The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation.
Klebsiella pneumoniae synthesized only b-type and d-type cytochromes under the wide range of growth conditions tested, and reaction with CO revealed two potential oxidases. The o-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 (less than 1 microM), 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 Mr 52,000 and 35,000), 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 (Km 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.
Smith
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A
Biochemistry Department, University of Southampton, UK.
Hill
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S
Anthony
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C
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000378:metabolism*; D009586:Nitrogen Fixation / Q000502:physiology*; D010088:Oxidoreductases / Q000302:isolation & purification / Q000502:physiology*; D013379:Substrate Specificity / Q000502:physiology
D000596:Amino Acids; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli