the egorbitals). As the axial coordination changes
compared to the planar coordination, the two eg
orbitals will lie at increasingly higher energy
than the other three orbitals (Figure 16.23). The
five electrons will fill the lower three orbitals,
resulting in two pairs of electrons and one
unpaired electron, forming a low-spin state. If the
axial coordination is similar then the five orbitals
have similar energies. The five electrons will
prefer to occupy different orbitals to minimize the
electron–electron repulsion energy. As a result,
all five electrons are unpaired and a high-spin
state with an effective spin of 5/2 is formed. For
the case of Fe^2 +, the six electrons will have values of 2 and 0 for the
high- and low-spin states respectively.
EPR has been used extensively to characterize the electronic structure
of metals and relate any changes to functional features. For example, cyto-
chrome cd 1 serves a respiratory nitrite reductase, with electrons delivered
to the enzyme from the cytochrome bc 1 complex via electron carriers such
as azurin, a small copper protein, in the nitrogen cycle (Allen et al. 2000;
Wasser et al. 2002; Stevens et al. 2004). This enzyme catalyzes the one-
electron reduction of nitrite to nitric oxide
in the nitrogen cycle. The protein has two
domains, one containing a heme cand the other
containing a heme d 1 , with nitric oxide being
released from the d 1 heme after reduction.
During this process both hemes undergo redox
changes with nitric oxide being tightly bound
for the Fe(II) state but not the Fe(III) state.
Coupled with the redox changes are structural
rearrangements of the protein, which involve
alteration of the heme coordination.
These events can be carefully monitored by
use of EPR as six-coordinated low-spin ferric
hemes are very sensitive to the chemical nature
and precise coordination of the axial ligands.
For example, the His–Fe–Hiscoordination has
an EPR spectrum that is distinctive from
a His–Fe–Met coordination (Figure 16.24).
In the presence of hydroxylamine, NH 2 OH,
cytochrome cd 1 is oxidized, thus providing a
mechanism to examine the time evolution
of the spectral features as the oxidation state
changes. Initially, the d 1 heme has the spectrum
of a low-spin heme, with gvalues of 2.94, 2.33,368 PART 2 QUANTUM MECHANICS AND SPECTROSCOPY
(a) High spin S 52 (b) Low spin S (^12)
Figure 16.23Energy-level diagram for
(a) high- and (b) low-spin Fe^3 +.
g 2.94
g 2.67
g 2.53
g 2.33
g 1.40
80 s
4 min
20 min
g 6.88 g 5.07
60 min
g 3.03
g 2.51
g 2.21 g 1.86
50 mT
Figure 16.24The EPR spectra of cytochrome cd 1
at different times after oxidation. Modified from
Allen et al. (2000).