Arnould S, Berthon J L, Hubert C, Dias M, Cibert C, Mornet R, Camadro J M
Département de Microbiologie, Institut Jacques-Monod, UMR CNRS 9922-Université Paris 7 Denis-Diderot, 2 Place Jussieu, F-75251 Paris Cedex 05, France.
Biochemistry. 1997 Aug 19;36(33):10178-84. doi: 10.1021/bi970549j.
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.
原卟啉原氧化酶是植物中血红素和叶绿素途径共同分支的最后一种酶，是二苯醚类除草剂的分子靶标。这些化合物相对于四吡咯底物原卟啉原IX竞争性抑制该酶。我们利用原卟啉原氧化酶的黄素性质来研究该酶对2,2'-二亚苯基碘鎓阳离子的反应性，2,2'-二亚苯基碘鎓阳离子是几种黄素蛋白的已知抑制剂。二亚苯基碘鎓与分子氧竞争性抑制膜结合的酵母原卟啉原氧化酶。典型的慢结合动力学表明，还原型黄素的酶迅速与抑制剂结合形成初始复合物，然后缓慢异构化为修饰的酶-抑制剂复合物（Ki = 6.75×10^(-8) M，Ki* = 4.1×10^(-9) M）。这种抑制作用强烈依赖于pH，在pH 8时最大。合成了取代的二亚苯基碘鎓，结果表明它们是比2,2'-二亚苯基碘鎓更好的抑制剂：4-甲基-2,2'-二亚苯基碘鎓的Ki = 4.4×10^(-8) M，Ki* = 1.3×10^(-9) M；6-甲基-2,2'-二亚苯基碘鎓的Ki = 2.2×10^(-8) M，Ki* = 1.1×10^(-9) M；4-硝基-2,2'-二亚苯基碘鎓的Ki = 6.4×10^(-9) M，Ki* = 1.2×10^(-12) M。4-硝基-2,2'-二亚苯基碘鎓是一种准不可逆抑制剂（k5/k6 > 5000）。二亚苯基碘鎓是一类新型的原卟啉原氧化酶抑制剂，其作用机制与二苯醚类除草剂非常不同，似乎是研究这种具有重要农学意义的酶的结构-功能关系的有前途的工具。
