An EPR study of the blue copper center in horse liver alcohol dehydrogenase.
Active-site specifically reconstituted Cu2+ horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) shows optical and EPR spectra similar to those of native blue copper (Type 1) proteins. EPR spectra at different temperatures and frequencies reveal a heterogeneity of the copper center: a minor 'non-blue' species with axial line shape (g parallel = 2.16, g perpendicular = 2.04; A parallel = 100 x 10(-4) cm-1), which accounts for approximately 10% of the total copper and is not accessible to ligands and a major blue species with rhombic line shape (g1 = 2.21, g2 = 2.06, g3 = 2.03, A1 = 50 x 10(-4) cm-1, A2 = 30 x 10(-4) cm-1, A3 = 76 x 10(-4) cm-1), which is accessible to ligands and participates in redox reactions. The major blue species in cupric horse liver alcohol dehydrogenase is metastable, since it is reduced in a process markedly accelerated in the presence of oxygen or hydrogen peroxide. In addition, the reduction depends on the presence of exogenous metal ligands or coenzymes. Whereas the binary complex enzyme-NAD+ is even more susceptible to bleaching than the free enzyme, the cupric center is stable towards bleaching in the binary complex enzyme-NADH. In the discussion the redox properties and coordination chemistry of Cu2+ in horse liver alcohol dehydrogenase and copper proteins are compared.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003300:Copper / Q000032:analysis*; D004578:Electron Spin Resonance Spectroscopy; D006736:Horses; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D009243:NAD; D011485:Protein Binding
D008024:Ligands; D009243:NAD; D003300:Copper; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase