(2)] core is dramatically illustrated by the oxidation of 9,10-dihydroanthracene by 2-methoxide, which has a second-order rate constant that is 3.6 × 10(7)-fold larger than that for the parent diamond core complex 2. These observations provide strong support for the DFT-based notion that an S = 2 Fe(IV)═O unit is much more reactive at H-atom abstraction than its S = 1 counterpart and suggest that core isomerization could be a viable strategy for the [Fe(IV)(2)(μ-O)(2)] diamond core of sMMO-Q to selectively attack the strong C-H bond of methane in the presence of weaker C-H bonds of amino acid residues that define the diiron active site pocket.     

DFT Calculations Suggest a New Type of Self-Protection and Self-Inhibition Mechanism in the Mammalian Heme Enzyme Myeloperoxidase: Nucleophilic Addition of a Functional Water rather than One-Electron Reduction

The mammalian heme enzyme myeloperoxidase (MPO) catalyzes the reaction of Cl(-) to the antimicrobial-effective molecule HOCl. During the catalytic cycle, a reactive intermediate "Compound?I" (Cpd?I) is generated. Cpd?I has the ability to destroy the enzyme. Indeed, in the absence of any substrate, Cpd?I decays with a half-life of 100?ms to an intermediate called Compound?II (Cpd?II), which is typically the one-electron reduced Cpd?I. However, the nature of Cpd?II, its spectroscopic properties, and the source of the additional electron are only poorly understood. On the basis of DFT and time-dependent (TD)-DFT quantum chemical calculations at the PBE0/6-31G* level, we propose an extended mechanism involving a new intermediate, which allows MPO to protect itself from self-oxidation or self-destruction during the catalytic cycle. Because of its similarity in electronic structure to Cpd?II, we named this intermediate Cpd?II'. However, the suggested mechanism and our proposed functional structure of Cpd?II' are based on the hypothesis that the heme is reduced by charge separation caused by reaction with a water molecule, and not, as is normally assumed, by the transfer of an electron. In the course of this investigation, we found a second intermediate, the reduced enzyme, towards which the new mechanism is equally transferable. In analogy to Cpd?II', we named it Fe(II') . The proposed new intermediates Cpd?II' and Fe(II') allow the experimental findings, which have been well documented in the literature for decades but not so far understood, to be explained for the first time. These encompass a)?the spontaneous decay of Cpd?I, b)?the unusual (chlorin-like) UV/Vis, circular dichroism (CD), and resonance Raman spectra, c)?the inability of reduced MPO to bind CO, d)?the fact that MPO-Cpd?II reduces SCN(-) but not Cl(-) , and e)?the experimentally observed auto-oxidation/auto-reduction features of the enzyme. Our new mechanism is also transferable to cytochromes, and could well be viable for heme enzymes in general.     

Role of Fe(IV)-oxo intermediates in stoichiometric and catalytic oxidations mediated by iron pyridine-azamacrocycles

