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Titolo:
H-1 NMR studies on the Cu-A center of nitrous oxide reductase from Pseudomonas stutzeri
Autore:
Holz, RC; Alvarez, ML; Zumft, WG; Dooley, DM;
Indirizzi:
Utah State Univ, Dept Chem & Biochem, Logan, UT 84322 USA Utah State UnivLogan UT USA 84322 pt Chem & Biochem, Logan, UT 84322 USA Montana State Univ, Dept Chem, Bozeman, MT 59717 USA Montana State Univ Bozeman MT USA 59717 Dept Chem, Bozeman, MT 59717 USA Univ Karlsruhe, Lehrstuhl Mikrobiol, D-76128 Karlsruhe, Germany Univ Karlsruhe Karlsruhe Germany D-76128 iol, D-76128 Karlsruhe, Germany
Titolo Testata:
BIOCHEMISTRY
fascicolo: 34, volume: 38, anno: 1999,
pagine: 11164 - 11171
SICI:
0006-2960(19990824)38:34<11164:HNSOTC>2.0.ZU;2-A
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEAR-MAGNETIC-RESONANCE; DINUCLEAR COPPER(II) CENTERS; ELECTRON-TRANSFER CENTER; SOLUBLE DOMAIN; N2O REDUCTASE; PARACOCCUS-DENITRIFICANS; TEMPERATURE-DEPENDENCE; RAMAN-SPECTROSCOPY; CYTOCHROME-OXIDASE; MULTICOPPER ENZYME;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
58
Recensione:
Indirizzi per estratti:
Indirizzo: Holz, RC Utah State Univ, Dept Chem & Biochem, Logan, UT 84322 USA Utah State Univ Logan UT USA 84322 Biochem, Logan, UT 84322 USA
Citazione:
R.C. Holz et al., "H-1 NMR studies on the Cu-A center of nitrous oxide reductase from Pseudomonas stutzeri", BIOCHEM, 38(34), 1999, pp. 11164-11171

Abstract

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, 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 H-1 NMR spectra recorded in H2O and D2O indicates that threesignals, c (27.5 ppm), c (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 (NH)-H-epsilon 2 (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 pll 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 H-1 NMR signals of mutant N2OR were recorded over the temperature range of 276-315 K. Both Curie and anti-Curie temperaturedependencies are observed for sets of hyperfine-shifted protons. Signals aand 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 (Delta E-L) between the ground (B-2(3u),) and excited (B-2(2u)) states. The temperature data obtained fur all of the observed hyperfine-shifted histidine ligand protons provided a Delta 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 separate experiment providing a Delta E-L value of 585 +/- 125 cm(-1). The differences between the Delta E-L Values determined by H-1 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 stales.

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Documento generato il 01/12/20 alle ore 13:37:37