DT-DIAPHORASE - REDOX POTENTIAL, STEADY-STATE, AND RAPID REACTION STUDIES

NAD(P)H:quinone oxidoreductase (DT-diaphorase) appears to be a 2-elect ron transfer flavoprotein, which catalyzes the conversion of quinones into hydroquinones. Upon photoreduction in the presence of dimethylfor mamide, the enzyme forms a red semiquinone. In the absence of dimethyl formamide, only 10% of the radical form is thermodynamically stabilize d. This indicates a redox potential of the enzyme-bound semiquinone/re duced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple, The 2-electron redox potential wa s determined to be -159 +/- 3 mV at 25 degrees C, pH 7.0, In the prese nce of benzoquinone or 3-aminopyridine adenine dinucleotide phosphate, as NADPH analogue, there is no change in the redox properties of the enzyme flavin. A significant decrease is observed in the presence of t he competitive inhibitor dicumarol (E(m) = -234 +/- 2 mV at pH 7.0). T he reaction mechanism of the flavoprotein has been investigated by ste ady-state and stopped-flow kinetic methods using NADPH, NADH, deamino- NADPH, and 3-acetylpyridine adenine dinucleotide reduced form (APADH) as electron donors and K3Fe(CN)(6), xo-1H-pyrrolo-[2,3-f]quinoline-2,7 ,9-tricarboxylic acid (PQQ), and ridinyl-3,6-bis(carboethoxyamino)-1,4 -benzoquinone (AZQ) as electron accepters in 50 mM phosphate buffer, p H 7.0, 25 degrees C. No evidence could be obtained to indicate that se miquinoid intermediates play a part in the catalytic mechanism of DT-d iaphorase with quinones as accepters. The rates of the reduction by NA DPH, NADH, deamino-NADPH, and APADH (1.3 x 10(9), 8.8 x 10(8), 8.3 x 1 0(8) and 9.8 x 10(6) M(-1) min(-1), respectively) as well as the rates of the reoxidation by PQQ and AZQ (9 x 10(4) and 2.8 x 10(6) M(-1) mi n(-1), respectively) are directly proportional to substrate concentrat ion, and there is no evidence of the formation of enzyme-substrate com plexes. If such complexes do indeed exist, the affinity of the enzyme for substrate must be extremely low. Using K3Fe(CN)(6) as electron acc eptor, the rate of oxidation of fully reduced enzyme is 4.6 x 10(7) M( -1) min(-1) and it is accurately proportional to ferricyanide concentr ation. This rate represents that of flavin semiquinone formation, with the subsequent oxidation of the semiquinone being much faster, since no spectral evidence for semiquinone formation could be obtained. Stud ies were also conducted attempting to use apo-DT-diaphorase reconstitu ted with PQQ as coenzyme. The lack of activity toward AZQ, K3Fe(CN)(6) , and menadione suggests that DT-diaphorase can use PQQ only as electr on acceptor and not as redox cofactor.