Cofactor‐independent oxygenation reactions catalyzed by soluble methane monooxygenase at the surface of a modified gold electrode

Abstract

Soluble methane monooxygenase (sMMO) is a three-component enzyme that catalyses dioxygen-and NAD(P)Hdependent oxygenation of methane and numerous other substrates. Oxygenation occurs at the binuclear iron active centre in the hydroxylase component (MMOH), to which electrons are passed from NAD(P)H via the reductase component (MMOR), along a pathway that is facilitated and controlled by the third component, protein B (MMOB). We previously demonstrated that electrons could be passed to MMOH from a hexapeptide-modified gold electrode and thus cyclic voltammetry could be used to measure the redox potentials of the MMOH active site. Here we have shown that the reduction current is enhanced by the presence of catalase or if the reaction is performed in a flow-cell, probably because oxygen is reduced to hydrogen peroxide, by MMOH at the electrode surface and the hydrogen peroxide then inactivates the enzyme unless removed by catalase or a continuous flow of solution. Hydrogen peroxide production appears to be inhibited by MMOB, suggesting that MMOB is controlling the flow of electrons to MMOH as it does in the presence of MMOR and NAD(P)H. Most importantly, in the presence of MMOB and catalase, the electrode-associated MMOH oxygenates acetonitrile to cyanoaldehyde and methane to methanol. Thus the electochemically driven sMMO showed the same catalytic activity and regulation by MMOB as the natural NAD(P)H-driven reaction and may have the potential for development into an economic, NAD(P)H-independent oxygenation catalyst. The significance of the production of hydrogen peroxide, which is not usually observed with the NAD(P)H-driven system, is also discussed.Keywords: electrochemical oxygenation; regulatory protein; soluble methane monooxygenase.Soluble methane monooxygenase (sMMO) catalyses the bacterial oxidation of methane to methanol using NAD(P)H as cofactor [1]. sMMO consists of three components: the hydroxylase (MMOH), the reductase (MMOR) and a regulatory protein (known as MMOB or protein B). The active site of the enzyme is located on the a subunit of MMOH and consists of a binuclear iron species located in a hydrophobic pocket approximately 12 Å beneath the protein surface [2]. MMOH, which has an (abc) 2 quaternary structure, interacts with the MMOR component from which it receives reductant to drive the reaction [3]:The regulatory protein (MMOB) plays several roles in the catalytic process including modulating the rate of electron transfer between MMOR and MMOH [4,5], altering the redox potential of the binuclear iron site [6], optimizing the interaction between MMOR and MMOH [7], controlling the access of large substrates to the active site [8] and affecting the regioselectivity of substrate oxygenation [9,10]. The 15.9-kDa MMOB also exists in a naturally occurring truncated form in which 12 amino acids are lost from the N-terminus. This truncated form (MMOB tru ) is completely inactive within the sMMO complex [11]. The sMMO reaction can also be driven using hydrogen peroxide via the peroxide shunt reaction [...

A Systematic Study of the Influence of Peptide Modification of a Gold Electrode on the Cyclic Voltammetry of Pseudoazurin from Alcaligenes faecalis Strain S‐6

“…In a further study, Astier et al [9] found that the same enzyme could, under some conditions, adsorb robustly but nondestructively to this peptide interface.…”

Propionate inactivation of butane monooxygenase activity in ‘Pseudomonas butanovora’: biochemical and physiological implications

“…) that rapidly and irreversibly inhibit the reductase subunit of the enzyme, preventing electron transfer to the hydroxylase subunit (Green et al, 1985;Jahng & Wood, 1996); and (iii) H 2 O 2 , which inactivates the enzyme through an unknown mechanism (Astier et al, 2003). Because propionate lacked structural similarity to any of the known inhibitors and inactivators of sMMO mentioned above, we thought it worthwhile to characterize the effect of propionate on BMO.…”

“…In the context of existing literature on sMMO, two possible mechanisms for propionate-dependent inactivation of BMO can be proposed. First, in vitro studies showed that when sMMO hydroxylase is electrically reduced at the surface of an electrode, O 2 is reduced to H 2 O 2 by the hydroxylase and subsequently inactivates it (Astier et al, 2003). We propose a model of BMO inactivation in which propionate stimulates the production of H 2 O 2 by BMO and causes oxidative enzyme damage.…”

“…Bacterial monooxygenase enzymes have been extensively studied because of their potential to degrade environmental pollutants, and to serve as biocatalysts for the creation of industrially important chemicals (Astier et al, 2003;Lipscomb, 1994;Smith et al, 2003;Smith & Dalton, 2004). In this connection we have studied the transcriptional and physiological regulation of butane monooxygenase (BMO) in 'Pseudomonas butanovora'.…”

“…The diferric active site, shown at the bottom of the model, is reduced and used to break one bond of O 2 , forming intermediate P. The scission of the second O 2 bond results in the formation of intermediate Q. Alternatively, the reaction of the reduced form of sMMO with O 2 can result in H 2 O 2 formation and a return of sMMO to a diferric resting state (Zhang & Lipscomb, 2006). Although the presence of the regulatory subunit (MMOB) eliminates the production of H 2 O 2 by sMMO hydroxylase, H 2 O 2 production resumed, as did enzyme inactivation, following the introduction of substrate to the reaction mixture (Astier et al, 2003). In this context, the aliphatic tail of propionate might resemble the alkane substrate of BMO sufficiently well to reduce the coupling of O 2 activation to substrate oxidation and result in H 2 O 2 formation.…”