PROBING THE GROUND-STATE OF THE PURPLE MIXED-VALENCE CU-A CENTER IN NITROUS-OXIDE REDUCTASE - A CW ENDOR (X-BAND) STUDY OF THE CU-65, N-15-HISTIDINE LABELED ENZYME AND INTERPRETATION OF HYPERFINE COUPLINGS BY MOLECULAR-ORBITAL CALCULATIONS

CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu( 1.5+)], S = 1/2, Cu-A Site in nitrous oxide reductase were obtained af ter insertion of Cu-65 or both Cu-65 and N-15-histidine. The N-14/N-15 isotopic substitution allowed for an unambiguous deconvolution of pro ton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 +/- 0.4 MHz for N-14 was detected. Its anisotropy was characteristic for imidazole bound to copper. A spin de nsity of 3-5% was estimated for the nitrogen donors to Cu-A, indicatin g that the ground state is B-2(3u). Proton hyperfine structure was det ected from four C-beta protons of coordinating cysteine residues. Thei r isotropic and anisotropic parts were deconvoluted by spectral simula tion. From the anisotropic couplings a spin density of 16-24% was esti mated for each of the cysteine thiolate donors of Cu-A. The [NHis-Cu(R S)(2)CuNHis](+) core structure of Cu-A in nitrous oxide reductase from Pseudomonas stutzeri is predicted to be similar to the crystallograph ically determined Cu-A structure (Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92:11955-11 959), but distinct from the Cu-A 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 couplin gs as a function of the electronic ground state was calculated by the INDO/S method. The Mulliken atomic-spin populations calculated by a gr adient-corrected density functional method and the semiempirical INDO/ S method were compared with experimentally derived spin populations, a nd good agreement between theory and experiment was found for both cal culations. The ground state of Cu-A is best represented by the resonan ce structures of the form [(CuS-S-CuII)-S-I <-> (CuSS-CuI)-S-I-S-. <-> (CuS-SCuI)-S-I-Cu-. <-> (CuS-S-CuI)-S-II]. It is proposed that the Cu 4s,p as well as sulfur 3d orbitals play a role in the stabilization o f this novel type of cluster.