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.

The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

“…herbicide, and diphenyleneiodonium (K i ϭ 10 nM and 0.1 M, respectively) (20). This shows that the structure and function of the active site, including the isoalloxazin moiety of the flavin, were not affected by cleavage of the polypeptide chain.…”

“…AF, acifluorfen; AFM, acifluorfen methyl; DPI, 2-2Ј diphenyleneiodonium. Ki is the inhibition constant in the E ϩ I ͗ϭ͘ EI reaction; Ki* is the inhibition constant in the EI ͗ϭ͘ EI* isomerization reaction (20).…”

Characteristics of Protoporphyrinogen Oxidase

“…It is highly probable that all the enzymes of the Hem Y family are flavoproteins as they all have the ~-a-~ ADP binding fold but this has not been formally demonstrated for other protoporphyrinogen oxidases. Arnould et al (1997) investigated the role of the flavin in catalysis by studying the reactivity of yeast protoporphyrinogen oxidase toward a potential inhibitor of flavoproteins, the diphenyleneiodonium cation. Diphenyleneiodonium and related bis(aryliodonium) species appear to be time-dependent, mechanism-based inhibitors of several flavoproteins such as mitochondrial NADH ubiquinone oxidoreductase (Gatley and Sherratt 1976;Ragan and Bloxham 1977), neutrophil NADPH oxidase (Doussiere and Vigna is 1992; O'Donnell et al 1993), xanthine oxidase (Doussiere and Vignais 1992), nitric oxide synthase (Stuehr et al 1991) and cytochrome P450 reductase (Tew 1993).…”

Molecular Characterization and Subcellular Localization of Protoporphyrinogen Oxidase in Spinach Chloroplasts

“…Protox has been reported to be widely distributed among plants, animals, and bacteria (Dailey, 1990;Camadro et al, 1999). Studies of the structure and function of Protox have been stimulated by the discovery that herbicides are very potent inhibitors of the Protox activities of yeast, mammal, and plant mitochondria and plant plastids in vitro (Matringe et al, 1992b;Che et al, 1993;Lee et al, 1993;Lee and Duke, 1994;Arnould et al, 1997;Birchfield et al, 1998). In mammals and yeast, Protox activity was detected in mitochondrial inner membrane (Dailey, 1990), while in plants, Protox activity has been observed in both plastids and mitochondria (Jacobs andJacobs, 1987, 1993;Matringe et al, 1992b;Smith et al, 1993).…”