An iron(II) complex with a pyridine-containing 14-membered macrocyclic (PyMAC) ligand L1 (L1 = 2,7,12-trimethyl-3,7,11,17-tetra-azabicyclo[11.3.1]heptadeca-1(17),13,15-triene), 1, was prepared and characterized. Complex 1 contains low-spin iron(II) in a pseudo-octahedral geometry as determined by X-ray crystallography. Magnetic susceptibility measurements (298 K, Evans method) and M?ssbauer spectroscopy (90 K, δ = 0.50(2) mm/s, ΔE(Q) = 0.78(2) mm/s) confirmed that the low-spin configuration of Fe(II) is retained in liquid and frozen acetonitrile solutions. Cyclic voltammetry revealed a reversible one-electron oxidation/reduction of the iron center in 1, with E(1/2)(Fe(III)/Fe(II)) = 0.49 V vs Fc(+)/Fc, a value very similar to the half-wave potentials of related macrocyclic complexes. Complex 1 catalyzed the epoxidation of cyclooctene and other olefins with H(2)O(2). Low-temperature stopped-flow kinetic studies demonstrated the formation of an iron(IV)-oxo intermediate in the reaction of 1 with H(2)O(2) and concomitant partial ligand oxidation. A soluble iodine(V) oxidant, isopropyl 2-iodoxybenzoate, was found to be an excellent oxygen atom donor for generating Fe(IV)-oxo intermediates for additional spectroscopic (UV-vis in CH(3)CN: λ(max) = 705 nm, ε ≈ 240 M(-1) cm(-1); M?ssbauer: δ = 0.03(2) mm/s, ΔE(Q) = 2.00(2) mm/s) and kinetic studies. The electrophilic character of the (L1)Fe(IV)═O intermediate was established in rapid (k(2) = 26.5 M(-1) s(-1) for oxidation of PPh(3) at 0 °C), associative (ΔH(?) = 53 kJ/mol, ΔS(?) = -25 J/K mol) oxidation of substituted triarylphosphines (electron-donating substituents increased the reaction rate, with a negative value of Hammet's parameter ρ = -1.05). Similar double-mixing kinetic experiments demonstrated somewhat slower (k(2) = 0.17 M(-1) s(-1) at 0 °C), clean, second-order oxidation of cyclooctene into epoxide with preformed (L1)Fe(IV)═O that could be generated from (L1)Fe(II) and H(2)O(2) or isopropyl 2-iodoxybenzoate. Independently determined rates of ferryl(IV) formation and its subsequent reaction with cyclooctene confirmed that the Fe(IV)-oxo species, (L1)Fe(IV)═O, is a kinetically competent intermediate for cyclooctene epoxidation with H(2)O(2) at room temperature. Partial ligand oxidation of (L1)Fe(IV)═O occurs over time in oxidative media, reducing the oxidizing ability of the ferryl species; the macrocyclic nature of the ligand is retained, resulting in ferryl(IV) complexes with Schiff base PyMACs. NH-groups of the PyMAC ligand assist the oxygen atom transfer from ferryl(IV) intermediates to olefin substrates.     

Functional mimic of dioxygen-activating centers in non-heme diiron enzymes: mechanistic implications of paramagnetic intermediates in the reactions between diiron(II) complexes and dioxygen

Two tetracarboxylate diiron(II) complexes, [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(C(5)H(5)N)(2)] (1a) and [Fe(2)(mu-O(2)CAr(Tol))(4)(4-(t)BuC(5)H(4)N)(2)] (2a), where Ar(Tol)CO(2)(-) = 2,6-di(p-tolyl)benzoate, react with O(2) in CH(2)Cl(2) at -78 degrees C to afford dark green intermediates 1b (lambda(max) congruent with 660 nm; epsilon = 1600 M(-1) cm(-1)) and 2b (lambda(max) congruent with 670 nm; epsilon = 1700 M(-1) cm(-1)), respectively. Upon warming to room temperature, the solutions turn yellow, ultimately converting to isolable diiron(III) compounds [Fe(2)(mu-OH)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (L = C(5)H(5)N (1c), 4-(t)BuC(5)H(4)N (2c)). EPR and M?ssbauer spectroscopic studies revealed the presence of equimolar amounts of valence-delocalized Fe(II)Fe(III) and valence-trapped Fe(III)Fe(IV) species as major components of solution 2b. The spectroscopic and reactivity properties of the Fe(III)Fe(IV) species are similar to those of the intermediate X in the RNR-R2 catalytic cycle. EPR kinetic studies revealed that the processes leading to the formation of these two distinctive paramagnetic components are coupled to one another. A mechanism for this reaction is proposed and compared with those of other synthetic and biological systems, in which electron transfer occurs from a low-valent starting material to putative high-valent dioxygen adduct(s).     

EPR, 1H and 2H NMR, and reactivity studies of the iron-oxygen intermediates in bioinspired catalyst systems

