Spontaneous spectral changes of the reduced cytochrome bd

Reduction of the membrane-bound cytochrome bd from Bacillus subtilis, Escherichia coli and Azotobacter vinelandii as well as of the purified enzyme from E. coli was followed by secondary absorption changes on a time scale of tens of minutes. The difference absorption spectra of these changes resembled those induced by CO binding with heme d2+ indicating interaction of the heme with an endogenous pi-acceptor ligand. The spontaneous spectral changes were prevented and reversed by CO binding with the reduced cytochrome bd. Bonding of heme d iron to an endogenous protein ligand at the sixth axial position upon reduction is proposed and several possible mechanisms of such a process are considered.