Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase

 A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO) is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol conversion by the bare FeO⁺ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH₄) complex with an OFe—CH₄ bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital interactions between a C _3v - or D _2d -distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered structure. The generated intermediate involves an HO—Fe—CH₃ moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments on regioselectivity of enzyme-catalyzed alkane hydroxylations. Received: 15 September 1997 / Accepted: 20 December 1997     

Dioxygen,an unexpected carbonic anhydrase ligand

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, grouped into seven different classes, which catalyze the reaction of CO₂ hydration to bicarbonate and protons. All of the fifteen human isoforms reported to date belong to the α-class and contain zinc as a cofactor. The structure of human Zn,Cu-CA II has been solved which contains a copper ion bound at its N-terminal, coordinated to His4 and His64. In the active site a dioxygen molecule is coordinated to the zinc ion. Since dioxygen is a rather unexpected CA ligand, molecular dynamics (MD) simulations were performed which suggested a superoxide character of the zinc bound O₂.     

Theoretical study of the catalytic mechanism of catechol oxidase

The mechanism for the oxidation of catechol by catechol oxidase has been studied using B3LYP hybrid density functional theory. On the basis of the X-ray structure of the enzyme, the molecular system investigated includes the first-shell protein ligands of the two metal centers as well as the second-shell ligand Cys92. The cycle starts out with the oxidized, open-shell singlet complex with oxidation states Cu₂(II,II) with a μ-η²:η² bridging peroxide, as suggested experimentally, which is obtained from the oxidation of Cu₂(I,I) by dioxygen. The substrate of each half-reaction is a catechol molecule approaching the dicopper complex: the first half-reaction involves Cu(I) oxidation by peroxide and the second one Cu(II) reduction. The quantitative potential energy profile of the reaction is discussed in connection with experimental data. Since no protons leave or enter the active site during the catalytic cycle, no external base is required. Unlike the previous density functional theory study, the dicopper complex has a charge of +2.     

Proton transport facilitating water-oxidation: the role of second sphere ligands surrounding the catalytic metal cluster

The ability of PSII to extract electrons from water, with molecular oxygen as a by-product, is a remarkable biochemical and evolutionary innovation. From an evolutionary perspective, the invention of PSII approximately 2.7 Ga led to the accelerated accumulation of biomass in the biosphere and the accumulation of oxygen in the atmosphere, a combination that allowed for the evolution of a much more complex and extensive biosphere than would otherwise have been possible. From the biochemical and enzymatic perspective, PSII is remarkable because of the thermodynamic and kinetic obstacles that needed to have been overcome to oxidize water as the ultimate photosynthetic electron donor. This article focuses on how proton release is an integral part of how these kinetic and thermodynamic obstacles have been overcome: the sequential removal of protons from the active site of H₂O-oxidation facilitates the multistep oxidation of the substrate water at the Mn₄CaO _x , the catalytic heart of the H₂O-oxidation reaction. As noted previously, the facilitated deprotonation of the Mn₄CaO _x cluster exerts a redox-leveling function preventing the accumulation of excess positive charge on the cluster, which might otherwise hinder the already energetically difficult oxidation of water. Using recent results, including the characteristics of site-directed mutants, the role of the second sphere of amino acid ligands and the associated network of water molecules surrounding the Mn₄CaO _x is discussed in relation to proton transport in other systems. In addition to the redox-leveling function, a trapping function is assigned to the proton release step occurring immediately prior to the dioxygen chemistry. This trapping appears to involve a yet-to-be clarified gating mechanism that facilitates to coordinated release of a proton from the neighborhood of the active site thereby insuring that the backward charge-recombination reaction does not out-compete the forward reaction of dioxygen chemistry during this final step of H₂O-oxidation.     

Land-Use Change and Biogeochemical Controls of Methane Fluxes in Soils of Eastern Amazonia

