title: 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 approximately 5 micro 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 approximately 100 micro 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: approximately 100 micro 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.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, Robert C. Byrd Health Sciences Center, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9142, USA. ahiemke@hsc.wvu.edu
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Shiemke
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Arp
authors.forename: Luis A
authors.initials: LA
authors.lastname: Sayavedra-Soto
mesh_terms: D001713:Biphenyl Compounds / Q000494:pharmacology*; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020619:Methylococcus capsulatus / Q000201:enzymology*; D009606:Nitrosomonas / Q000201:enzymology*; D009861:Onium Compounds / Q000494:pharmacology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D010105:Oxygenases / Q000037:antagonists & inhibitors*; D011808:Quinone Reductases / Q000502:physiology; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D001713:Biphenyl Compounds; D004791:Enzyme Inhibitors; D009861:Onium Compounds; C031291:diphenyliodonium; C009842:allylthiourea; D010088:Oxidoreductases; D010105:Oxygenases; C021555:methane monooxygenase; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; C045582:ammonia monooxygenase; D013890:Thiourea