The Cu(A) center is a dinuclear Cu2S2(Cys) electron transfer center found in cytochrome c oxidase and nitrous oxide reductase. In a previous investigation of the equatorial histidine ligands' effect on the reduction potential, electron transfer and spectroscopic properties of the Cu(A) center, His120 in the engineered Cu(A) azurin was mutated to Asn, Asp, and Ala. The identical absorption and EPR spectra of these mutants indicate that a common ligand is bound to the copper center. To identify this replacement ligand, the His120Gly Cu(A) azurin mutant was constructed and purified. Absorption and X-band EPR spectra show that His120Gly is similar to the other His120X (X=Asn, Asp, Ala) mutant proteins. Titrations with chloride, imidazole, and azide suggest that the replacement ligand is not exchangeable with exogenous ligands. The possibility of an internal amino acid acting as the replacement ligand for His120 in the His120X mutant proteins was investigated by analyzing the Cu(A) azurin crystal structure and then converting the likely internal ligand, Asn119, to Asp, Ser, or Ala in the His120Gly mutant. The double mutants H120G/Asn119X (X=Asp, Ser, or Ala) displayed UV-Vis absorption and EPR spectra that are identical to His120Gly and the other His120X mutants, indicating that Asn119 is not the internal ligand replacing His120 in the His120X mutant proteins. These results demonstrate the remarkable stability of the dinuclear His120 mutants of Cu(A) azurin. Copyright (C) 1999 Elsevier Science Inc.
Bibliographical noteFunding Information:
The authors thank Rich Milberg of the University of Illinois Mass Spectrometry Lab for collecting the mass spectrometry data. This work is supported by the National Science Foundation under Award No. CHE 95-02421 to Y.L. (CAREER Award and Special Creativity Extension). S.M.B. thanks the NIH for financial support through a biophysics training grant. Y.L. is a Sloan Research Fellow of the Alfred Sloan Foundation, a Cottrell Scholar of the Research Corporation, a Camille Dreyfus Teacher-Scholar of the Camille and Henry Dreyfus Foundation, and a Beckman Young Investigator of the Arnold and Mabel Beckman Foundation.
- Cytochrome c oxidase
- Electron paramagnetic resonance spectroscopy
- Ligand substitution
- Nitrous oxide reductase