Active-site cobalt(II)-substituted horse liver alcohol dehydrogenase: characterization of intermediates in the oxidation and reduction processes as a function of pH

Substitution of Co(I1) for the catalytic site Zn(I1) of horse liver alcohol dehydrogenase (LADH) yields an active enzyme derivative, Co"E, with characteristic Co(I1) charge-transfer and d-d electronic transitions that are sensitive to the events which take place during catalysis (Koerber, S. C., MacGibbon, A. K. H., Dietrich, H., Zeppezauer, M., & Dum, M. F. (1983) Biochemistry 22, 3424-34311. In this study, UV-visible spectroscopy and rapid-scanning stopped-flow (RSSF) kinetic methods are used to detect and identify intermediates in the LADH catalytic mechanism. In the presence of the inhibitor isobutyramide, the pre-steady-state phase of alcohol (RCH,OH) oxidation at p H above 7 is characterized by the formation and decay of an intermediate with A,,, = 570, 640, and 672 nm for both aromatic and aliphatic alcohols (benzyl alcohol, p-nitrobenzyl alcohol, anisyl alcohol, ethanol, and methanol). By comparison with the spectrum of the stable ternary complex formed with oxidized nicotinamide adenine dinucleotide (NAD+) and 2,2',2''-trifluoroethoxide ion (TFE-), Co"E(NAD+,TFE-), the intermediate which forms is proposed to be the alkoxide ion (RCH20-) complex, Co"E(NAD+,RCH,O-). The timing of reduced nicotinamide adenine dinucleotide (NADH) formation indicates that intermediate decay is limited by the interconversion of ternary complexes, Le., Co"E(NAD+,RCH20-) * Co"E(NADH,RCHO). From competition experiments, we infer that, at p H values below 5, NAD+ and alcohol form a Co"E(NAD+,RCH20H) ternary complex. RSSF studies carried out as a function of pH indicate that the apparent pK, values for the ionization of alcohol within the ternary complex, Le., Co"E(NAD+,RCH20H ) Co"E(NAD+,RCH20-) + H+, fall in the range 5-7.5. Using pyrazole as the dead-end inhibitor, we find that the single-turnover time courses for the reduction of benzaldehyde, p-nitrobenzaldehyde, anisaldehyde, and acetaldehyde at pH above 7 all show evidence for the formation and decay of an intermediate. Via spectral comparisons with Co"E- (NAD+,TFE-) and with the intermediate formed during alcohol oxidation, we identify the intermediate as the same Co"E(NAD+,RCH20-) ternary complex detected during alcohol oxidation.