KINETICS OF PROTOPORPHYRINOGEN OXIDASE INHIBITION BY DIPHENYLENEIODONIUM DERIVATIVES

Protoporphyrinogen oxidase, the last: enzyme of the common branch of t he heme and chlorophyll pathways in plants, is the molecular target of diphenyl ether-type herbicides. These compounds inhibit the enzyme co mpetitively with respect to the tetrapyrrole substrate, protoporphyrin ogen IX, Mie used the flavinic nature of protoporphyrinogen oxidase to investigate the reactivity of the enzyme toward the 2,2'-diphenylenei odonium cation, a known inhibitor of several flavoproteins, Diphenylen eiodonium inhibited the membrane-bound yeast protoporphyrinogen oxidas e competitively with molecular oxygen. The typical slow-binding kineti cs suggested that the enzyme with a reduced flavin rapidly combined wi th the inhibitor to form an initial complex which then slowly isomeriz ed to a modified enzyme-inhibitor complex (K-i = 6.75 x 10(-8) M, K-i = 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: K-i = 4.4 x 10(-8) M and K-i = 1.3 x 10(-9) M for 4-methyl-2,2'-diphenyl eneiodonium K-i = 2.2 x 10(-8) M and K-i = 1.1 x 10(-9) M for 6-methy l-2,2'-diphenyleneiodonium, and K-i = 6.4 x 10(-9) M and K-i = 1.2 x 10(-12) M for 4-nitro-2,2'-diphenyleneiodonium. The 4-nitro-2,2'-diphe nyleneiodonium was a quasi irreversible inhibitor (k(5)/k(6) > 5000). Diphenyleneiodoniums are a new class of protoporphyrinogen oxidase inh ibitors 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 e nzyme.