Tropical soils account for 10%–20% of the 15–35 Tg of atmospheric methane (CH₄) consumed annually by soils, although tropical deforestation could be changing the soil sink. The objectives of this study were (a) to quantify differences in soil CH₄ fluxes among primary forest, secondary forest, active pasture, and degraded pasture in eastern Amazonia; and (b) to investigate controlling mechanisms of CH₄ fluxes, including N availability, gas-phase transport, and soil respiration. At one ranch, Fazenda Vitória, annual uptake estimates (kg CH₄ha⁻¹ y⁻¹) based on monthly measurements were: primary forest, 2.1; secondary forest, 1.0; active pasture, 1.3; degraded pasture, 3.1. The lower annual uptake in the active pasture compared with the primary forest was due to CH₄ production during the wet season in the pasture soils, which is consistent with findings from other studies. In contrast, the degraded pasture was never a CH₄ source. Expressing uptake as a negative flux and emission as a positive flux, CH₄ fluxes were positively correlated with CO₂ fluxes, indicating that root and microbial respiration in the productive pastures, and to a lesser extent in the primary forest, contributed to the formation of anaerobic microsites where CH₄ was produced, whereas this productivity was absent in the degraded pasture. In all land uses, uptake rates of atmospheric CH₄ were greater in the dry season than in the wet season, indicating the importance of soil water content and gas transport on CH₄ fluxes. These clay soils had low annual uptake rates relative to reported rates on sandy soils, which also is consistent with gas transport within the soil being a limiting factor. Nitrogen availability indices did not correlate with CH₄ fluxes, indicating that inhibition of CH₄ oxidation was not an important mechanism explaining differences among land uses. At another ranch, Fazenda Agua Parada, no significant effect of pasture age was observed along a chronosequence of pasture ages. We conclude that land-use change can either increase or decrease the soil sink of CH₄, depending on the duration of wet and dry seasons, the effects of seasonal precipitation on gas-phase transport, and the phenology and relative productivity of the vegetation in each land use.     

Catecholase activity of a μ-hydroxodicopper(II) macrocyclic complex: structures, intermediates and reaction mechanism

The monohydroxo-bridged dicopper(II) complex (1), its reduced dicopper(I) analogue (2) and the trans-μ-1,2-peroxo-dicopper(II) adduct (3) with the macrocyclic N-donor ligand [22]py4pz (9,22-bis(pyridin-2′-ylmethyl)-1,4,9,14,17,22,27,28,29,30- decaazapentacyclo -[22.2.1^14,7.1^11,14.1^17,20]triacontane-5,7(28),11(29),12,18,20(30), 24(27),25-octaene), have been prepared and characterized, including a 3D structure of 1 and 2. These compounds represent models of the three states of the catechol oxidase active site: met, deoxy (reduced) and oxy. The dicopper(II) complex 1 catalyzes the oxidation of catechol model substrates in aerobic conditions, while in the absence of dioxygen a stoichiometric oxidation takes place, leading to the formation of quinone and the respective dicopper(I) complex. The catalytic reaction follows a Michaelis–Menten behavior. The dicopper(I) complex binds molecular dioxygen at low temperature, forming a trans-μ-1,2-peroxo-dicopper adduct, which was characterized by UV–Vis and resonance Raman spectroscopy and electrochemically. This peroxo complex stoichiometrically oxidizes a second molecule of catechol in the absence of dioxygen. A catalytic mechanism of catechol oxidation by 1 has been proposed, and its relevance to the mechanisms earlier proposed for the natural enzyme and other copper complexes is discussed. Electronic Supplementary Material Supplementary material is available for this article at     

Coordination and binding geometry of methyl-coenzyme M in the red1m state of methyl-coenzyme M reductase

Methane formation in methanogenic Archaea is catalyzed by methyl-coenzyme M reductase (MCR) and takes place via the reduction of methyl-coenzyme M (CH₃-S-CoM) with coenzyme B (HS-CoB) to methane and the heterodisulfide CoM-S–S-CoB. MCR harbors the nickel porphyrinoid coenzyme F₄₃₀ as a prosthetic group, which has to be in the Ni(I) oxidation state for the enzyme to be active. To date no intermediates in the catalytic cycle of MCR_red1 (red for reduced Ni) have been identified. Here, we report a detailed characterization of MCR_red1m (“m” for methyl-coenzyme M), which is the complex of MCR_red1a (“a” for absence of substrate) with CH₃-S-CoM. Using continuous-wave and pulse electron paramagnetic resonance spectroscopy in combination with selective isotope labeling (¹³C and ²H) of CH₃-S-CoM, it is shown that CH₃-S-CoM binds in the active site of MCR such that its thioether sulfur is weakly coordinated to the Ni(I) of F₄₃₀. The complex is stable until the addition of the second substrate, HS-CoB. Results from EPR spectroscopy, along with quantum mechanical calculations, are used to characterize the electronic and geometric structure of this complex, which can be regarded as the first intermediate in the catalytic mechanism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

The first step of the dioxygenation reaction carried out by tryptophan dioxygenase and indoleamine 2,3-dioxygenase as revealed by quantum mechanical/molecular mechanical studies

Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two heme-containing enzymes which catalyze the conversion of l-tryptophan to N-formylkynurenine (NFK). In mammals, TDO is mostly expressed in liver and is involved in controlling homeostatic serum tryptophan concentrations, whereas IDO is ubiquitous and is involved in modulating immune responses. Previous studies suggested that the first step of the dioxygenase reaction involves the deprotonation of the indoleamine group of the substrate by an evolutionarily conserved distal histidine residue in TDO and the heme-bound dioxygen in IDO. Here, we used classical molecular dynamics and hybrid quantum mechanical/molecular mechanical methods to evaluate the base-catalyzed mechanism. Our data suggest that the deprotonation of the indoleamine group of the substrate by either histidine in TDO or heme-bound dioxygen in IDO is not energetically favorable. Instead, the dioxygenase reaction can be initiated by a direct attack of heme-bound dioxygen on the C₂=C₃ bond of the indole ring, leading to a protein-stabilized 2,3-alkylperoxide transition state and a ferryl epoxide intermediate, which subsequently recombine to generate NFK. The novel sequential two-step oxygen addition mechanism is fully supported by our recent resonance Raman data that allowed identification of the ferryl intermediate (Lewis-Ballester et al. in Proc Natl Acad Sci USA 106:17371–17376, 2009). The results reveal the subtle differences between the TDO and IDO reactions and highlight the importance of protein matrix in modulating stereoelectronic factors for oxygen activation and the stabilization of both transition and intermediate states.     

On the Dynamical Behavior of the Cysteine Dioxygenase‐l‐Cysteine Complex in the Presence of Free Dioxygen and l‐Cysteine

In this work, viable models of cysteine dioxygenase (CDO) and its complex with l ‐cysteine dianion were built for the first time, under strict adherence to the crystal structure from X‐ray diffraction studies, for all atom molecular dynamics (MD). Based on the CHARMM36 FF, the active site, featuring an octahedral dummy Fe(II) model, allowed us observing water exchange, which would have escaped attention with the more popular bonded models. Free dioxygen (O₂) and l ‐cysteine, added at the active site, could be observed being expelled toward the solvating medium under Random Accelerated Molecular Dynamics (RAMD) along major and minor pathways. Correspondingly, free dioxygen (O₂), added to the solvating medium, could be observed to follow the same above pathways in getting to the active site under unbiased MD. For the bulky l ‐cysteine, 600 ns of trajectory were insufficient for protein penetration, and the molecule was stuck at the protein borders. These models pave the way to free energy studies of ligand associations, devised to better clarify how this cardinal enzyme behaves in human metabolism.     

Photosynthetic water oxidation vs. mitochondrial oxygen reduction: distinct mechanistic parallels

The photosynthetic oxygen evolving complex (PSII-OEC) and the mitochondrial cytochrome c oxidase (CcO) not only catalyze anti-parallel reactions (the OEC oxidizes water to dioxygen, whereas CcO reduces dioxygen to water), they also share a number of uncanny molecular and mechanistic similarities. Both feature a redox-active polymetallic cluster that includes a key tyrosine, and both utilize a two-phase mechanism. In one phase the polymetallic cluster undergoes four sequential one-electron transfers: In the PSII-OEC, four successive photooxidations of the photosystem II reaction center P680 (to P680⁺) allows acceptance of 4 × 1e- from the Mn₄Ca cluster; in CcO, four reduced cytochrome c Fe²⁺ cations donate 4 × 1e- to the bimetallic center. In the second phase for each enzyme, the polymetallic cluster undergoes a single four-electron transfer with the O₂/2 H₂O redox couple. Intriguing mechanistic similarities between these two complex redox enzymes first delineated over a decade ago by Hoganson/Proshlyakov/Babcock et al. are updated and expanded in this article.     

Structural and spectroscopic studies shed light on the mechanism of oxalate oxidase

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.     

Vegetation heterogeneity and ditches create spatial variability in methane fluxes from peatlands drained for forestry

