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

Ryszard J. Gurbiel, Yang Cheng Fann, Melanie M. Werst, Peter E. Doan, Brian M. Hoffman*, Kristene K. Surerus, James A. Fee, Siegfried M. Musser, Sunney I. Chan

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

To study the ligation of the CuA 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 CuA 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 CuA 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 CuA 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 CuA binding site. The CuA sites in the three enzymes are further compared through 63,65Cu ENDOR.

Original languageEnglish (US)
Pages (from-to)10888-10894
Number of pages7
JournalJournal of the American Chemical Society
Volume115
Issue number23
DOIs
StatePublished - Nov 1 1993

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry

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