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Article
Proton NMR Spectroscopy as a Probe of Dinuclear Copper(II) Active Sites in Metalloproteins. Characterization of the Hyperactive Copper(II)-Substituted Aminopeptidase from Aeromonas proteolytica
Journal of the American Chemical Society
  • Richard C. Holz, Marquette University
  • Brian Bennett, Marquette University
  • Guanjing Chen, Utah State University
  • Li-June Ming, University of South Florida
Document Type
Article
Language
eng
Format of Original
7 p.
Publication Date
7-1-1998
Publisher
American Chemical Society
Original Item ID
doi: 10.1021/ja971589d
Disciplines
Abstract
Proton NMR spectra of the hyperactive Cu(II)-substituted aminopeptidase from Aeromonas proteolytica (AAP) were recorded in both H2O and D2O buffered solution at pH 6.7. Several remarkably sharp, well resolved hyperfine shifted 1H NMR signals were observed in the 70 to −20 ppm chemical shift range. That hyperfine shifted signals were observed is due to spin-coupling of the two Cu(II) ions. Comparison of the spectra recorded in H2O and D2O buffered solutions indicated that the signals at 44.6, 43.3, and 17.7 ppm were solvent exchangeable. The two most strongly downfield shifted signals were assigned to imidazole N−H protons of the two coordinated histidine residues, while the remaining exchangeable signal was assigned to a peptidyl N−H proton that is in close proximity to the dicopper(II) center. One-dimensional NOE studies at pH 6.7 revealed two Y−CH2−CH< moieties that were assigned to coordinated aspartic acid and histidine residues. In addition, a Y−CH2−CH2−CH< moiety was also identified and was assigned to the coordinated glutamic acid residue, Glu152. All of the hyperfine shifted signals for [CuCu(AAP)] sharpened and shifted toward the diamagnetic region as the temperature was increased following Curie behavior. Fits of these data and those of a series of magnetically diverse μ-phenoxo and μ-alkoxo dicopper(II) model complexes to the population distribution of the ground and first excited states, provided information on the magnetic properties of dicopper(II) clusters. These fits indicated that the two Cu(II) ions in AAP are ferromagnetically coupled with a 2J value of 50 + 40 cm-1. These data provide the first structural information regarding the hyperactive [CuCu(AAP)] enzyme and are discussed in terms of the previously proposed mechanism of action for AAP.
Comments

Accepted version. Journal of the American Chemical Society, Vol. 120, No. 25 (July 1998): 6329-6335 DOI. © 1998 American Chemical Society. Used with permission.

Brian Bennett and Richard Holz were affiliated with Utah State University at the time of publication.

Citation Information
Richard C. Holz, Brian Bennett, Guanjing Chen and Li-June Ming. "Proton NMR Spectroscopy as a Probe of Dinuclear Copper(II) Active Sites in Metalloproteins. Characterization of the Hyperactive Copper(II)-Substituted Aminopeptidase from Aeromonas proteolytica" Journal of the American Chemical Society (1998) ISSN: 0002-7863
Available at: http://works.bepress.com/richard_holz/47/