Active trihydroxynaphthalene reductase (3HNR) is essential for the biosynthesis of fungal melanin by Magnaporthe grisea and is a focus of inhibitor design studies directed toward control of blast disease in rice. Tricyclazole, a preventative fungicide against rice blast, has been previously characterized as inhibiting 3HNR noncompetitively [Viviani, F., Vors, J. P., Gaudry, M., & Marquet, A. (1993) Bull. Soc. Chem. Fr. 136, 395-404] with respect to its naphthol substrate. Our steady-state kinetic and fluorescence titration studies show that instead the inhibitor binds competitively with respect to the naphthol substrate and that it binds to 3HNR forms with the preferences 3HNR.NADPH > 3HNR.NADP+ > 3HNR (unliganded): Kt = 15 nM, 0.56 microM, and Kd = 8.5 microM, respectively. Analysis of the frontier molecular orbitals of tricyclazole and NADP(H) provides a basis for the affinity differences of tricyclazole for 3HNR.NADP(H) enzyme forms. Fluorescence titrations show that NADPH and naphthol substrates form binary complexes with 3HNR [Kd(NADP+) = 38 microM and Kd(U7278, an alternate naphthol-like substrate) = 220 microM]. However, the overwhelmingly preferred order of productive binding is NADPH followed by naphthol substrate, as shown by the uncompetitive inhibition of 3HNR by tricyclazole with respect to NADPH. Consistent with this mechanism, the K(m)'s for the naphthol substrates U7278 and scytalone (5 and 6 microM, respectively) are much lower than the Kd's of the binary complexes. The partition ratio of U7278 and a physiological substrate (scytalone) was 95:1 and unchanged on varying 3HNR.NADP+/3HNR(unliganded), which is also consistent with the ordered mechanism. The pH dependence of the hydride transfer rate from U7278 to NADP+ was measured, as was the pH dependence of Kcat/K(m)(NADP+). Hydride transfer had a pH dependence which suggests a single deprotonated residue (pKa = 6.0) is required for catalysis. Khyd, the rate constant for hydride transfer, was 9-fold larger than Kcat with U7278 as a substrate. A burst in the pre-steady-state suggests that release of one or both of the products is rate limiting to Kcat at pH 7.0. The pH dependence of Kcat/K(m)(NADP+) indicates a requirement for a single deprotonated group and this ionization is assigned to the 2' phosphate of NADP+. 3HNR was found to be 800-fold more specific for NADP+ relative to NAD+. Analysis of sequence and structure [Anderson, A., Jordan, D. B., Schneider. G., & Lindqvist. Y. (1996) Structure 4, 1161-1170] reveals that 3HNR is a member of the short-chain dehydrogenase superfamily of enzymes.