The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron-sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[ 3 H]pyridaben ([ 3 H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron-sulfur cluster N2 to quinone.

Using this combinatorial library approach, a family of potent 2,2-dimethylbenzopyran-based inhibitors was developed with IC(50) values in the range of 18-55 nM. Cell-based assays revealed that these inhibitors were rather non-cytotoxic in the MCF-7 cell line; however, they were quite cytostatic in a panel of cancer cell lines suggesting their potential as chemotherapeutic/chemopreventive candidates.

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Bidrin, 3-(dimethoxyphosphinyloxy)-N,N-dimethyl-c/s-crotonamide, is metabolized to yield trace amounts of 3-(dimethoxyphosphinyloxy)-N-methyl-N-hydroxymethyl-c/s-crotonamide and larger amounts of 3-(dimethoxyphosphinyloxy)-N-methyl-c/s-crotonamide (SD 9129). SD 9129 is further metabolized to yield 3-(dimethoxyphosphinyloxy)-N-hydroxymethyl-c/s-crotonamide and 3-(dimethoxyphosphinyloxy)-c/s-crotonamide. The toxicity to both insects and mammals increased upon successive N-demethylation. Balance studies on the fate of the P32 and C14 from Bidrin-P32, Bidrin-N-methyl-C14, SD 9129-P32, and SD 9129-N-methyl-C14 are considered. Studies on milk residues, urinalysis, and metabolism in houseflies (Musca domestica L.) and bean plants are reported. An unusual pattern of synergism of the toxicity of the Bidrin metabolites in houseflies by sesamex [2-(2-ethoxyethoxy)ethyl-3,4-(methylenedioxy)phenyl acetal of acetaldehyde] was noted.NO. 2 MAR.-APR.

ing line, the upper part of II hich gives a distinct green color by spraying with aqueous 10% sodium hydroxide. The lower limit for obtaining this green spot was 5 y .

This investigation was supported in part by research grants from the Shell Development Co. and the Division of Biology and Medicine,

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