1H
NMR spectra of the CuA center of N2OR from Pseudomonas stutzeri,
and a mutant enzyme that contains only CuA, were
recorded
in both H2O- and D2O-buffered
solution at pH 7.5. Several sharp, well-resolved hyperfine-shifted 1H
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
1H
NMR spectra recorded in H2O and D2O
indicates that three signals, c (27.5 ppm), e (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
Ne2H
(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 CuA 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 pH 7.5 allowed the histidine ring protons to be definitively
assigned,
while the remaining signals were assigned by comparison to previously
reported
spectra from CuA centers. The temperature
dependence
of the observed hyperfine-shifted
1H 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 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 (DEL)
between the ground (2B3u) and excited
(2B2u)
states. The temperature data obtained for all of the observed
hyperfine-shifted
histidine ligand protons provided a DEL
value of 62 ± 35 cm-1. The temperature dependence of
the observed cysteine CbH
and CaH
protons (a and h) were fit in a separate experiment
providing
a DEL
value of 585 ± 125 cm-1. The differences between the
DEL
values determined by 1H 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 states.