Interaction of cytochrome bd with carbon monoxide at low and room temperatures: evidence that only a small fraction of heme b595 reacts with CO.
Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b(595) with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b(595). However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b(595) is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b(595) and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b(595) in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b(595) for CO is about 20-fold lower than that of heme d.
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
V B
VB
Borisov
S E
SE
Sedelnikova
R K
RK
Poole
A A
AA
Konstantinov
D002248:Carbon Monoxide / Q000378:metabolism*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D006418:Heme / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D013696:Temperature
D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D006418:Heme; D002248:Carbon Monoxide; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli