DETECTION OF 2 HISTIDYL LIGANDS TO CU-A OF CYTOCHROME-OXIDASE BY 35-GHZ ENDOR - N-14,15 AND CU-63,65 ENDOR STUDIES OF THE CU-A SITE IN BOVINE HEART CYTOCHROME-AA(3) AND CYTOCHROME-CAA(3) AND CYTOCHROME-BA(3) 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 H-1 and N-14 resonances and permit analysis of the field dependence of the N-14 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-[delta,epsilon-N-15(2)]histidine. The 35-GHz Cu(A) ENDOR spectrum of this protein includes N-15 ENDOR resonances whose frequencies confirm the presence of two nitrogenous ligands; comparison with the N-14 ENDOR spectra further shows that the ligand with the larger hyperfine coupling (N1) displays well-resolved N-14 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 Cu(A) 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 Cu-63, Cu-65 ENDOR.