H-1 NMR studies on the Cu-A center of nitrous oxide reductase from Pseudomonas stutzeri

H-1 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 H-1 NMR signals were observed in the 60 to -10 ppm chemical shift range, C omparison of the native and mutant N2OR spectra recorded in H2O-buffered so lutions indicated that several additional signals are present in the native protein spectrum. These signals are attributed to a dinuclear copper(II) c enter. At least two of the observed hyperfine-shifted signals associated wi th the dinuclear center, those at 23.0 and 13.2 ppm, are lost upon replacem ent of H2O buffer with D2O buffer. These data indicate that at least two hi stidine residues are ligands of a dinuclear Cu(II) center. Comparison of th e mutant N2OR H-1 NMR spectra recorded in H2O and D2O indicates that three signals, c (27.5 ppm), c (23.6 ppm), and i (12.4 ppm), are solvent exchange able. The two most strongly downfield-shifted signals (c and e) are assigne d to the two (NH)-H-epsilon 2 (N-H) protons of the coordinated histidine re sidues, 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 dec rease in intensity as the temperature was increased, indicating that proton e resides on a more solvent-exposed histidine residue. One-dimensional nOe studies at pll 7.5 allowed the histidine ring protons to be definitively a ssigned, while the remaining signals were assigned by comparison to previou sly reported spectra from Cu-A centers. The temperature dependence of the o bserved hyperfine-shifted H-1 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 increase s with increasing temperatures), while signals b-g, i, and j (histidine pro tons) follow Curie behavior (contact shift decreases with increasing temper atures). Fits of the temperature dependence of the observed hyperfine-shift ed signals provided the energy separation (Delta E-L) between the ground (B -2(3u),) and excited (B-2(2u)) states. The temperature data obtained fur al l of the observed hyperfine-shifted histidine ligand protons provided a Del ta E-L value of 62 +/- 35 cm(-1) The temperature dependence of the observed cysteine (CH)-H-beta and (CH)-H-alpha protons (a and h) were fit in a sepa rate experiment providing a Delta E-L value of 585 +/- 125 cm(-1). The diff erences between the Delta E-L Values determined by H-1 NMR spectroscopy and those determined by EPR or MCD likely arise from coupling between relative ly low-frequency vibrational states and the ground and excited electronic s tales.