Inhibition of Membrane-Bound Methane Monooxygenase and Ammonia Monooxygenase by Diphenyliodonium: Implications for Electron Transfer

Abstract

Diphenyliodonium (DPI) is known to irreversibly inactivate flavoproteins. We have found that DPI inhibits both membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus and ammonia monooxygenase (AMO) of Nitrosomonas europaea. The effect of DPI on NADH-dependent pMMO activity in vitro is ascribed to inactivation of NDH-2, a flavoprotein which we proposed catalyzes reduction of the quinone pool by NADH. DPI is a potent inhibitor of type 2 NADH:quinone oxidoreductase (NDH-2), with 50% inhibition occurring at Ϸ5 M. Inhibition of NDH-2 is irreversible and requires NADH. Inhibition of NADH-dependent pMMO activity by DPI in vitro is concomitant with inhibition of NDH-2, consistent with our proposal that NDH-2 mediates reduction of pMMO. Unexpectedly, DPI also inhibits pMMO activity driven by exogenous hydroquinols, but with Ϸ100 M DPI required to achieve 50% inhibition. Similar concentrations of DPI are required to inhibit formate-, formaldehyde-, and hydroquinol-driven pMMO activities in whole cells. The pMMO activity in DPI-treated cells greatly exceeds the activity of NDH-2 or pMMO in membranes isolated from those cells, suggesting that electron transfer from formate to pMMO in vivo can occur independent of NADH and NDH-2. AMO activity, which is known to be independent of NADH, is affected by DPI in a manner analogous to pMMO in vivo: Ϸ100 M is required for 50% inhibition regardless of the nature of the reducing agent. DPI does not affect hydroxylamine oxidoreductase activity and does not require AMO turnover to exert its inhibitory effect. Implications of these data for the electron transfer pathway from the quinone pool to pMMO and AMO are discussed.Ammonia monooxygenase (AMO) and the membranebound form of methane monooxygenase (pMMO) are two of the three members of a novel family of membrane-bound monooxygenases capable of oxidizing small hydrocarbons (3, 33). These two enzymes are similar in their putative subunit composition, inhibitor profiles, and the DNA sequence of the genes encoding the subunits (19,20,22). The membranebound butane monooxygenase from Nocardioides sp. strain CF8 was recently identified as the third member of this class of enzymes, sharing many of the characteristics of pMMO and AMO (18). These enzymes are known to require copper, because inhibition by metal chelators is reversed only by addition of copper (14,17). In addition, all three are irreversibly inhibited by acetylene, and when radiolabeled acetylene is used, a Ϸ27-kDa peptide is specifically labeled (5, 18, 26, 37).Enzymes of this class can oxidize small alkanes and halogenated solvents, hence their potential application in bioremediation (9,25,29,45). Although there are other enzymes that are able to oxidize small alkanes (32,46,47), they are distinct in structure and properties from AMO and pMMO. These two enzymes thus represent a novel mechanism for alkane oxidation, one that has yet to be elucidated due to the difficulty in isolating pure samples of either enzyme with high activity (4,17,28,31,49,51).AMO and pM...

Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath

“…Stimulation of pMMO activity by mixothiazol and stigmatellin has been interpreted as resulting from either the preferential shuttling of electrons to the pMMO or that the pMMO has a quinone-or semiquinonebinding site . Most of the available evidence supports the second interpretation (Basu et al, 2003;Choi et al, 2003;DiSpirito et al, 2004;Shiemke et al, 1995Shiemke et al, , 2004Zahn & DiSpirito, 1996).…”

Effects of Zinc on Particulate Methane Monooxygenase Activity and Structure

“…Only quinols yield activity for solubilized pMMO (61), and improved activity has been reported for pMMO in the presence of a type 2 NADH:quinone oxidoreductase (62)(63)(64), which may provide reduced quinones directly to pMMO. In one possible model, a quinol could bind near the zinc site and supply electrons to the active site.…”

The Role of One- and Two-Electron Transfer Reactions in Forming Thermodynamically Unstable Intermediates as Barriers in Multi-Electron Redox Reactions

“…As in the case of methane mono-oxygenases, nitrifying bacteria produce ammonia monooxygenases, which use metals including copper to activate oxygen (Keener et al 1998;Shiemke et al 2004;Whitaker et al 2000). Ammonia, NH 3 , is the physiological substrate.…”