EPR and kinetic analysis of the interaction of halides and phosphate with nitrate reductase.
Electron paramagnetic resonance spectra obtained during turnover of the Mo center of NADH:nitrate reductase at pH 8 were comprised of two Mo(V) species, signal A (g1 = 1.996, g2 = 1.969, g3 = 1.967, A1H = 1.25 mT, A2H = 1.18 mT, and A3H = 1.63 mT) and signal B (g1 = 1.996, g2 = 1.969, and g3 = 1.967), the former exhibiting superhyperfine interaction due to strong coupling with a single, exchangeable proton. Binding of halides and nitrite to the Mo center increased the proportion of signal A whereas phosphate had no effect on the EPR line shape. Halides decreased and phosphate increased the rates of enzyme activities involving the Mo center (NADH:nitrate reductase and reduced methyl viologen:nitrate reductase), but neither had any effect on activities involving FAD (NADH:ferricyanide reductase) or heme (NADH:cytochrome c reductase), indicating specific binding of halides to the Mo center. Halides were found to be weak, mixed competitive-noncompetitive inhibitors (Cl- KI = 39 mM, mu = 0.2 M, pH 8) of nitrate reductase forming a catalytically inactive ternary halide-nitrate-enzyme complex. Inhibition patterns changed from nearly noncompetitive (F-) to nearly competitive (I-). The weakening of nitrate binding due to halide binding correlated with increased halide electronegativity rather than ionic radius. In contrast, phosphate (Kd = 7.4 mM, mu = 0.2 M, pH 8) and arsenate were determined to be nonessential activators, characterized by a constant value of (Vmax/Km)app, increasing nitrate reductase activity by weakening nitrate binding without affecting the stability of the transition state. Phosphate had no effect on product inhibition by nitrite (KI = 0.33 mM) or the oxidation-reduction midpoint potentials of the Mo center.
Kay
C J
CJ
Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa 33612.
Barber
M J
MJ
D001149:Arsenates / Q000494:pharmacology; D002708:Chlorella / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D006219:Halogens / Q000494:pharmacology; D007700:Kinetics; D008982:Molybdenum; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology
D001149:Arsenates; D006219:Halogens; D010710:Phosphates; D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C025657:arsenic acid