ELECTRON PARAMAGNETIC RESONANCE 131
coli (labeled PdII in Figure 3.23 ). Figure 3.23 shows the experimental EPR
spectra of N 2 OR V (spectrum A), PdII (spectrum B), and purple CyoA (spec-
trum D). Also shown are simulated spectra for PdII (spectrum C) and purple
CyoA (spectrum E). The experimental spectra are axial with gz , g|| , > gx and gy ,
g⊥ (see Figure 3.22B ). The seven - line splitting pattern, to be expected from the
coupling of one unpaired electron ( S = 1/2) with two I = 3/2 spin copper nucleus
(number of lines = 2 I + 1 = 2(6/2) + 1), are very similar for N 2 OR and COX,
while the purple CyoA protein shows a more complex pattern. For the simu-
lated cytochrome c oxidase subunit II spectra (PdII, spectrum C in Figure
3.23 ), gmax = 2.19, gmid = 2.03, and gmin = 1.99. The small hyperfi ne splitting con-
stantA is simulated at 3.1 mT and results in a classic seven - line splitting
pattern with an intensity ratio of 1 : 2 : 3 : 4 : 3 : 2 : 1. The small 3.1 mT splitting
indicates a high degree of delocalization of the electron between the copper
ions and onto the copper ligands as well.
The reference 31 authors conclude that the Cu A center in both N 2 OR and
COX should be described as a Cu(1.5) – Cu(1.5) dimer rather than as a mixed -
valence Cu(II) – Cu(I) dimer. X - ray crystallographic and EXAFS studies on
cytochrome c oxidase, as discussed in Section 7.8, have defi ned two bridging
cysteine (Cu 2 S 2 nearly planar core) and two terminal histidine ligands, coordi-
nated through their N δ 1 nitrogen atoms (as well as methionine sulfur and glu-
tamate main chain oxygen ligands) for the Cu A center. The structural data
agree with Cu A ’ s EPR identifi cation as a bimetallic, delocalized, one - unpaired -
electron center in its reduced state with the unpaired electron delocalized over
the two copper ions as well as the cysteine sulfur ligands.
The ENDOR technique, described briefl y in Section 3.5.3 , has been applied
to Cu A centers in nitrous oxide reductase (N 2 OR) and the recombinant water -
soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase
(COX), called M160T9, and a mutant M160QT0, where the weak axial sulfur
ligand of methionine (M) at position 160 was replaced by a glutamine (Q).^32
The reference 32 authors employed high - fi eld (95 - GHz)^1 H ENDOR spectros-
copy in a comparative study of the Cu A site present in the proteins, for instance,
comparing their sulfur and copper spin densities. Comparison of their results
to known X - ray crystallographic structures allowed some correlations between
the hyperfi ne parameters of weakly (primarily involved with the histidine
ligands) and strongly coupled protons (those involved with the cysteine
ligands), the spin density distribution, and structural properties of the various
CuA sites. In other words, the Cu A ENDOR experiment formed relationships
between the EPR spin couplings and couplings involving the nearby protons
of the ligand atoms. For instance, the isotropic hyperfi ne coupling ( Aiso ) of the
strongly coupled cysteine β - protons (protons attached to cysteine ’ s β carbon
atom) were an important source of structural information because they
depended on the H – C – S – S dihedral angle in Cu A (the angle formed by the β
proton, the β - carbon atom, and the sulfur atom of one cysteine ligand, and the
sulfur atom of the other bridging cysteine ligand). Similarly, the weaker hyper-
fi ne coupling of the histidine protons, specifi cally of the H ε 1 protons (protons