Complexes [(BPMEN)Fe(II)(CH(3)CN)(2)](ClO(4))(2) (1, BPMEN = N,N'-dimethyl-N,N'-bis(2-pyridylmethyl)-1,2-diaminoethane) and [(TPA)Fe(II)(CH(3)CN)(2)](ClO(4))(2) (2, TPA = tris(2-pyridylmethyl)amine) are among the best nonheme iron-based catalysts for bioinspired oxidation of hydrocarbons. Using EPR and (1)H and (2)H NMR spectroscopy, the iron-oxygen intermediates formed in the catalyst systems 1,2/H(2)O(2); 1,2/H(2)O(2)/CH(3)COOH; 1,2/CH(3)CO(3)H; 1,2/m-CPBA; 1,2/PhIO; 1,2/(t)BuOOH; and 1,2/(t)BuOOH/CH(3)COOH have been studied (m-CPBA is m-chloroperbenzoic acid). The following intermediates have been observed: [(L)Fe(III)(OOR)(S)](2+), [(L)Fe(IV)═O(S)](2+) (L = BPMEN or TPA, R = H or (t)Bu, S = CH(3)CN or H(2)O), and the iron-oxygen species 1c (L = BPMEN) and 2c (L = TPA). It has been shown that 1c and 2c directly react with cyclohexene to yield cyclohexene oxide, whereas [(L)Fe(IV)═O(S)](2+) react with cyclohexene to yield mainly products of allylic oxidation. [(L)Fe(III)(OOR)(S)](2+) are inert in this reaction. The analysis of EPR and reactivity data shows that only those catalyst systems which display EPR spectra of 1c and 2c are able to selectively epoxidize cyclohexene, thus bearing strong evidence in favor of the key role of 1c and 2c in selective epoxidation. 1c and 2c were tentatively assigned to the oxoiron(V) intermediates.     

Gauging the relative oxidative powers of compound I, ferric-hydroperoxide, and the ferric-hydrogen peroxide species of cytochrome P450 toward C-H hydroxylation of a radical clock substrate

Density functional calculations were performed in response to the controversies regarding the identity of the oxidant species in cytochrome P450. The calculations were used to gauge the relative C-H hydroxylation reactivity of three potential oxidant species of the enzyme, the high-valent oxo-iron species Compound I (Cpd I), the ferric hydroperoxide Compound 0 (Cpd 0), and the ferric-hydrogen peroxide complex Fe(H(2)O(2)). The results for the hydroxylation of a radical probe substrate, 1, show the following trends: (a) Cpd I is the most reactive species; in its presence the other two reagents will be silent. (b) In the absence of Cpd I, substrate oxidation by Cpd 0 and Fe(H(2)O(2)) will take place via a stepwise mechanism that involves initial O-O homolysis followed by H-abstraction from 1. (c) Cpd 0 will undergo mostly porphyrin hydroxylation and only approximately 15% of substrate oxidation producing mostly the rearranged alcohol, 3 (Scheme 2). (d) Fe(H(2)O(2)) will generate mostly free hydrogen peroxide (uncoupling). A small fraction will perform substrate oxidation and lead mostly to 3. Reactivity probes for these reagents are kinetic isotope effect (KIE) and the product ratio of unrearranged to rearranged alcohols, [2/3]. Thus, for substrate oxidation by Cpd 0 or Fe(H(2)O(2)) KIE will be small, approximately 2, while Cpd I will have large KIE values. Typically both Cpd 0 and Fe(H(2)O(2)) will lead to a [2/3] ratio < 1, while Cpd I will lead to ratios > 1. In addition, the product isotope effect (KIE(2)/KIE(3) not equal 1) is expected from the reactivity of Cpd I.     

ENDOR studies of the ligation and structure of the non-heme iron site in ACC oxidase

Ethylene is a plant hormone involved in all stages of growth and development, including regulation of germination, responses to environmental stress, and fruit ripening. The final step in ethylene biosynthesis, oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) to yield ethylene, is catalyzed by ACC oxidase (ACCO). In a previous EPR and ENDOR study of the EPR-active Fe(II)-nitrosyl, [FeNO],(7) complex of ACCO, we demonstrated that both the amino and the carboxyl moieties of the inhibitor d,l-alanine, and the substrate ACC by analogy, coordinate to the Fe(II) ion in the Fe(II)-NO-ACC ternary complex. In this report, we use 35 GHz pulsed and CW ENDOR spectroscopy to examine the coordination of Fe by ACCO in more detail. ENDOR data for selectively (15)N-labeled derivatives of substrate-free ACCO-NO (E-NO) and substrate/inhibitor-bound ACCO-NO (E-NO-S) have identified two histidines as protein-derived ligands to Fe; (1,2)H and (17)O ENDOR of samples in D(2)O and H(2)(17)O solvent have confirmed the presence of water in the substrate-free Fe(II) coordination sphere (E-NO). Analysis of orientation-selective (14,15)N and (17)O ENDOR data is interpreted in terms of a structural model of the ACCO active site, both in the presence (E-NO-S) and in the absence (E-NO) of substrate. Evidence is also given that substrate binding dictates the orientation of bound O(2).     

Heme compound II models in chemoselectivity and disproportionation reactions

Heme compound II models bearing electron-deficient and -rich porphyrins, [Fe^IV(O)(TPFPP)(Cl)]⁻ (1a) and [Fe^IV(O)(TMP)(Cl)]⁻ (2a), respectively, are synthesized, spectroscopically characterized, and investigated in chemoselectivity and disproportionation reactions using cyclohexene as a mechanistic probe. Interestingly, cyclohexene oxidation by 1a occurs at the allylic C–H bonds with a high kinetic isotope effect (KIE) of 41, yielding 2-cyclohexen-1-ol product; this chemoselectivity is the same as that of nonheme iron(iv)-oxo intermediates. In contrast, as observed in heme compound I models, 2a yields cyclohexene oxide product with a KIE of 1, demonstrating a preference for C C epoxidation. The latter result is interpreted as 2a disproportionating to form [Fe^IV(O)(TMP⁺˙)]⁺ (2b) and Fe^III(OH)(TMP), and 2b becoming the active oxidant to conduct the cyclohexene epoxidation. In contrast to 2a, 1a does not disproportionate under the present reaction conditions. DFT calculations confirm that compound II models prefer C–H bond hydroxylation and that disproportionation of compound II models is controlled thermodynamically by the porphyrin ligands. Other aspects, such as acid and base effects on the disproportionation of compound II models, have been discussed as well.

Disproportionation of Cpd II models depends on the electron-richness of the porphyrin ligand; Cpd II with an electron-deficient ligand is difficult to disproportionate, whereas Cpd II with an electron-rich ligand readily disproportionates to form Cpd I as a true oxidant.     

Cryoreduction EPR and 13C, 19F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant

We recently used cryoreduction EPR/ENDOR techniques to show that a substrate can modulate the properties of both the monooxygenase active-oxygen intermediates and of the proton-delivery network which encompasses them. In the present report we use Q-band pulsed 19F ENDOR (Mims 3-pulse sequence) to examine the substrate binding geometries of camphor, through use of the 5,5'--difluorocamphor, and 13C ENDOR to examine the binding of 5-methylenyl camphor labeled with 13C at C11. These probes are examined in multiple states of the catalytic cycle of P450cam and its T252A mutant. As part of this investigation we further report a new cryoreduction reaction, the reduction of a ferroheme to the EPR-visible Fe(I) state, and use it to probe the substrate binding to the EPR-silent ferroheme state. Finally we report the solvent kinetic isotope effect on the decay of the camphor complex of the hydroperoxo-ferric intermediate, the first such measurement on an individual step within the P450cam reaction cycle. Following reduction of oxyferrous-P450cam, this step is the rate-limiting step in camphor hydroxylation, and its solv-KIE of 1.8 at 190 K establishes that it involves activation of the hydroperoxo moiety by transfer of the 'second' proton of catalysis. We suggest that the finding that the heme pocket can exist in multiple substates, including multiple substrate binding locations, even in P450cam, along with the established possibility that the hydroperoxo-ferriheme intermediate can react with substrate, may explain the formation of multiple products by P450s.     

