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 afterinsertion 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 Cb
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 [NHis-
Cu(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 semi-empirical 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.