The Dinuclear Center of Cytochrome bo 3 from Escherichia coli

For the study of the dinuclear center of heme-copper oxidases cytochrome bo₃ from Escherichia coli offers several advantages over the extensively charactererized bovine cytochrome c oxidase. The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies. Cytochrome bo₃ is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in CcO. The absence of Cu_A and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o₃. Careful analysis allows us to distinguish between small molecules that bind to heme o₃, those which are ligands of Cu_B, and those which react to yield higher oxidation states of heme o₃. Here we review results from our studies of the reactions of fast cytochrome bo₃ with formate, fluoride, chloride, azide, cyanide, NO, and H₂O₂.