¹H NMR Studies on the CuA Center of Nitrous Oxide Reductase from Pseudomonas stutzeri

Authors

Richard C. Holz, Marquette UniversityFollow
Marcela L. Alvarez, Montana State University - Bozeman
Walter G. Zumft, Universität Karlsruhe
David M. Dooley, Utah State University

Document Type

Article

Publication Date

8-5-1999

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

¹H NMR spectra of the Cu_A center of N₂OR from Pseudomonas stutzeri, and a mutant enzyme that contains only Cu_A, 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 N^ε2H (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 Cu_A 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 Cu_A 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 (ΔE_L) between the ground (²B_3u) and excited (²B_2u) states. The temperature data obtained for all of the observed hyperfine-shifted histidine ligand protons provided a ΔE_L value of 62 ± 35 cm^-1. The temperature dependence of the observed cysteine C^βH and C^αH protons (a and h) were fit in a separate experiment providing a ΔE_L value of 585 ± 125 cm^-1. The differences between the ΔE_L 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.

Comments

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.

Recommended Citation

Holz, Richard C.; Alvarez, Marcela L.; Zumft, Walter G.; and Dooley, David M., "¹H NMR Studies on the CuA Center of Nitrous Oxide Reductase from Pseudomonas stutzeri" (1999). Chemistry Faculty Research and Publications. 305.
https://epublications.marquette.edu/chem_fac/305