Pulsed ENDOR determination of relative orientation of g-frame and molecular frame of imidazole-coordinated heme center of iNOS

Mammalian nitric oxide synthase (NOS) is a flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide. Information about the relative alignment of the heme and FMN domains of NOS is important for understanding the electron transfer between the heme and FMN centers, but no crystal structure data for NOS holoenzyme are available. In our previous work [Astashkin, A. V.; Elmore, B. O.; Fan, W.; Guillemette, J. G.; Feng, C. J. Am. Chem. Soc. 2010, 132, 12059-12067], the distance between the imidazole-coordinated low-spin Fe(III) heme and FMN semiquinone in a human inducible NOS (iNOS) oxygenase/FMN construct has been determined by pulsed electron paramagnetic resonance (EPR). The orientation of the Fe-FMN radius vector, R(Fe-FMN), with respect to the heme g-frame was also determined. In the present study, pulsed electron-nuclear double resonance (ENDOR) investigation of the deuterons at carbons C2 and C5 in the deuterated coordinated imidazole was used to determine the relative orientation of the heme g-frame and molecular frame, from which R(Fe-FMN) can be referenced to the heme molecular frame. Numerical simulations of the ENDOR spectra showed that the g-factor axis corresponding to the low-field EPR turning point is perpendicular to the heme plane, whereas the axis corresponding to the high-field turning point is in the heme plane and makes an angle of about 80° with the coordinated imidazole plane. The FMN-heme domain docking model obtained in the previous work was found to be in qualitative agreement with the combined experimental results of the two pulsed EPR works.     

Probing the 5f orbital contribution to the bonding in a U(V) ketimide complex

Reaction of UCl(4) with 5 equiv of Li(N═C(t)BuPh) generates the homoleptic U(IV) ketimide complex [Li(THF)(2)][U(N═C(t)BuPh)(5)] (1) in 71% yield. Similarly, reaction of UCl(4) with 5 equiv of Li(N═C(t)Bu(2)) affords [Li(THF)][U(N═C(t)Bu(2))(5)] (2) in 67% yield. Oxidation of 2 with 0.5 equiv of I(2) results in the formation of the neutral U(V) complex U(N═C(t)Bu(2))(5) (3). In contrast, oxidation of 1 with 0.5 equiv of I(2), followed by addition of 1 equiv of Li(N═C(t)BuPh), generates the octahedral U(V) ketimide complex [Li][U(N═C(t)BuPh)(6)] (4) in 68% yield. Complex 4 can be further oxidized to the U(VI) ketimide complex U(N═C(t)BuPh)(6) (5). Complexes 1-5 were characterized by X-ray crystallography, while SQUID magnetometry, EPR spectroscopy, and UV-vis-NIR spectroscopy measurements were also preformed on complex 4. Using this data, the crystal field splitting parameters of the f orbitals were determined, allowing us to estimate the amount of f orbital participation in the bonding of 4.     

Electron inventory, kinetic assignment (E(n)), structure, and bonding of nitrogenase turnover intermediates with C2H2 and CO

Improved 1H ENDOR data from the S(EPR1) intermediate formed during turnover of the nitrogenase alpha-195Gln MoFe protein with C2(1,2)H2 in (1,2)H2O buffers, taken in context with the recent study of the intermediate formed from propargyl alcohol, indicate that S(EPR1) is a product complex, likely with C2H4 bound as a ferracycle to a single Fe of the FeMo-cofactor active site. 35 GHz CW and Mims pulsed 57Fe ENDOR of 57Fe-enriched S(EPR1) cofactor indicates that it exhibits the same valencies as those of the CO-bound cofactor of the lo-CO intermediate formed during turnover with CO, [Mo4+, Fe3+, Fe6(2+), S9(2-)(d43)](+1), reduced by m = 2 electrons relative to the resting-state cofactor. Consideration of 57Fe hyperfine coupling in S(EPR1) and lo-CO leads to a picture in which CO bridges two Fe of lo-CO, while the C2H4 of S(EPR1) binds to one of these. To correlate these and other intermediates with Lowe-Thorneley (LT) kinetic schemes for substrate reduction, we introduce the concept of an "electron inventory". It partitions the number of electrons a MoFe protein intermediate has accepted from the Fe protein (n) into the number transmitted to the substrate (s), the number that remain on the intermediate cofactor (m), and the additional number delivered to the cofactor from the P clusters (p): n = m + s - p (with p = 0 here). The cofactors of lo-CO and S(EPR1) both are reduced by m = 2 electrons, but the intermediates are not at the same LT reduction stage (E(n)): (n = 2; m = 2, s = 0) for lo-CO; (n = 4; s = 2, m = 2) for S(EPR1). This is the first proposed correlation of an LT E(n) kinetic state with a well-defined chemical state of the enzyme.     

