An EPR study of the blue copper center in horse liver alcohol dehydrogenase

Abstract

Active-site specifically reconstituted Cu 2+ 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∥ = 2.16, g⊥ =2.04; A∥ = 100·10 −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 ( g 1 = 2.21, g 2 = 2.06, g 3 = 2.03, A 1 = 50·10 −4 cm −1, A 2 = 30·10 − 4 cm −1, A 3 = 76·10 −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 Cu 2+ in horse liver alcohol dehydrogenase and copper proteins are compared.