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algorithm provides two important parameters of fitting quality. One is a standard deviation that, when
compared to the actual contribution of a signal to the to the spectrum, indicates the significance of including
that particular signal in the fit [Figure 2C]; a “confidence level” can also be readily calculated. The second is a
correlation matrix [Figure 2D] that indicates cases where the calculated contribution of one signal is heavily
dependent on the contribution of another. The highly correlated pairs that are seen in tissues are cyt b -cyt
L
c ; N3-N4; S1-S2; S3-aconitase; and, less so, N1b-N4 and N1b-N2. The N4 and N3 signals each have a small
1
but isolated resonance corresponding to a particular principal orientation and can thus be fitted manually
rather than iteratively and constrained thereafter. Cyts b and c are rarely observed with high intensity and
1
L
signals from either or both can be taken as markers of unusually elevated redox potential. [FeS] clusters
S1 and S2 can be deconvoluted with care by exploiting differences in relaxation behavior but this is hardly
worth the effort as they essentially provide the same information in almost all cases and are treated together
as (S1 + S2); similarly, N1b and N2 can be considered together [84,85] . The most important coupled pair, then,
is S3-aconitase, which overlaps extensively across their narrow field envelopes and yet provide very different
information that renders their deconvolution important. Fortunately, these are readily distinguished by their
[80]
very different temperature dependences . Where disease tissue is available with a reliable control, high
sensitivity to changes can be accomplished by generating difference spectra (i.e., disease minus control).
One could, in principle, merely fit the difference spectrum, in which many of the signals from the individual
spectra, those that are not affected by disease, would have cancelled out. A good measure of the effectiveness
of the fitting procedure, however, is that the difference spectra of the two individual calculated simulations
are indistinguishable from fitting to the difference spectrum directly and provide excellent reproduction of
[79]
the difference of the experimental spectra .

APPLICATION OF EPR TO MITOCHONDRIAL DISEASE
EPR as a stand-alone technique provides three pieces of information relevant to MD and other diseases
and conditions with metabolic components including cancer, neurological diseases, and cardiac dysfuncti
on [79,82,106,108-111,115] . The first is a measure of the redox potential across the MRC by quantitation of signals due
to oxidized and reduced redox centers. This is important because the redox potential is the thermodynamic
driving force for catalysis and electron transport and, ultimately, represents the potential energy available for
conversion to chemical energy via ATP synthesis. The characteristic sharp distinct signal observed at g = 1.92
is largely due to the g features of complex I reduced FeS clusters N1b and N2, with additional contributions
x,y
from g of N3 and N4 and from g of complex II S1 and S2. The dominant N1b and N2 clusters exhibit
x,y
y
[84]
midpoint potentials of -205 mV to -270 mV , and diminution of the g = 1.92 signal compared with healthy
cells or tissue therefore represents a significant departure of the redox potential from the expected -320 mV,
+
dictated by the NAD /NADH couple [116] . More sensitive still are the N4 (g = 1.88; E = -280 mV) and N3
m
3
(g = 1.86; E = -325 mV) clusters, with the isolated g features upfield of the g = 1.92 signal. Care must be
m
3
3
2+
taken when Mn is present, particularly prevalent in the liver, as the N3 g feature overlaps with the often
3
5
more-intense high-field M = - / resonance of the DM = / manifold of S = / Mn 2+[79,108] . The absence of the
5
1
2
2
S
I
2
N3 and N4 signals in spectra where the g = 1.92 is well-developed is indicative of either reduced metabolic
potential (elevated redox potential), due to inefficient primary metabolism, a compromised MRC, or a
membrane that allows reducing equivalents to non-productively drain from the MRC. Another possible
cause, though less likely, is specific breaks in the Complex I intramolecular electron transport chain.
The second useful piece of information from EPR is the intensity of the [3Fe4S] signal due to aconitase,
+
that reports on the instantaneous oxidative stress burden due to ROS production as a result of MD. Upon
reaction with O , a non-covalently-bound iron, Fe , is lost from the active EPR-silent [4Fe4S] cluster
•–
2+
a
2
+
and the resulting catalytically inactive but EPR-active [3Fe4S] cluster exhibits a sharp and distinct, almost-
isotropic signal centered at g = 2.018 [90,117-120] . The magnitude of this signal has been observed to increase
dramatically in tumor tissue, where excess ROS has been confirmed by other techniques, whereas in a mouse
MD model the increase was moderate (125% of control) [115,79] .