title: Kinetics of protoporphyrinogen oxidase inhibition by diphenyleneiodonium derivatives.
abstract: Protoporphyrinogen oxidase, the last enzyme of the common branch of the heme and chlorophyll pathways in plants, is the molecular target of diphenyl ether-type herbicides. These compounds inhibit the enzyme competitively with respect to the tetrapyrrole substrate, protoporphyrinogen IX. We used the flavinic nature of protoporphyrinogen oxidase to investigate the reactivity of the enzyme toward the 2,2'-diphenyleneiodonium cation, a known inhibitor of several flavoproteins. Diphenyleneiodonium inhibited the membrane-bound yeast protoporphyrinogen oxidase competitively with molecular oxygen. The typical slow-binding kinetics suggested that the enzyme with a reduced flavin rapidly combined with the inhibitor to form an initial complex which then slowly isomerized to a modified enzyme-inhibitor complex (Ki = 6.75 x 10(-8) M, Ki* = 4.1 x 10(-9) M). This inhibition was strongly pH-dependent and was maximal at pH 8. Substituted diphenyleneiodoniums were synthesized and shown to be even better inhibitors than 2,2'-diphenyleneiodonium: Ki = 4.4 x 10(-8) M and Ki* = 1.3 x 10(-9) M for 4-methyl-2,2'-diphenyleneiodonium, Ki = 2.2 x 10(-8) M and Ki * = 1.1 x 10(-9) M for 6-methyl-2,2'-diphenyleneiodonium, and Ki = 6.4 x 10(-9) M and Ki* = 1.2 x 10(-1)2 M for 4-nitro-2,2'-diphenyleneiodonium. The 4-nitro-2,2'-diphenyleneiodonium was a quasi irreversible inhibitor (k5/k6 > 5000). Diphenyleneiodoniums are a new class of protoporphyrinogen oxidase inhibitors that act via a mechanism very different from that of diphenyl ether-type herbicides and appear to be promising tools for studies on the structure-function relationships of this agronomically important enzyme.
authors.affiliation: Département de Microbiologie, Institut Jacques-Monod, UMR CNRS 9922-Université Paris 7 Denis-Diderot, 2 Place Jussieu, F-75251 Paris Cedex 05, France.
authors.forename: S
authors.initials: S
authors.lastname: Arnould
authors.forename: J L
authors.initials: JL
authors.lastname: Berthon
authors.forename: C
authors.initials: C
authors.lastname: Hubert
authors.forename: M
authors.initials: M
authors.lastname: Dias
authors.forename: C
authors.initials: C
authors.lastname: Cibert
authors.forename: R
authors.initials: R
authors.lastname: Mornet
authors.forename: J M
authors.initials: JM
authors.lastname: Camadro
mesh_terms: D002412:Cations; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D009861:Onium Compounds / Q000737:chemistry / Q000494:pharmacology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase
chemical_list: D002412:Cations; D004791:Enzyme Inhibitors; D009861:Onium Compounds; C007517:diphenyleneiodonium; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase