SUBUNIT FUNCTIONAL-STUDIES OF NAD(P)H-QUINONE OXIDOREDUCTASE WITH A HETERODIMER APPROACH

NAD(P)H:quinone oxidoreductase (NQOR; EC 1.6.99.2) is a homodimeric en zyme which catalyzes the reduction of quinones, azo dyes, and other el ectron accepters by NADPH or NADH, To pursue subunit functional studie s, we expressed a wild-type/mutant heterodimer of NQOR in Escherichia coil, The wild-type subunit of the heterodimer was tagged with polyhis tidine and the other subunit contained a His-194 --> Ala mutation (H19 4A), a change known to dramatically increase the K-m for NADPH. This a pproach enabled us to efficiently purify the heterodimer (H194A/HNQOR) from the homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate column under nondenaturing conditions, The compositi on of the purified heterodimer was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis and immunoblot analysis, The enzyme kinetics of the purified heterodimer were studied with two two-electr on accepters, 2,6-dichloroindophenol and menadione, and a four-electro n acceptor, methyl red, as the substrates. With two-electron accepters , the K-m(NADPH) and K-m(NADH) values of the heterodimer H194A/HNQOR w ere virtually identical to those of the wild-type homodimer, but the k (cat(NADPH)) and k(cat(NADH)) values were only about 50% those of the wild-type homodimer. With the four-electron acceptor, the K-m and k(ca t) values of H194A/HNQOR for NADPH and NADH were similar to those of t he low-efficiency mutant homodimer. These results suggest that the sub units of NQOR function independently with two-electron accepters, but dependently with a four-electron acceptor. This heterodimer approach m ay have general applications for studying the functional and structura l relationships of subunits in dimeric or oligomeric proteins.