Rat liver cytosolic azoreductase. Electron transport properties and the mechanism of dicumarol inhibition of the purified enzyme.

A highly purified cytosolic azoreductase isolated from the livers of 3-methylcholanthrene-treated rats has been characterized with respect to its electron- accepting and -donating properties, the mechanism of its inhibition by dicumarol, and its similarity to DT- diaphorase. Anaerobic titration of the enzyme with NADPH as the electron donor indicates that each mole of enzyme can accept 4 reducing eq, or 2 electrons/mol of enzyme-bound FAD. The purified azoreductase cat- alyzes the reduction of methyl red and under anaerobic conditions, 2 mol of NADPH are required for the reduc- tion of 1 mol of methyl red which is consistent with the expected stoichiometry for reduction of the azo linkage of methyl red by NADPH. Dicumarol strongly inhibits the azoreductase-catalyzed reduction of methyl red. Spectrophotometric measurements indicate that dicu- marol binds to the purified azoreductase at a ratio of approximately 1 mol of dicumarol to 1 mol of enzyme- bound FAD. Dicumarol is a competitive inhibitor of methyl red reduction with respect to NADPH and ap- pears to inhibit the reaction by interfering with elec- tron transfer from NADPH to enzyme-bound FAD. Pu- rified rat azoreductase from rat liver appears to be identical with rat DT-diaphorase. This conclusion is based on the co-purification of both enzyme activities as well as the apparent homogeneity of the final enzyme preparation. Other properties of both enzymes are also consistent with this conclusion. The enzyme responsible for the metabolism of methyl red (2’-carboxy-4-N,N-dimethylaminoazobenzene) is localized mainly in the liver 105,000 x g supernatant fraction. This azoreductase activity can be induced 7- to lo-fold by pretreat- ment of rats with 3-methylcholanthrene (1, 2). We have re- cently reported the purification to apparent homogeneity of an azoreductase from the liver 105,000 x g supernatant frac- tion of 3-methylcholanthrene-treated rats (3,4). The purified azoreductase has an apparent molecular weight of 52,000 as determined by gel filtration on Sephadex G-100. Polyacryl- amide gel electrophoresis in the presence of sodium dodecyl sulfate gives a minimal molecular weight of 30,000 suggesting that the native enzyme exists as a dimer. The enzyme is a flavoprotein and contains 2 mol of FAD/mol of enzyme (4). This azoreductase actively catalyzes the reduction of methyl red and a structure activity study revealed that the 2’-carboxyl * The