The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe. We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b of the fully reduced enzyme with low affinity. EPR spectrum of the NO complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g = 2.041 and g = 1.993 and a clear triplet of triplet (or a triplet of doublet for the NO complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue. Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b. This indicates that heme d exists in close proximity to heme b forming a binuclear center. Another axially symmetric EPR signal with g-values at g = 2.108 and g = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b-NO complex.

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