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

DeWitt, Jane G.; Bentsen, James G.; Rosenzweig, Amy C.; Hedman, Britt; Green, Jeffrey; Pilkington, S.J.; Papaefthymiou, Georgia C.; Dalton, Howard; Hodgson, Keith O.; Lippard, Stephen J.

doi:10.1021/ja00024a031

Cited by 219 publications

(226 citation statements)

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“…The organism Methylococcus capsulatus (Bath) and the soluble enzyme proteins from it were grown and purified as previously described [16]. The methanes CD, and CH,D, (99.9%) were purchased from MSD isotopes while fully deuterated benzene (99.5%), toluene (99-t %) and styrene (98+%) were from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects

Wilkins

Dalton

Samuel

et al. 1994

European Journal of Biochemistry

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The data from the deuterium isotope experiments in this study show that the primary kinetic isotope effect for methane oxidation catalysed by soluble methane monooxygenase from Methylococcus capsulatus (Bath) is very small, <2. In contrast, the primary kinetic isotope effect for -CH, group oxidation in toluene is large, > 7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferry1 species is believed to operate for CH,, the toluene -CH, group and similar alkanes. Direct oxygen atom addition, rather than H atom abstraction, is indicated for aromatic ring oxidations in benzene and toluene and for styrene oxide formation from styrene. Thus, more than one mechanistic pathway appears to operate in soluble methanemonooxygenase-catalysed reactions and, in some cases, the pathway chosen may be dictated by the substrate.In the soluble methane-monooxygenase-catalysed oxidation of toluene the rates of: (a) substrate dissociation from the enzyme-substrate complex, (b) product formation and (c) product release (benzyl alcohol and p-cresol) from the enzyme-product complex are comparable in magnitude. Therefore all three of these steps are partially rate-determining in the soluble methane monooxygenase catalytic cycle for toluene oxidation.The active site of the enzyme soluble methane monooxygenase [l] is just one example of the bridged diiron motif now known to occur in a number of non-heme iron proteins of vastly different functions [2]. In the case of soluble methane monooxygenase the function of the diiron site is to activate 0, so that one of the oxygen atoms is inserted into a methane C-H bond to form methanol. The other oxygen atom ends up in the second product, water. The enzyme consists of three components : the hydroxylase, the reductase and a regulatory protein, component B. The p-OH bridged diiron site [3, 41 resides in the hydroxylase and it is the fully reduced, Fe(II)Fe(II), form which is catalytically active [5]. 25).National Institutes of Health.high-valent iron-oxo intermediate, forming a substrate radical which then rapidly reacts with iron-bound OH to produce the product [9-111. This mechanism is, in many respects, similar to that proposed for cytochrome P-450 (121. It has been shown that P-450-catalysed hydroxylation of toluene produces benzyl alcohol plus o-, m-and p-cresols with benzyl alcohol as the major product [13 1. With fully deuterated toluene the product ratio was reversed and the cresols dominated. We had earlier observed that catalysis of toluene oxidation by soluble methane monooxygenase produced benzyl alcohol and only p-cresol (not ortho and metu) [8,9, 141. Recently Rataj et al. [15] reported that benzyl alcohol alone and no p-cresol was produced in this oxidation of toluene using soluble methane monooxygenase from Methylosinus trichosporium and crude extract from Methylococcus cupsulutus (Bath). They were also unable to detect any styrene oxide when styrene was used as substrate, a product we had observed previously with the M. cupsulutus ...

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Section: Methodsmentioning

confidence: 99%

Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects

Wilkins

Dalton

Samuel

et al. 1994

European Journal of Biochemistry

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“…The averaged data were processed as described previously 39 by fitting a second-order polynomial to the post-edge region and subtracting this background from the entire spectrum. A three-region cubic spline was used to model the smooth background above the edge.…”

Section: Xas Data Collection and Analysismentioning

confidence: 99%

Synthesis, Characterization, and Reactivities of Manganese(V)−Oxo Porphyrin Complexes

et al. 2007

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The reactions of manganese(III) porphyrin complexes with terminal oxidants, such as mchloroperbenzoic acid, iodosylarenes, and H 2 O 2 , produced high-valent manganese(V)-oxo porphyrins in the presence of base in organic solvents at room temperature. The manganese(V)-oxo porphyrins have been characterized with various spectroscopic techniques, including UV-vis, EPR, 1 H and 19 F NMR, resonance Raman, and X-ray absorption spectroscopy. The combined spectroscopic results indicate that the manganese(V)-oxo porphyrins are diamagnetic low-spin (S = 0) species with a longer, weaker Mn-O bond than in previously reported Mn(V)-oxo complexes of non-porphyrin ligands. This is indicative of double bond character between the manganese(V) ion and the oxygen atom, and may be attributed to the presence of a trans-axial ligand. The Figure S2), UV-vis spectrum of (TM-2-PyP)Mn V =O ( Figure S3), UV-vis and EPR spectra of (TDCPP)Mn IV =O (2a) (Figure S4), time traces for the natural decay of 1a in the presence of different amounts of base ( Figure S5), and LC-ESI MS of PPh 3 O obtained in isotope labeling experiment ( Figure S6). NIH Public Access

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“…These studies and direct protein isolation and characterization have shown that MMO is composed of three protein components, each of which is required for efficient substrate hydroxylation coupled to NADH oxidation: a 245-kDa hydroxylase (MMOH), a 40-kDa reductase (MMOR), and a 15-kDa protein called component B (MMOB). The hydroxylase has a dimeric protomer structure, with each protomer containing three subunits (a,p,y) (Rosenzweig et al, 1993;Froland et al, 1994) and a hydroxo-bridged dinuclear iron cluster (Fox et al, 1988(Fox et al, , 1993DeWitt et al, 1991;DeRose et al, 1993;Thomann et al, 1993). MMOH and its diiron clusters are essential for catalysis by MMO (Fox et al, 1989).…”

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Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

et al. 1997

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Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the 02-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 11, 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 I1 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 I1 Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A and 2.7 8, resolution, respectively, both determined at 18 "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 H20), as well as both carboxylate oxygens of Glu a144. This bis-yhydroxo-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 "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 p 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 Da209E, a ligand of one of the irons.

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

Cited by 219 publications

References 5 publications

Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects

Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects

Synthesis, Characterization, and Reactivities of Manganese(V)−Oxo Porphyrin Complexes

Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

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