ELECTRON PARAMAGNETIC RESONANCE 129
distinguish between hyperfi ne and superhyperfi ne coupling in a bioinorganic
system where both may be present. Palmer discusses the solutions to this
problem on pages 163 – 165 of reference 29.
3.5.3 Electron Nuclear Double Resonance (ENDOR) and Electron
Spin - Echo Envelope Modulation (ESEEM)
Electron nuclear double resonance (ENDOR) and electron spin - echo enve-
lope modulation (ESEEM) are two of a variety of pulsed EPR techniques that
are used to study paramagnetic metal centers in metalloenzymes. The tech-
niques are discussed in Chapter 4 of reference 1a and will not be discussed in
any detail here. The techniques can defi ne electron – nuclear hyperfi ne interac-
tions too small to be resolved within the natural width of the EPR line. For
instance, as a paramagnetic transition metal center in a metalloprotein inter-
acts with magnetic nuclei such as^1 H,^2 H,^13 C,^14 N,^15 N,^17 O,^31 P, or^33 S, these
interactions may be detected by ENDOR or ESEEM analysis. Using these
techniques, the nucleus of a particular ligand atom complexed to the metal
may be identifi ed. In favorable circumstances, metal – ligand bond distances
and bond angles may be determined as well.
3.5.4 Descriptive Examples,
The enzyme cytochrome c oxidase provides an example of the utility of EPR
in studying paramagnetic metal sites in metalloenzymes. The enzyme cyto-
chrome c oxidase (CcO) is a member of an enzyme superfamily that couples
oxidation of ferrocytochrome c to the 4 e − /4 H + reduction of molecular oxygen
in eukaryotes. Bacterial CcO complexes consist of two, or at most three, sub-
units — I, II, III — these contain the catalytic sites for all CcOs. Subunit I con-
tains the ligating amino acid residues for the iron - containing heme a and heme
a 3 sites and a monometallic copper (Cu B ) site. CcO ’ s subunit II features a 10 -
strand β barrel that contains the ligating aa residues for the bimetallic copper
(CuA ) site. Much more detail on cytochrome c oxidase is found in Section 7.8.
The metal ions in all sites in the enzyme undergo oxidation and reduction as
CcO goes through its catalytic cycle. The bimetallic Cu A site cycles between
Cu(II) – Cu(II), Cu(II) – Cu(I), and, most importantly for this example, a
Cu(1.5) – Cu(1.5), mixed - valence (MV) state in which the copper ions share an
unpaired electron. Researchers P. M. H. Kroneck, W. E. Antholine, and co -
workers fi rst proposed the mixed - valence state for the bimetallic copper site,
CuA , on the basis of their EPR measurements in the early 1990s.^30 Although
controversial at the time it was fi rst proposed, X - ray crystallography subse-
quently confi rmed the bimetallic Cu A site, as will be discussed in Section 7.8.
In 1996, Kroneck and co - workers published an extended study of the dinuclear
copper electron transfer center in several enzymes, including cytochrome c
oxidase (referred to as COX by these and other authors), nitrous oxide reduc-
tase (N 2 OR), and a bimetallic copper site engineered into the soluble domain
of subunit II of quinol oxidase (QOX) in Escherichia coli ( E. coli ), generating