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

Abstract

Liver alcohol dehydrogenase (LADH) is a Znll-depen- dent 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 H- substituted LADH complex with NADH and dimethyl sulfoxide (DMSO). The structure was solved by molecular replacement. The space group is P21o with cell dimensions a -- 44.4, b = 180.6, c = 50.8 A and /5---- 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 2.1A. 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 ~,. These are considerably longer than the corre- sponding distances for the Zn H enzyme, 2.19 and 2.15,4,. The Cun 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 prob- ably accounts for the similarity of the spectral character- istics of Cun-LADH and type I copper proteins.