The o-type oxidase of the acidophilic methylotroph Acetobacter methanolicus

Abstract

Membranes of the acidophilic methylotroph Acetobucter methanolicus contained only b-and c-type cytochrome and a CO-binding b-type cytochrome. An azide-sensitive oxidase that oxidizes cytochrome c and ascorbatel TMPD was solubilized from the membrane with a mixture of CHAPS and Zwittergent3-12 (1.7 fold increase in specific activity with 32% yield). The solubilized oxidase is unusually stable with respect to high ionic strength (200 mM-NaC1) and stable between pH 4.0 and 6.8. Of the two soluble c-type cytochromes from A. methanoficus only the typical class I cytochrome (cytochrome cH) was a good substrate, as was equine cytochrome c. The oxidase was partially purified by anion-exchange chromatography but further purification proved impossible. The yield with respect to equine cytochrome c oxidation was l8%, with a 22-fold purification, but during purification most of the activity with respect to cytochrome cH and TMPD was lost. Neutral phospholipids had little effect on activity of the oxidase but the charged phospholipids phosphatidylglycerol and phosphatidylserine stimulated activity up to about fourfold. It proved impossible to incorporate the oxidase into active lipoprotein vesicles. During the purification process the pH optimum for oxidation of cytochrome cH was unchanged (pH 5.6) whereas that for oxidation of equine cytochrome changed from pH 9.5 to 7.5 and the sensitivity of the oxidase to azide changed from non-competitive to competitive during the purification process. The partially-purified oxidase contained only btype cytochrome, some of which was CO-reactive. It is proposed that the oxidase is a cytochrome co type of oxidase that loses its cytochrome c component during the purification process and is only able to oxidize c-type cytochromes if these can be formed into a 'reconstituted' cytochrome co with the partially-purified oxidase. IntroductionThe present paper describes work on an unusual o-type oxidase from the acidophilic methylotroph Acetobacter methanolicus, which grows at pH 4 on methanol as its sole carbon and energy source (Steudel & Babel, 1982;Steudel et al., 1980). As in other methylotrophic bacteria, methanol is oxidized by way of the quinoprotein methanol dehydrogenase, its specific electron acceptor cytochrome cL, and a typical class I c-type cytochrome which is oxidized by a membrane oxidase (Anthony, 1982(Anthony, , 1986Elliott & Anthony, 1988). Except for the oxidase all these proteins are periplasmic and so operate at pH4, the pH of the growth medium. Preliminary observations suggested that this oxidase may be more stable to extremes of pH and salt concentration compared with cytochrome oxidases in other bacteria.The bacterial o-type oxidases are defined loosely as oxidases having a CO-binding cytochrome b, which for convenience is usually referred to as cytochrome o and which is assumed to be the oxygen-reactive site. Cytochrome o was first described in Staphylococcus aureus (Chance, 1953 ;Castor & Chance, 1959) and the 0-type oxidases have since been shown to be the most widely...

The interaction of methanol dehydrogenase and cytochrome cL in the acidophilic methylotroph Acetobacter methanolicus

“…It was kindly donated by Professor W. Babel (Institut fur Biotechnologie, Leipzig, Germany). Methods for growth on methanol at pH 4.0 and preparation of extracts were exactly as described previously (Chan & Anthony, 1991).…”

“…These methods are all as described by Chan & Anthony (1991), molecular masses being determined with Sigma Dalton mark VII standard proteins.…”

“…A more important consideration, however, with respect to protein-protein interactions is that it is an acidophilic bacterium and so its MDH, cytochrome CL and cytochrome CH interact in the periplasm at pH values between pH 4 and 5.5. As with other bacteria growing on methanol, it has a basic MDH (pl 7.8) and acidic cytochrome CL (pl about 4.8), both of these proteins being stable down to pH 3 (Elliott & Anthony, 1988;Chan & Anthony, 1991). Neither protein reacts with antibodies to the corresponding proteins from M. extorquens or Methylophilus methylotrophus.…”

Physiology of Acetic Acid Bacteria in Light of the Genome Sequence of <i>Gluconobacter oxydans</i>

“…Acetobacter species additionally contain an ubiquinol oxidase of the a 1 type. Only A. methanolica contains a cytochrome c oxidase when grown on methanol [Chan and Anthony, 1991]. Gluconobacter, Gluconacetobacter as well as Acidomonas , Kozakia and Asaia use ubiquinones of the Q-10 type as electron carrier in their cytoplasmic membranes.…”

The periplasmic modifier protein for methanol dehydrogenase in the methylotrophs Methylophilus methylotrophus and Paracoccus denitrificans

“…Dehydrogenases for glucose 6-phosphate and 6-phosphogluconate were determined as described by Lawton & Anthony (1989, and malate dehydrogenase was determined as described by Alefounder & Ferguson (1981). Cytochromes were measured as described by Cross & Anthony (19806) and the o-type oxidase was determined as described by Chan & Anthony (1991).…”