Interaction of cytochrome bd with carbon monoxide at low and room temperatures - Evidence that only a small fraction of heme b(595) reacts with CO

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotole rant 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 re activity of the high spin heme b(595) with ligands. Using purified cytochro me 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 corre sponds to the reaction with CO of only about 5% of the heme. CO binding wit h a minor fraction of heme b(595) is also revealed at room temperature by t ime-resolved studies of CO recombination, The data resolve the apparent dis crepancies between conclusions drawn from room and low temperature spectros copic studies of the CO reaction with cytochrome bd, The results are consis tent with the proposal that hemes b(595) and d form a diheme oxygen-reducin g center with a binding capacity for a single exogenous ligand molecule tha t partitions between the hemes d and b(595) in accordance with their intrin sic affinities for the ligand, In this model, the affinity of heme b(595) f or CO is about 20-fold lower than that of heme d.