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Probing the ground state of the purple mixed valence CuA center in nitrous oxide reductase: a CW ENDOR (X-band) study of the 65Cu, 15N-histidine labeled enzyme and interpretation of hyperfine couplings by molecular orbital calculations

  • Neese, F.1
  • Kappl, R.2
  • Hüttermann, J.2
  • Zumft, W. G.3
  • Kroneck, P. M. H.1
  • 1 Fakultät für Biologie, Universität Konstanz, Postfach 5560 M665, D-78457 Konstanz, Germany Tel.: +49-7531-882103; Fax: +49-7531-882966; e-mail: [email protected], DE
  • 2 Fachrichtung 3.6 Biophysik und Physikalische Grundlagen der Medizin, Universität des Saarlandes, D-66421 Homburg, Germany Tel.: +49-6841-166200; Fax: +49-6841-166227; e-mail: [email protected], DE
  • 3 Lehrstuhl für Mikrobiologie, Universität Fridericiana, D-76128 Karlsruhe, Germany, DE
Published Article
JBIC Journal of Biological Inorganic Chemistry
Publication Date
Feb 01, 1998
DOI: 10.1007/PL00010649
Springer Nature


CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, CuA site in nitrous oxide reductase were obtained after insertion of 65Cu or both 65Cu and 15N-histidine. The 14N/15N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for 14N was detected. Its anisotropy was characteristic for imidazole bound to copper. A spin density of 3–5% was estimated for the nitrogen donors to CuA, indicating that the ground state is 2B3u. Proton hyperfine structure was detected from four Cβ protons of coordinating cysteine residues. Their isotropic and anisotropic parts were deconvoluted by spectral simulation. From the anisotropic couplings a spin density of 16–24% was estimated for each of the cysteine thiolate donors of CuA. The [NHisCu(RS)2CuNHis]+ core structure of CuA in nitrous oxide reductase from Pseudomonas stutzeri is predicted to be similar to the crystallographically determined CuA* structure (Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92 : 11955–11959), but distinct from the CuA structure of Paracoccus denitrificans cytochrome c oxidase (Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Nature 376 : 660–669). The angular dependence of the isotropic couplings as a function of the electronic ground state was calculated by the INDO/S method. The Mulliken atomic-spin populations calculated by a gradient-corrected density functional method and the semiempirical INDO/S method were compared with experimentally derived spin populations, and good agreement between theory and experiment was found for both calculations. The ground state of CuA is best represented by the resonance structures of the form [CuIS–S–CuII↔ CuIS•S–CuI↔ CuIS–S•CuI↔ CuIIS–S–CuI]. It is proposed that the Cu 4s,p as well as sulfur 3d orbitals play a role in the stabilization of this novel type of cluster.

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