The 'methanol oxidase' system in an acidophilic methylotroph, Acetobacter methanolicus

Chan, Hak Tak Claude (1990) The 'methanol oxidase' system in an acidophilic methylotroph, Acetobacter methanolicus. University of Southampton, Doctoral Thesis.

Abstract

This thesis investigates two of the electron transfer proteins, namely methanol dehydrogenase (MDH) and cytochrome oxidase, in an acidophilic methylotroph Acetobacter methanolicus during growth on methanol. The presence of a small subunit of MDH (β-subunit) arranged in a α₂β_2 configuration was demonstrated. The amino acid sequence of the small subunit was determined and the predicted secondary structure showed that the lysine-rich amiphipathic helix at the C-terminal of the β-subunits (the proposed cytochrome c_L binding site) of other methylotrophs is absent in A.methanolicus; only 8% of the amino acids were lysine (compared with 13% and 20% in P.denitrificans and M.extorquens). These observations suggested that either the previous proposal was incorrect or a different protein-protein interaction mechanism is operating in A.methanolicus. A new assay for MDH was developed which uses cytochrome c_L to mediate electron transfer to an excess of the dye PIP. The reaction was extremely sensitive to the ionic strength of the buffer, indicating that the interaction of MDH and cytochrome c_L involves electrostatic forces, although there is an evident lack of charged amino acid residues in the β-subunit. A detectable MDH/cytochrome c_L complex whose formation is inhibited by NaCl was observed, confirming this conclusion. The extent of electrostatic forces responsible for the MDH/cytochrome c_L interaction is however not clear. During growth on methanol, membranes of A.methanolicus contained only b- and c-type cytochrome and a CO-binding b-type cytochrome. An azide-sensitive oxidase that oxidizes cytochrome c and ascorbate/TMPD was solubilized from the membrane with a mixture of CHAPS and Zwittergent_3-12 (1.7-fold increase in specific activity with 32% yield). The solubilized oxidase is unusually stable with respect to high ionic strength (0.2 M-NaCl) and stable between pH 4.0 and 6.8. Of the two soluble c-type cytochromes from A.methanolicus only the class I cytochrome c_H 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 18%, with a 22-fold purification, but during purification most of the activity with respect to cytochrome c_H 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 4-fold. During the purification process the pH optimum for cytochrome c_H was unchanged (pH 5.5) but that for 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 b-type 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 it is able to `reconstitute' a cytochrome co with them.

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