Membranes of the acidophilic methylotroph contained only - and -type cytochrome and a CO-binding -type cytochrome. An azide-sensitive oxidase that oxidizes cytochrome and ascorbate/TMPD was solubilized from the membrane with a mixture of CHAPS and Zwittergent (1.7 fold increase in specific activity with 32% yield). The solubilized oxidase is unusually stable with respect to high ionic strength (200 mM-NaCl) and stable between pH 4.0 and 6.8. Of the two soluble -type cytochromes from only the typical class I cytochrome (cytochrome ) was a good substrate, as was equine cytochrome The oxidase was partially purified by anion-exchange chromatography but further purification proved impossible. The yield with respect to equine cytochrome oxidation was 18%, with a 22-fold purification, but during purification most of the activity with respect to cytochrome 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 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 -type cytochrome, some of which was CO-reactive. It is proposed that the oxidase is a cytochrome type of oxidase that loses its cytochrome component during the purification process and is only able to oxidize -type cytochromes if these can be formed into a ‘reconstituted’ cytochrome with the partially-purified oxidase.


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