HYDROXYLATED NAPHTHOQUINONES AS SUBSTRATES FOR ESCHERICHIA-COLI ANAEROBIC REDUCTASES

We have used two hydroxylated naphthoquinol menaquinol analogues, redu ced plumbagin (PBH2, 5-hydroxy-2-methyl-1,4-naphthoquinol) and reduced lapachol [LPCH2, hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol], a s substrates for Escherichia coli anaerobic reductases. These compound s have optical, solubility and redox properties that make them suitabl e for use in studies of the enzymology of menaquinol oxidation. Oxidiz ed plumbagin and oxidized lapachol have well resolved absorbances at 4 19 nm (epsilon = 3.95 mM(-1).cm(-1)) and 481 nm (epsilon = 2.66 mM(-1) .cm(-1)) respectively (in Mops/KOH buffer, pH 7.0). PBH2 is a good sub strate for nitrate reductase A (K-m = 282 +/- 28 mu M, k(cat) = 120 +/ - 6 s(-1)) and fumarate reductase (K-m = 155 +/- 24 mu M, k(cat) = 30 +/- 2 s(-1)), but not for DMSO reductase. LPCH2 is a good substrate fo r nitrate reductase A (K-m = 57 +/- 35 mu M, k(cat) = 68 +/- 13 s(-1)) , fumarate reductase (K-m = 85 +/- 27 mu M, k(cat) = 74 +/- 6 s(-1)) a nd DMSO reductase (K-m = 238 +/- 30 mu M, k(cat) = 191 +/- 21 s(-1)). The sensitivity of enzymic LPCH2 and PBH2 oxidation to 2-n-heptyl-4-hy droxyquinoline N-oxide inhibition is consistent with their oxidation o ccurring at sites of physiological quinol binding.