Fourier transform infrared spectroscopic studies of proton transfer processes and the dissociation of Zn2+-bound water in alcohol dehydrogenases.

ABSTRACT

The following complexes were investigated by Fourier transform difference spectroscopy binary complexes of alcohol dehydrogenases from yeast (YADH) and horse liver (LADH) with nicotinamide adenine dinucleotide (NAD+) and adenosine (5')-diphospho(5)-beta-D-ribose (ADP-Rib); the binary complex of Zn2+-free YADH with NAD+, the ternary complex of LADH with NAD+ and 2,2,2-trifluoroethanol. After addition of NAD+ to YADH and LADH, protonation of the N1 atom of the adenine ring of NAD+ is observed. It is shown that this proton arises from the dissociation of the Zn2+-bound water. The interaction of the Zn2+ ion with water is very strong, since this interaction is not just an electrostatic interaction. If the Zn2+ ions are in a tetrahedral environment, a large covalent contribution also occurs. If ADP-Rib is present instead of NAD+, no protonation of the N1 atom of the adenine ring of ADP-Rib is found, which demonstrates that the positively charged nicotinamide ring favors the conduction of the positive charge. All these results confirm the mechanism of Brändén et al. (1975) the Zn2+-bound water is split and the arising (OH)- deprotonates the alcohol. In the case of the ternary complex of LADH with NAD+ and 2,2,2-trifluoroethanol, we demonstrate that the alcohol is deprotonated and the alcoholate ion is bound directly to the Zn2+ ion. The conduction of the proton from the active site to the N1 atom of adenine occurs via a hydrogen-bonded chain with large proton polarizability due to collective proton motion. The nature and mechanism of this pathway are discussed on the basis of data from previous studies.