Refined structure of Cu-substituted alcohol dehydrogenase at 2.1 Å resolution

Liver alcohol dehydrogenase (LADH) is a Zn^II-dependent dimeric enzyme. LADH with the active-site Zn^II substituted by Cu^II resembles blue (type I) copper proteins by its spectroscopic characteristics. In this work we present the X-ray structure of the active site Cu^II-substituted LADH complex with NADH and dimethyl sulfoxide (DMSO). The structure was solved by molecular replacement. The space group is P2₁ with cell dimensions a = 44.4, b = 180.6, c = 50.8 Å and β = 108°. There is one dimer of the enzyme in the asymmetric unit. The refinement was carried out to a crystallographic R-factor of 16.1% for 41 119 unique reflections in the resolution range 12.0 to 2.1 Å. The coordination geometry of Cu^II in LADH is compared with the active-site metal coordination in the Zn–LADH–NADH–DMSO complex and blue-copper proteins. The distances from the metal to the protein ligands (Cys46, His67 and Cys174) are similar for the Zn^II and Cu^II ions. The distances of the O atom of the inhibitor DMSO to the Cu^II ion in the two subunits of the dimer are 3.19 and 3.45 Å. These are considerably longer than the corresponding distances for the Zn^II enzyme, 2.19 and 2.15 Å. The Cu^II ion is positioned nearly in the plane of the three protein ligands (NS₂) with a geometry similar to the trigonal arrangement of the three strongly bound ligands (N₂S) in blue-copper proteins. This coordination probably accounts for the similarity of the spectral characteristics of Cu^II–LADH and type I copper proteins.