An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

ABSTRACT Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO 2 , and H 2 , together with minor amounts of ethanol. The organism also generates H 2 S in the presence of elemental sulfur (S 0 ). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S 0 . The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (∼1.0 g-atom/subunit) and zinc (∼1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent K m s (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.