A Haemoprotein Is Not Involved in the Control by Oxygen of Enteric Nitrogenase Synthesis

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Methylobacterium extorquens AM1 contains a novel c-type cytochrome, called cytochrome c-553, previously thought to be a precursor of the electron acceptor (cytochrome cL) for methanol dehydrogenase. Its amino acid composition and serological characteristics show that it has no structural relationship to cytochrome cL. It usually comprises less than 5% of the total c-type cytochromes. In a moxD mutant, which contains neither methanol dehydrogenase nor cytochrome cL, it comprises 30% of the soluble cytochrome and it has been purified and characterized from that mutant. Cytochrome c-553 is large ( M , 23000), acidic and monohaem, with a redox potential of 194 mV. It reacts rapidly and completely with CO but is not autoxidizable. It is not autoreducible, and it is not an electron acceptor from methanol dehydrogenase or methylamhe dehydrogenase, nor an important electron donor to the oxidase. It is able to accept electrons from cytochrome cL and to donate electrons to cytochrome cH. It is present in the soluble fraction (presumably periplasmic) and membrane fraction of wild-type bacteria during growth on a wide range of growth substrates, but its function in these bacteria or in the moxD mutant has not been determined.

Section: Methodsmentioning

confidence: 99%

Characterization of a novel soluble c-type cytochrome in a moxD mutant of Methylobacterium extorquens AM1

Day

Nunn²,

Anthony³

1990

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“…Proteins were assayed by the bicinchoninic acid protein assay (Smith et al, 1988) with bovine serum albumin standards in detergent buffers. Haem was determined as the pyridine haemochrome as described by Fuhrhop & Smith (1975).…”

Section: Methodsmentioning

confidence: 99%

The o-type oxidase of the acidophilic methylotroph Acetobacter methanolicus

Chan

Anthony

1991

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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...

“…Protein was measured by the bicichoninic acid method adapted for use on microtitre plates as described by Smith et al (1988). For determination of its amino acid composition 1OOpg of purified oxidase was precipitated from solution with an equal volume of ice-cold acetone, washed twice in acetone, suspended in 100 p1 of AnalaR water (BDH) and transferred to a hydrolysis tube.…”

Section: Methodsmentioning

confidence: 99%

“…K. pneumoniae (oxytoca) (NCIB 12204) was maintained as described for E. coli by Smith et al (1988). The growth medium contained Na2HP04 (73 mM), KH2P0, (18 mM), nitriloacetic acid (300 VM) and the following (final concentration in mg 1-I): MgSO,.…”

Section: Methodsmentioning

confidence: 99%

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The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation

Smith

Hill

Anthony

1990

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Klebsiella pneumoniae synthesized only 6-type and d-type cytochromes under the wide range of growth conditions tested, and reaction with CO revealed two potential oxidases. The 0-type oxidase was produced only in the presence of O2 and appeared to be repressed by glucose. The d-type oxidase was, by contrast, produced only in the absence of measurable O2 (< 1 p~), and was the only oxidase expressed in nitrogen-fixing conditions. It was extracted from the membrane, purified and shown to be similar to that from E. coli in being a heterodimer (subunits of M, 52000 and 35000), in containing two distinguishable b haems and haem d (one or two molecules per molecule of oxidase), and in being able to react with O2 to give a stable oxygenated intermediate. The purified d-type cytochrome oxidase had a very high affinity for O2 (K, 20 nM; measured by the spectral properties of leghaemoglobin). It is proposed that this provides a role for this oxidase in lowering the O2 concentration to allow nitrogenase synthesis and function, and to provide a terminal oxidase to permit electron-transport-coupled ATP synthesis which supports the increase in efficiency of nitrogen fixation observed under microaerobic conditions.