Direct evidence of nitrogen coupling in the copper(II) complex of bovine serum albumin by S-band electron spin resonance technique.
ESR spectra of the tight binding Cu(II) complex of bovine serum albumin (BSA) has been studied using S-band. At physiological pH, only one form of copper binding to BSA was detected from the ESR spectra. From previous X-band ESR spectra, nitrogen superhyperfine splittings were observable in the g perpendicular region; however, the resolution of the g parallel region was not sufficient to confirm the exact donor atoms of the complex. Using low-frequency ESR (2-4 GHz) at 77 K, we have resolved the nitrogen superhyperfine structure in the g parallel region. A computer simulation method has been developed for distinguishing between three and four nitrogen donor atoms. The Hyde-Froncisz theory of g and A strain broadening has been modified to use a field-swept calculation for the line shape. The observed intensity pattern and the computer simulation of such spectra positively confirm the structure of Cu(II) ion coordinated to four in-plane nitrogen atoms in frozen aqueous solutions of Cu(II)-BSA complexes at physiological pH. This is the first time that this binding site has been confirmed on the protein instead of a protein fragment or model compound. This work is another example of the usefulness of the S-band ESR technique for characterizing the metal-protein interactions when random variation in g factors cause line broadening in conventional X-band ESR spectra.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003300:Copper / Q000032:analysis*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D009584:Nitrogen / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis
D012710:Serum Albumin, Bovine; D003300:Copper; D009584:Nitrogen