Reactions of the membrane-bound cytochrome bo terminal oxidase of Escherichia coli with carbon monoxide and oxygen.
The cytochrome 'bo' quinol oxidase of Escherichia coli contains 2 mol of haem, one or both of which are 'haem O'. One of the haems forms, with the single copper present, a binuclear site for ligand binding and oxygen reduction. Cytoplasmic membranes from a strain of E. coli lacking the alternative cytochrome bd quinol oxidase, and having amplified levels of cytochrome bo, were used to study oxygen and carbon monoxide reactivity with this oxidase. The high-spin ligand-binding haem was identified from its contribution to the Soret region and the shift in midpoint potential from +211 to +477 mV in the presence of CO. Oxidative titration of a CO-liganded sample was accompanied by a decrease in the contribution from a photodissociable CO-binding haem. The photodissociation spectrum was typical of a high-spin haem. Photolysis of CO-liganded, reduced membranes in the presence of O2 at sub-zero temperatures revealed O2 binding and cytochrome oxidation characterized by differential absorbance changes in the alpha-spectral region. Monitoring by epr spectroscopy of the same reaction sequence at -80 degrees C revealed a slight increase in g = 6 signal intensity immediately after photolysis attributable to cytochrome o oxidation prior to Cu oxidation. Subsequent decline in the g = 6 signal and appearance of a g = 3 signal indicated sequential electron flow from low-spin to high-spin haems and copper oxidation, suggesting that a second haem carries electrons from ubiquinol to the binuclear centre.
Division of Life Sciences, King's College London, UK.
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B
Bolgiano
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Salmon
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RK
Poole
D002248:Carbon Monoxide / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010782:Photolysis; D011199:Potentiometry; D013696:Temperature
D003573:Cytochrome b Group; D002248:Carbon Monoxide; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; D010100:Oxygen