The Purification and Characterization of the o-type Cytochrome Oxidase from Methylophilus methylotrophus, and its Reconstitution into a 'Methanol Oxidase' Electron Transport Chain

Froud, Stephen J.; Anthony, Christopher

doi:10.1099/00221287-130-9-2201

Cited by 15 publications

(31 citation statements)

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“…Much less is known about the second class of o-type oxidase, cytochrome co, although it has been purified from a number of bacteria, including Methylophilus rnethylotrophus (Carver & Jones, 1983;Froud & Anthony, 1984), Azotobacter vinelandii (Yang & Jurtshuk, 1978;Jurtshuk et al, 1981;Hunter et al, 1989), Rhodopseudornonas palustris (King & Drews, 1976), and Pseudornonas aeruginosa (Matsushita et al, 1982). Cytochrome co is sensitive to low concentrations of azide and cyanide, and it oxidizes the high-potential substrates cytochrome c and ascorbate/TMPD, but not the lowpotential substrate ubiquinol.…”

Section: H T C Chan and C Anthonymentioning

confidence: 99%

“…The kinetic constants for this inhibition, and the relative rates of oxidation of TMPD and cytochrome, are similar to those previously published for the cytochrome co of M . methylotrophus (Froud & Anthony, 1984).…”

Section: Characterization Of the Membranes Of A Methanolicusmentioning

confidence: 99%

“…By contrast, the substrate for cytochrome co is a class I periplasmic cytochrome c, whose oxidation is usually particularly sensitive to azide, the Ki for azide being less than 5 p .~ (Froud & Anthony, 1984;Auton & Anthony, 1989). Cytochrome co may also be assayed by taking advantage of its ability to oxidize TMPD (usually together with substrate amounts of ascorbate).…”

Section: Introductionmentioning

confidence: 99%

“…Cytochrome co may also be assayed by taking advantage of its ability to oxidize TMPD (usually together with substrate amounts of ascorbate). One of the first to be purified and shown to be a cytochrome c oxidase was the cytochrome co from the obligate methylotroph Methylophilus rnethylotrophus (Froud & Anthony, 1984). This has been purified to homogeneity and shown to consist of equal amounts of band c-type cytochromes, corresponding to the two types of protein subunit seen on SDS-PAGE; these had molecular masses of 3 1.5 kDa and 23.8 kDa respectively.…”

Section: Introductionmentioning

confidence: 99%

“…It was also shown that the cytochrome c subunit did not correspond to either of the two soluble cytochromes c from M . rnethylotrophus (Froud & Anthony, 1984;Anthony, 1986). The oxidase was found to be very difficult to purify reproducibly and it was unstable (Carver & Jones, 1983;Froud & Anthony, 1984).…”

Section: Introductionmentioning

confidence: 99%

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The o-type oxidase of the acidophilic methylotroph Acetobacter methanolicus

Chan

Anthony

1991

Journal of General Microbiology

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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...

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Section: H T C Chan and C Anthonymentioning

confidence: 99%

Section: Characterization Of the Membranes Of A Methanolicusmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The o-type oxidase of the acidophilic methylotroph Acetobacter methanolicus

Chan

Anthony

1991

Journal of General Microbiology

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Cytochromes c‐dependent aerobic respiration of Paracoccus denitrificans

Kučera

1993

J. Basic Microbiol.

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Terminal parts of the respiratory chain of Paracoccus denitrificans containing cytochromes c have been investigated through the use of NNN′Nprime;‐tetramethyl‐p‐phenylenediamine (TMPD) as an electron donor. The cells have been shown to possess two major membrane bound TMPD oxidases characterized by their different sensitivities to cyanide. The more sensitive one, identified as cytochrome aa3, appears to be produced under oxygen‐rich growth conditions, whereas a less sensitive and as yet uncharacterized enzyme accompanied with only moderate amounts of cytochrome aa3 functions when oxygen becomes limiting. The soluble periplasmic cytochrome cd1 does not contribute significantly to the total oxygen consumption rate, although its maximal catalytic capacity may be high. The fact thap the activity of alternative TMPD oxidase is not proportional to cytochrome o content is discussed in relation to possible occurrence of two distinct cytochrome o‐type terminal oxidases.

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The mechanism of inhibition by EDTA and EGTA of methanol oxidation by methylotrophic bacteria

Chan

Anthony

1992

FEMS Microbiology Letters

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Ethyleneglycol (aminoethylether) tetra-acetic acid (EGTA) was shown to be a potent competitive inhibitor of electron transfer between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. Addition of Ca2+ ions relieved the inhibition by removal of the inhibitory EGTA. Removal of EGTA by gel filtration completely relieved the inhibition. EGTA did not remove the tightly bound Ca2+ present in the MDH. Indo-1, a fluorescent analogue of EGTA, bound tightly to MDH in a 1:1 ratio but not to cytochrome cL; binding was prevented by EGTA. It was concluded that EGTA inhibits methanol oxidation by binding to lysyl or arginyl residues on MDH thus preventing docking with cytochrome cL.

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The Purification and Characterization of the o-type Cytochrome Oxidase from Methylophilus methylotrophus, and its Reconstitution into a 'Methanol Oxidase' Electron Transport Chain

Cited by 15 publications

References 36 publications

The o-type oxidase of the acidophilic methylotroph Acetobacter methanolicus

The o-type oxidase of the acidophilic methylotroph Acetobacter methanolicus

Cytochromes c‐dependent aerobic respiration of Paracoccus denitrificans

The mechanism of inhibition by EDTA and EGTA of methanol oxidation by methylotrophic bacteria

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