Nitrate reductase from Penicillium chrysogenum : The reduced flavin-adenine dinucleotide-dependent reaction

Nitrate reductase from Penicillium chrysogenum catalyzes the FADH 2 -dependent reduction of nitrate in the absence of excess dithionite. At pH 7.2 (0.1 m potassium phosphate buffer) the FADH 2 /nitrate reciprocal plot patterns were parallel. Nitrite was competitive with nitrate and an uncompetitive inhibitor with respect to FADH 2. FAD was competitive with FADH 2 and uncompetitive with nitrate. FMNH 2 and riboflavin H 2 also served as reducing agents and yielded the same kinetic patterns. The results are consistent with a nonclassical, two-site model in which nitrate and nitrite bind to a site different from the site at which FADH 2 and FAD bind. The following results suggest that the site at which FADH 2 binds to reduce the enzyme (and at which FAD binds in competition with FADH 2 to inhibit the reaction) is not the same high-affinity site to which FAD binds as a cosubstrate in the NADPH + FAD-dependent reaction: (a) The K m for FAD in the NADPH + FAD-dependent reaction at pH 7.2 is only 0.17 μ m, while the K i, for FAD in the FADH 2 -dependent reaction is 65 μ m. (b) FAD is the preferred flavin in the NADPH + oxidized flavin-dependent reaction. (The K m values for FMN and riboflavin are 15 and 41 μ m, respectively.) But all three oxidized flavins have about the same K i in the reduced flavin-dependent reaction (65, 80, and 165 μ m, respectively). Furthermore, (c) the K m values for FADH 2, FMNH 2, and riboflavinH 2 in the reduced flavin-dependent reaction are all about the same (ca. 900 μM), (d) V values (specific activities in units per milligram of protein) of the NADPH + oxidized flavin-dependent reaction in 0.1 m phosphate, pH 7.2, are in the order FAD (225) > FMN (150) > riboflavin (60), but the values in the reduced flavin-dependent reaction are in the order FADH 2 (100) < FMNH 2 (150) < riboflavinH 2 (500). Dithionite is known to reduce the cytochrome b component of nitrate reductase. But in the absence of a flavin, the specific activity of the dithionite-dependent reaction is only about 7—extremely low compared to the range of 60–500 for the nucleotide-dependent reactions. This result, together with the intersecting FAD/nitrate reciprocal plot patterns, suggests two possible models for the NADPH + FAD-dependent reactions: (a) FAD binds to two different high-affinity sites. At the “upstream” site, FAD mediates the transfer of electrons from NADPH to the cytochrome b prosthetic group. At the “downstream” site FAD plays either an additional electron transfer role or an activator role promoting the flow of electrons to nitrate. (b) There is only one high-affinity FAD site. FAD must occupy this site (even after the cytochrome is reduced) in order for electrons to flow to nitrate.