HUMAN PROTOPORPHYRINOGEN OXIDASE - RELATION BETWEEN THE HERBICIDE BINDING-SITE AND THE FLAVIN COFACTOR

Protoporphyrinogen IX oxidase (protox) catalyzes the oxidation of prot oporphyrinogen IX to protoporphyrin IX in the penultimate step of heme and chlorophyll biosynthesis in animals and plants, Protox is the tar get of light-dependent peroxidizing herbicides and is inhibited at nan omolar levels by several chemical classes including tetrahydrophthalim ides (discussed below) and diphenyl ethers (e.g., acifluorfen) usually with little selectivity between the mammalian and plant enzymes. The herbicide binding site is examined here with a photoaffinity radioliga nd optimized on the basis of structure-activity relationships. A radio synthetic procedure is described for this new herbicidal probe, 2-fluo rophenyl)-3,4,5,6-[H-3]tetrahydrophthalimide ([H-3]AzTHP), resulting i n high specific activity (2.6 TBq/mmol). Human protox expressed in Esc herichia coli and purified by affinity chromatography is used with [H- 3]AzTHP to characterize the herbicide/substrate binding site. Specific binding of [H-3]AzTHP to human protox is rapid, completely reversible in the absence of Light with a K-d Of 93 nM, and competitively inhibi ted by the 5-propargyloxy analogue and by acifluorfen, which are known to bind at the substrate (protoporphyrinogen) site. The B-max establi shes one [H-3]AzTHP binding site per FAD. Diphenylene iodonium, propos ed to inhibit protox by interaction with the FAD cofactor, inhibits en zyme activity by 48% at 100 mu M without affecting [H-3]AzTHP binding in the presence or absence of substrate, suggesting that the herbicide binding site may not be proximal to FAD, The first step has been take n in photoaffinity labeling the herbicide/substrate site with [H-3]AzT HP resulting in apparent covalent derivatization of 13% of the herbici de binding site.