Detection of Two Histidyl Ligands to Cu_A of Cytochrome Oxidase by 35-GHz ENDOR: ^14,15N and ^63,65Cu ENDOR Studies of the Cu_A Site in Bovine Heart Cytochrome aa₃ and Cytochromes caa₃ and ba₃ from Thermus thermophilus

To study the ligation of the Cu_A site of heme-copper terminal oxidases, we have performed ENDOR measurements at X-band (9-GHz) and 35-GHz microwave frequencies on the three titled enzymes. The 35-GHz measurements provide complete spectral separation of the ¹H and ¹⁴N resonances and permit analysis of the field dependence of the ¹⁴N ENDOR for each enzyme. The results indicate that two nitrogenous ligands were quite unequal hyperfine couplings are ligated to Cu_A in each of the enzymes studied. We have also examined cytochrome caa₃ isolated from His⁻ Thermus cells grown in the presence of d,l-[δ,ϵ-¹⁵N₂] histidine. The 35-GHz Cu_A ENDOR spectrum of this protein includes ¹⁵N ENDOR resonances whose frequencies confirm the presence of two nitrogenous ligands; comparison with the ¹⁴N ENDOR spectra further shows that the ligand with the larger hyperfine coupling (N1) displays well-resolved ¹⁴N quadrupole splitting. The theory for simulating frozen-solution ENDOR spectra as refined here permits a determination of both hyperfine and quadrupole tensors for N1 of all three enzymes. These indicate that the bonding parameters and geometry of Cu_A are well conserved. These measurements demonstrate unequivocally that two histidylimidazole N atoms are coordinated to CUA in the oxidized form of the enzyme and provide a first indication of the site geometry. On the basis of the previously established sequence homology among these heme-copper oxidases, it is likely that the two histidine residues conserved in all subunit II and subunit IIc sequences form part of the Cu_A binding site. The Cu_A sites in the three enzymes are further compared through ^63,65Cu ENDOR.