Hydrogen atom abstraction by a mononuclear ferric hydroxide complex: insights into the reactivity of lipoxygenase

The lipoxygenase mimic [Fe(III)(PY5)(OH)](CF3SO3)2 is synthesized from the reaction of [Fe(II)(PY5)(MeCN)](CF3SO3)2 with iodosobenzene, with low-temperature studies suggesting the possible intermediacy of an Fe(IV) oxo species. The Fe(III)-OH complex is isolated and identified by a combination of solution and solid-state methods, including EPR and IR spectroscopy. [Fe(III)(PY5)(OH)](2+) reacts with weak X-H bonds in a manner consistent with hydrogen-atom abstraction. The composition of this complex allows meaningful comparisons to be made with previously reported Mn(III)-OH and Fe(III)-OMe lipoxygenase mimics. The bond dissociation energy (BDE) of the O-H bond formed upon reduction to [Fe(II)(PY5)(H2O)]2+ is estimated to be 80 kcal mol(-1), 2 kcal mol(-1) lower than that in the structurally analogous [Mn(II)(PY5)(H2O)]2+ complex, supporting the generally accepted idea that Mn(III) is the thermodynamically superior oxidant at parity of coordination sphere. The identity of the metal has a large influence on the entropy of activation for the reaction with 9,10-dihydroanthracene; [Mn(III)(PY5)(OH)]2+ has a 10 eu more negative DeltaS++ value than either [Fe(III)(PY5)(OH)]2+ or [Fe(III)(PY5)(OMe)]2+, presumably because of the increased structural reorganization that occurs upon reduction to [Mn(II)(PY5)(H2O)]2+. The greater enthalpic driving force for the reduction of Mn(III) correlates with [Mn(III)(PY5)(OH)]2+ reacting more quickly than [Fe(III)(PY5)(OH)]2+. Curiously, [Fe(III)(PY5)(OMe)]2+ reacts with substrates only about twice as fast as [Fe(III)(PY5)(OH)]2+, despite a 4 kcal mol(-1) greater enthalpic driving force for the methoxide complex.     

Synthesis, characterization, reactivity and catalytic activity of oxidovanadium(IV), oxidovanadium(V) and dioxidovanadium(V) complexes of benzimidazole modified ligands

The reaction between [V(IV)O(acac)(2)] and the ONN donor Schiff base obtained by the condensation of pyridoxal and 2-aminoethylbenzimidazole (Hpydx-aebmz, I) or 2-aminomethylbenzimidazole (Hpydx-ambmz, II) in equimolar amounts results in the formation of [V(IV)O(acac)(pydx-aebmz)] 1 and [V(IV)O(acac)(pydx-ambmz)] 2, respectively. The aerobic oxidation of the methanolic solution of 1 yielded [V(V)O(2)(pydx-aebmz)] 3 and its reaction with aqueous H(2)O(2) gave the oxidoperoxidovanadium(v) complex, [V(V)O(O(2))(pydx-aebmz)] 4. The formation of 4 in solution is also established by titrations of methanolic solutions of 1 with H(2)O(2). By titrating solutions of 3 and of 4 with aqueous H(2)O(2) several distinct V(V)-pydx-aebmz species also containing the peroxido ligand are detected. The full geometry optimization of all species envisaged was done using DFT methods for suitable model complexes. The (51)V NMR chemical shifts (δ(V)) have also been calculated, the theoretical data being used to support assignments of the experimental chemical shifts. The (51)V hyperfine coupling constants are calculated for 1, the obtained values being in good agreement with the experimental EPR data. Reaction between the V(IV)O(2+) exchanged zeolite-Y and Hpydx-aebmz and Hpydx-ambmz in refluxing methanol, followed by aerial oxidation results in the formation of the encapsulated V(V)O(2)-complexes, abbreviated herein as [V(V)O(2)(pydx-aebmz)]-Y 5 and [V(V)O(2)(pydx-ambmz)]-Y 6. The molecular structure of 1, determined by single crystal X-ray diffraction, confirms its distorted octahedral geometry with the ONN binding mode of the tridentate ligand, with one acetylacetonato group remaining bound to the V(IV)O-centre. Oxidation of styrene is investigated using some of these complexes as catalyst precursors with H(2)O(2) as oxidant. Under optimised reaction conditions for the conversion of styrene in acetonitrile, a maximum of 68% conversion of styrene (with [V(V)O(2)(pydx-aebmz)]-Y) and 65% (with [V(V)O(2)(pydx-ambmz)]-Y) is achieved in 6 h of reaction time. The selectivity of the various products is similar for both catalysts and follows the order: benzaldehyde (ca. 55%) > 1-phenylethane-1,2-diol > benzoic acid > styrene oxide > phenyl acetaldehyde. Speciation of the systems and plausible intermediates involved in the catalytic oxidation processes are established by UV-Vis, EPR, (51)V NMR and DFT studies. Both non-radical (Sharpless) and radical mechanisms of the olefin oxidations were theoretically studied, and the radical pathway was found to be even more favorable than the Sharpless mechanism.     