Drainage of peatlands for forestry starts a succession of ground vegetation in which mire species are gradually replaced by forest species. Some mire plant communities vanish quickly following the water-level drawdown; some may prevail longer in the moister patches of peatland. Drainage ditches, as a new kind of surface, introduce another component of spatial variation in drained peatlands. These variations were hypothesized to affect methane (CH₄) fluxes from drained peatlands. Methane fluxes from different plant communities and unvegetated surfaces, including ditches, were measured at the drained part of Lakkasuo mire, Central Finland. The fluxes were found to be related to peatland site type, plant community, water-table position and soil temperature. At nutrient-rich fen sites fluxes between plant communities differed only a little: almost all plots acted as CH₄ sinks (−0.9 to −0.4 mg CH₄ m⁻² d⁻¹), with the exception of Eriophorum angustifolium Honck. communities, which emitted 0.9 g CH₄ m⁻² d⁻¹. At nutrient-poor bog site the differences between plant communities were clearer. The highest emissions were measured from Eriophorum vaginatum L. communities (29.7 mg CH₄ m⁻² d⁻¹), with a decreasing trend to Sphagna (10.0 mg CH₄ m⁻² d⁻¹) and forest moss communities (2.6 mg CH₄ m⁻² d⁻¹). CH₄ emissions from different kinds of ditches were highly variable, and extremely high emissions (summertime averages 182–600 mg CH₄ m⁻² d⁻¹) were measured from continuously water-covered ditches at the drained fen. Variability in the emissions was caused by differences in the origin and movement of water in the ditches, as well as differences in vegetation communities in the ditches. While drainage on average greatly decreases CH₄ emissions from peatlands, a great spatial variability in fluxes is emerged. Emissions from ditches constantly covered with water, may in some cases have a great impact on the overall CH₄ emissions from drained peatlands.     

An improved purification procedure for the soluble [NiFe]-hydrogenase of Ralstonia eutropha: new insights into its (in)stability and spectroscopic properties

Infrared (IR) spectra in combination with chemical analyses have recently shown that the active Ni–Fe site of the soluble NAD⁺-reducing [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanide groups and one carbon monoxide as ligands. Experiments presented here confirm this result, but show that a variable percentage of enzyme molecules loses one or two of the cyanide ligands from the active site during routine purification. For this reason the redox conditions during the purification have been optimized yielding hexameric enzyme preparations (HoxFUYHI₂) with aerobic specific H₂–NAD⁺ activities of 150–185 μmol/min/mg of protein (up to 200% of the highest activity previously reported in the literature). The preparations were highly homogeneous in terms of the active site composition and showed superior IR spectra. IR spectro-electrochemical studies were consistent with the hypothesis that only reoxidation of the reduced enzyme with dioxygen leads to the inactive state, where it is believed that a peroxide group is bound to nickel. Electron paramagnetic resonance experiments showed that the radical signal from the NADH-reduced enzyme derives from the semiquinone form of the flavin (FMN-a) in the hydrogenase module (HoxYH dimer), but not of the flavin (FMN-b) in the NADH-dehydrogenase module (HoxFU dimer). It is further demonstrated that the hexameric enzyme remains active in the presence of NADPH and air, whereas NADH and air lead to rapid destruction of enzyme activity. It is proposed that the presence of NADPH in cells keeps the enzyme in the active state.     

Unveiling the Pathways of Dioxygen Through the C2 Component of the Environmentally Relevant Monooxygenase p‐Hydroxyphenylacetate Hydroxylase from Acinetobacter baumannii: A Molecular Dynamics Investigation

In this work, models of the homotetrameric C2 component of the monooxygenase p‐hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, in complex with dioxygen (O₂) and, or not, the substrate p‐hydroxyphenylacetate (HPA) were built. Both models proved to be amenable to random‐acceleration molecular dynamics (RAMD) simulations, whereby a tiny randomly oriented external force, acting on O₂ at the active site in front of flavin mononucleotide (FMNH^?), accelerated displacement of O₂ toward the bulk solvent. This allowed us to carry out a sufficiently large number of RAMD simulations to be of statistical significance. The two systems behaved very similarly under RAMD, except for O₂ leaving the active site more easily in the absence of HPA, but then finding similar obstacles in getting to the gate as when the active site was sheltered by HPA. This challenges previous conclusions that HPA can only reach the active center after that the C4aOOH derivative of FMNH^? is formed, requiring uptake of O₂ at the active site before HPA. According to these RAMD simulations, O₂ could well get to FMNH^? also in the presence of the substrate at the active site.     

