Document Type


Publication Date


Source Publication


Source ISSN



1H NMR spectra of the CuA center of N2OR from Pseudomonas stutzeri, and a mutant enzyme that contains only CuA, were recorded in both H2O- and D2O-buffered solution at pH 7.5. Several sharp, well-resolved hyperfine-shifted 1H NMR signals were observed in the 60 to −10 ppm chemical shift range. Comparison of the native and mutant N2OR spectra recorded in H2O-buffered solutions indicated that several additional signals are present in the native protein spectrum. These signals are attributed to a dinuclear copper(II) center. At least two of the observed hyperfine-shifted signals associated with the dinuclear center, those at 23.0 and 13.2 ppm, are lost upon replacement of H2O buffer with D2O buffer. These data indicate that at least two histidine residues are ligands of a dinuclear Cu(II) center. Comparison of the mutant N2OR 1H NMR spectra recorded in H2O and D2O indicates that three signals, c (27.5 ppm), e (23.6 ppm), and i (12.4 ppm), are solvent exchangeable. The two most strongly downfield-shifted signals (c and e) are assigned to the two Nε2H (N-H) protons of the coordinated histidine residues, while the remaining exchangeable signal is assigned to a backbone N-H proton in close proximity to the CuA cluster. Signal e was found to decrease in intensity as the temperature was increased, indicating that proton e resides on a more solvent-exposed histidine residue. One-dimensional nOe studies at pH 7.5 allowed the histidine ring protons to be definitively assigned, while the remaining signals were assigned by comparison to previously reported spectra from CuA centers. The temperature dependence of the observed hyperfine-shifted 1H NMR signals of mutant N2OR were recorded over the temperature range of 276−315 K. Both Curie and anti-Curie temperature dependencies are observed for sets of hyperfine-shifted protons. Signals a and h (cysteine protons) follow anti-Curie behavior (contact shift increases with increasing temperatures), while signals b−g, i, and j (histidine protons) follow Curie behavior (contact shift decreases with increasing temperatures). Fits of the temperature dependence of the observed hyperfine-shifted signals provided the energy separation (ΔEL) between the ground (2B3u) and excited (2B2u) states. The temperature data obtained for all of the observed hyperfine-shifted histidine ligand protons provided a ΔEL value of 62 ± 35 cm-1. The temperature dependence of the observed cysteine CβH and CαH protons (a and h) were fit in a separate experiment providing a ΔEL value of 585 ± 125 cm-1. The differences between the ΔEL values determined by 1H NMR spectroscopy and those determined by EPR or MCD likely arise from coupling between relatively low-frequency vibrational states and the ground and excited electronic states.


Accepted version. Biochemistry, Vol. 38, No. 34 (August 5, 1999): 11164-11171. DOI. © 1999 American Chemical Society Publications. Used with permission.

Richard Holz was affiliated with the Utah State University at the time of publication.

holz_6333acc.docx (573 kB)
ADA Accessible Version

Included in

Chemistry Commons