Abstract
Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. ADH is a membrane-bound quinohemoprotein-cytochrome c complex which consists of subunits I (78 kDa), II (48 kDa), and III (14 kDa) and contains several hemes c as well as pyrroloquinoline quinone as prosthetic groups. To understand the role of the heme c moieties in the intramolecular electron transport and the ubiquinone reduction, the ADH complex of Gluconobacter suboxydans was separated into a subunit I/III complex and subunit II, then reconstituted into the complex. The subunit I/III complex, probably subunit I, contained 1 mol each of pyrroloquinoline quinone and heme c and exhibited significant ferricyanide reductase, but no Q1 reductase activities. Subunit II was a triheme cytochrome c and had no enzyme activity, but it enabled the subunit I/III complex to reproduce the Q1 and ferricyanide reductase activities. Hybrid ADH consisting of the subunit I/III complex of G. suboxydans ADH and subunit II of Acetobacter aceti ADH was constructed and it had showed a significant Q1 reductase activity, indicating that subunit II has a ubiquinone-binding site. Inactive ADH from G. suboxydans exhibiting only 10% of the Q1 and ferricyanide reductase activities of the active enzyme has been isolated separately from active ADH (Matsushita, K., Yakushi, T., Takaki, Y., Toyama, H., and Adachi, O (1995) J. Bacteriol. 177, 6552-6559). Using these active and inactive ADHs and also isolated subunit I/III complex, we performed kinetic studies which suggested that ADH contains four ferricyanide-reacting sites, one of which was detected in subunit I and the others in subunit II. One of the three ferricyanide-reacting sites in subunit II was defective in inactive ADH. The ferricyanide-reacting site remained inactive even after alkali treatment of inactive ADH and also after reconstituting the ADH complex from the subunits, in contrast to the restoration of Q1 reductase activity and the other ferricyanide reductase activities. Thus, the data suggested that the heme c in subunit I and two of the three heme c moieties in subunit II are involved in the intramolecular electron transport of ADH into ubiquinone, where one of the two heme c sites may work at, or close to, the ubiquinone-reacting site and another between that and the heme c site in subunit I. The remaining heme c moiety in subunit II may have a function other than the electron transfer from ethanol to ubiquinone in ADH.
Highlights
None, where one of the two heme c sites may work at, or close to, the ubiquinone-reacting site and another between that and the heme c site in subunit I
Alcohol dehydrogenase (ADH) is a quinohemoprotein-cytochrome c complex bound to the periplasmic side of the cytoplasmic membrane and functions as the primary dehydrogenase in the ethanol oxidase respiratory chain, where ADH oxidizes ethanol by transferring electrons to ubiquinone embedded in the membrane phospholipids
The results indicated that subunit I of ADH is a quinohemoprotein which contains one molecule each of pyrroloquinoline quinone (PQQ) and heme c, that subunit II contains three heme c moieties which are responsible for ubiquinone reduction and that the four heme c sites of ADH are separately involved in the various ferricyanide reductase activities of the ADH complex
Summary
Monoclonal antibodies against the subunit I of ADH of G. suboxydans were prepared as described [17]. The detergent was omitted from the system at this point Another ADH fraction having relatively low enzyme activity was eluted with a linear gradient consisting of 5-bed volumes each of 100 and 200 mM acetate buffer (pH 5.0) followed by 5-bed volumes of 200 mM buffer. Solid ammonium sulfate was added to the fraction at a final concentration of 30% saturation, and the suspension was applied to a Phenyl-Sepharose column (about 5 mg of protein per 1-ml bed volume) equilibrated with 50 mM KPB (pH 6.0) containing 30% saturated ammonium sulfate and 2 mM CaCl2. Q1 reductase activity of ADH was measured spectrophotometrically by following the decrease of absorbance at 275 nm at 25 °C in a reaction mixture (1 ml) consisting of appropriate amounts of enzyme, 10 mM ethanol, 50 M Q1, and McIlvaine buffer (pH 4.5), as described [1]. One unit of these activities was defined as the amount of enzyme oxidizing 1 mol of ethanol per min
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