Theoretical study of the hydroxylation of phenols mediated by an end-on bound superoxo–copper(II) complex

Peptidylglycine α-amidating monooxygenase and dopamine β-monooxygenase are copper-containing proteins which catalyze essential hydroxylation reactions in biological systems. There are several possible mechanisms for the reductive O₂-activation at their mononuclear copper active site. Recently, Karlin and coworkers reported on the reactivity of a copper(II)–superoxo complex which is capable of inducing the hydroxylation of phenols with incorporated oxygen atoms derived from the Cu(II)-O₂ ^·− moiety. In the present work the reaction mechanism for the abovementioned superoxo complex with phenols is studied. The pathways found are analyzed with the aim of providing some insight into the nature of the chemical and biological copper-promoted oxidative processes with 1:1 Cu(I)/O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and spectroscopic properties of Co(salen) derivatives containing pendant thioether groups and their dioxygen adducts

Two Co(salen) derivatives, Co(sal-ipsen) and Co(sal-bsen), containing pendant (CH₂)₂S(i-C₃H₇) and (CH₂)₂SC₆H₅ groups were synthesized. Electronic and ESR spectra in methylene chloride show that the former is five-coordinate with pendant thioether coordination at 198 K or below whereas the latter is four-coordinate at 198 K and becomes a mixture of the four- and five-coordinate species at liquid nitrogen temperature. Upon oxygenation at low temperatures, both complexes form dioxygen adducts in which the pendant thioether groups are coordinated to the trans position to dioxygen. Resonance Raman spectra show that Co(sal-ipsen) yields an equilibrium mixture of the 1:1 and 1:2(O₂/ Co) adducts at 190 K while Co(sal-bsen) forms only the 1:1 adduct under similar conditions. These differences between Co(sal-ipsen) and Co(sal-bsen) can be attributed to the variance in basicity of their pendant sulfur atoms.     

Interaction of the active site of the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors

The study of Ni–Fe–Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O₂. In this work we studied these inhibition processes for the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni–Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni–Fe–Se hydrogenase is more protected against the attack by O₂. The inhibition of the Ni–Fe–Se hydrogenase activity by O₂ could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O₂ on the bridging site between Ni and Fe.     

How does the protein environment optimize the thermodynamics of thiol sulfenylation? Insights from model systems to QM/MM calculations on human 2-Cys peroxiredoxin

Protein thiol/sulfenic acid oxidation potentials provide a tool to select specific oxidation agents, but are experimentally difficult to obtain. Here, insights into the thiol sulfenylation thermodynamics are obtained from model calculations on small systems and from a quantum mechanics/molecular mechanics (QM/MM) analysis on human 2-Cys peroxiredoxin thioredoxin peroxidase B (Tpx-B). To study thiol sulfenylation in Tpx-B, our recently developed computational method to determine reduction potentials relatively compared to a reference system and based on reaction energies reduction potential from electronic energies is updated. Tpx-B forms a sulfenic acid (R-SO^?) on one of its active site cysteines during reactive oxygen scavenging. The observed effect of the conserved active site residues is consistent with the observed hydrogen bond interactions in the QM/MM optimized Tpx-B structures and with free energy calculations on small model systems. The ligand effect could be linked to the complexation energies of ligand L with CH₃S^? and CH₃SO^?. Compared to QM only calculations on Tpx-B’s active site, the QM/MM calculations give an improved understanding of sulfenylation thermodynamics by showing that other residues from the protein environment other than the active site residues can play an important role.     

Using synthetic chemistry to understand copper protein active sites: a personal perspective

The results of studies performed in the author’s laboratory are surveyed, with particular emphasis on demonstrating the value of a multidisciplinary synthetic modeling approach for discovering new and unusual chemistry helpful for understanding the properties of the active sites of copper proteins or assessing the feasibility of mechanistic pathways they might follow during catalysis. The discussion focuses on the progress made to date toward comprehending the nitrite reductase catalytic site and mechanism, the electronic structures of copper thiolate electron transfer centers, the sulfido-bridged “Cu_Z” site in nitrous oxide reductase, and the processes of dioxygen binding and activation by mono- and dicopper centers in oxidases and oxygenases.     

Catechol oxidase and phenoxazinone synthase activity of a manganese(II) isoindoline complex

The mononuclear [Mn(6′Me₂indH)(H₂O)₂(CH₃CN)](ClO₄)₂ (6′Me₂indH: 1,3-bis(6′-methyl-2′-pyridylimino)isoindoline) complex has been prepared and characterized by various techniques such as elemental analysis, IR, UV-visible and ESR spectroscopy. The title compound was suitable as catalyst for the catalytic oxidation of 3,5-di-tert-butylcatechol (3,5-DTBCH₂) to 3,5-di-tert-butyl-1,2-benzoquinone (3,5-DTBQ) (catecholase activity), and o-aminophenol (OAPH) to 2-aminophenoxazine-3-one (APX) (phenoxazinone synthase activity) with dioxygen at ambient condition in good yields. Kinetic measurements revealed first-order dependence on the catalyst and dioxygen concentration and saturation type behavior with respect to the corresponding substrate. It was also found that the added triethylamine in both systems accelerates the reaction.