Water oxidation intermediates applied to catalysis: benzyl alcohol oxidation

Four distinct intermediates, Ru(IV)═O(2+), Ru(IV)(OH)(3+), Ru(V)═O(3+), and Ru(V)(OO)(3+), formed by oxidation of the catalyst [Ru(Mebimpy)(4,4'-((HO)(2)OPCH(2))(2)bpy)(OH(2))](2+) [Mebimpy = 2,6-bis(1-methylbenzimidazol-2-yl) and 4,4'-((HO)(2)OPCH(2))(2)bpy = 4,4'-bismethylenephosphonato-2,2'-bipyridine] on nanoITO (1-PO(3)H(2)) have been identified and utilized for electrocatalytic benzyl alcohol oxidation. Significant catalytic rate enhancements are observed for Ru(V)(OO)(3+) (~3000) and Ru(IV)(OH)(3+) (~2000) compared to Ru(IV)═O(2+). The appearance of an intermediate for Ru(IV)═O(2+) as the oxidant supports an O-atom insertion mechanism, and H/D kinetic isotope effects support net hydride-transfer oxidations for Ru(IV)(OH)(3+) and Ru(V)(OO)(3+). These results illustrate the importance of multiple reactive intermediates under catalytic water oxidation conditions and possible control of electrocatalytic reactivity on modified electrode surfaces.     

DFT Calculations Suggest a New Type of Self‐Protection and Self‐Inhibition Mechanism in the Mammalian Heme Enzyme Myeloperoxidase: Nucleophilic Addition of a Functional Water rather than One‐Electron Reduction

The mammalian heme enzyme myeloperoxidase (MPO) catalyzes the reaction of Cl^? to the antimicrobial‐effective molecule HOCl. During the catalytic cycle, a reactive intermediate “Compound I” (Cpd I) is generated. Cpd I has the ability to destroy the enzyme. Indeed, in the absence of any substrate, Cpd I decays with a half‐life of 100 ms to an intermediate called Compound II (Cpd II), which is typically the one‐electron reduced Cpd I. However, the nature of Cpd II, its spectroscopic properties, and the source of the additional electron are only poorly understood. On the basis of DFT and time‐dependent (TD)‐DFT quantum chemical calculations at the PBE0/6‐31G* level, we propose an extended mechanism involving a new intermediate, which allows MPO to protect itself from self‐oxidation or self‐destruction during the catalytic cycle. Because of its similarity in electronic structure to Cpd II, we named this intermediate Cpd II′. However, the suggested mechanism and our proposed functional structure of Cpd II′ are based on the hypothesis that the heme is reduced by charge separation caused by reaction with a water molecule, and not, as is normally assumed, by the transfer of an electron. In the course of this investigation, we found a second intermediate, the reduced enzyme, towards which the new mechanism is equally transferable. In analogy to Cpd II′, we named it Fe^II′. The proposed new intermediates Cpd II′ and Fe^II′ allow the experimental findings, which have been well documented in the literature for decades but not so far understood, to be explained for the first time. These encompass a) the spontaneous decay of Cpd I, b) the unusual (chlorin‐like) UV/Vis, circular dichroism (CD), and resonance Raman spectra, c) the inability of reduced MPO to bind CO, d) the fact that MPO‐Cpd II reduces SCN^? but not Cl^?, and e) the experimentally observed auto‐oxidation/auto‐reduction features of the enzyme. Our new mechanism is also transferable to cytochromes, and could well be viable for heme enzymes in general.     

