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

Abstract

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 ...

Trichloroethylene oxidation by purified toluene 2-monooxygenase: products, kinetics, and turnover-dependent inactivation

“…Conversely, methane monooxygenase oxidizes toluene to benzyl alcohol, ortho-cresol, meta-cresol, and para-cresol. The ratios of products differ depending on the source of enzyme (22,39). Both enzymes are 3.…”

Carbon isotope fractionation during aerobic biodegradation of n-alkanes and aromatic compounds in unsaturated sand

“…The smaller isotope enrichment factors are likely related to the difference in reaction mechanism. In contrast to degradation of n-alkanes that involves a C-H bond cleavage in the initial step, aromatic rings are transformed via formation of an arene oxide without breaking the C-H bonds (Tomaszewski et al, 1975;Wilkins et al, 1994). The mechanism without H abstraction alters the bonds strength to carbon atoms less during formation of the transition state and hence the isotope effect is smaller than for n-alkanes.…”

The geochemistry of methane isotopologues

“…Values for the primary isotope effect (corresponding to D α p = 0.73) and secondary isotope effect ( D α s = 0.93) have been reported for methane oxidation by sMMO (Wilkins et al, 1994). The overall D α calculated from these values (0.88 via Eqn.…”

“…Pudzianowski and Loew (1983) note, however, that the transition state of the CH 4 /CH 3 D + O( 3 P) reaction they modeled has only qualitative similarity to the transition state of the methane hydrogen abstraction/hydroxylation reaction performed by methane monooxygenase. Such fundamental differences between the two processes may explain the difference between their calculated fractionation and the experimental observations.Multiple experimental determinations of the kinetic isotope effects for H or D abstraction have been reported (e.g., Green and Dalton, 1989;Rataj et al, 1991;Wilkins et al, 1994, and (Fig. 4.3b).…”

“…Multiple experimental determinations of the kinetic isotope effects for H or D abstraction have been reported (e.g., Green and Dalton, 1989;Rataj et al, 1991;Wilkins et al, 1994, and (Fig. 4.3b).…”