The 'methanol oxidase' system in an acidophilic methylotroph, Acetobacter methanolicus
University of Southampton, 1990
Online
Hochschulschrift
Zugriff:
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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The 'methanol oxidase' system in an acidophilic methylotroph, Acetobacter methanolicus
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Autor/in / Beteiligte Person: | Chan, Hak Tak Claude |
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Veröffentlichung: | University of Southampton, 1990 |
Medientyp: | Hochschulschrift |
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