Distinct reaction pathways followed upon reduction of oxy-heme oxygenase and oxy-myoglobin as characterized by Mössbauer spectroscopy

Activation of O(2) by heme-containing monooxygenases generally commences with the common initial steps of reduction to the ferrous heme and binding of O(2) followed by a one-electron reduction of the O(2)-bound heme. Subsequent steps that generate reactive oxygen intermediates diverge and reflect the effects of protein control on the reaction pathway. In this study, M?ssbauer and EPR spectroscopies were used to characterize the electronic states and reaction pathways of reactive oxygen intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of oxy-heme oxygenase (HO) and oxy-myoglobin (Mb). The results confirm that one-electron reduction of (Fe(II)-O(2))HO is accompanied by protonation of the bound O(2) to generate a low-spin (Fe(III)-O(2)H(-))HO that undergoes self-hydroxylation to form the alpha-meso-hydroxyhemin-HO product. In contrast, one-electron reduction of (Fe(II)-O(2))Mb yields a low-spin (Fe(III)-O(2)(2-))Mb. Protonation of this intermediate generates (Fe(III)-O(2)H(-))Mb, which then decays to a ferryl complex, (Fe(IV)=O(2-))Mb, that exhibits magnetic properties characteristic of the compound II species generated in the reactions of peroxide with heme peroxidases and with Mb. Generation of reactive high-valent states with ferryl species via hydroperoxo intermediates is believed to be the key oxygen-activation steps involved in the catalytic cycles of P450-type monooxygenases. The M?ssbauer data presented here provide direct spectroscopic evidence supporting the idea that ferric-hydroperoxo hemes are indeed the precursors of the reactive ferryl intermediates. The fact that a ferryl intermediate does not accumulate in HO underscores the determining role played by protein structure in controlling the reactivity of reaction intermediates.     

Water oxidation by a mononuclear ruthenium catalyst: characterization of the intermediates

A detailed characterization of intermediates in water oxidation catalyzed by a mononuclear Ru polypyridyl complex [Ru(II)-OH(2)](2+) (Ru = Ru complex with one 4-t-butyl-2,6-di-(1',8'-naphthyrid-2'-yl)-pyridine ligand and two 4-picoline ligands) has been carried out using electrochemistry, UV-vis and resonance Raman spectroscopy, pulse radiolysis, stopped flow, and electrospray ionization mass spectrometry (ESI-MS) with H(2)(18)O labeling experiments and theoretical calculations. The results reveal a number of intriguing properties of intermediates such as [Ru(IV)═O](2+) and [Ru(IV)-OO](2+). At pH > 2.9, two consecutive proton-coupled one-electron steps take place at the potential of the [Ru(III)-OH](2+)/[Ru(II)-OH(2)](2+) couple, which is equal to or higher than the potential of the [Ru(IV)═O](2+)/[Ru(III)-OH](2+) couple (i.e., the observation of a two-electron oxidation in cyclic voltammetry). At pH 1, the rate constant of the first one-electron oxidation by Ce(IV) is k(1) = 2 × 10(4) M(-1) s(-1). While pH-independent oxidation of [Ru(IV)═O](2+) takes place at 1420 mV vs NHE, bulk electrolysis of [Ru(II)-OH(2)](2+) at 1260 mV vs NHE at pH 1 (0.1 M triflic acid) and 1150 mV at pH 6 (10 mM sodium phosphate) yielded a red colored solution with a Coulomb count corresponding to a net four-electron oxidation. ESI-MS with labeling experiments clearly indicates that this species has an O-O bond. This species required an additional oxidation to liberate an oxygen molecule, and without any additional oxidant it completely decomposed slowly to form [Ru(II)-OOH](+) over 2 weeks. While there remains some conflicting evidence, we have assigned this species as (1)[Ru(IV)-η(2)-OO](2+) based on our electrochemical, spectroscopic, and theoretical observations alongside a previously reported analysis by T. J. Meyer's group (J. Am. Chem. Soc. 2010, 132, 1545-1557).