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Page 40 Bennett. J Transl Genet Genom 2020;4:36-49 I https://doi.org/10.20517/jtgg.2020.17
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environment. The N1b [2Fe2S] and N2 [4Fe4S] clusters have higher midpoint potentials, around -205 mV
to -270 mV, depending on the overall redox status of other clusters, and exhibit essentially axial EPR spectra
with g^ ~ 1.92.

The signal from Complex II (succinate dehydrogenase) is dominated by two overlapping signals due to
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reduced [2Fe2S] (S1) and [4Fe4S] (S2) clusters. The precise g-values for these signals are dependent on
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the extent of reduction and the temperature, as the influence of the spin-spin interaction between them is
dependent on both. However, they both give rise to an intense derivative feature at g ~1.92, adding to the
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contribution from Complex I signals [87-89] . A third complex II signal due to an oxidized [3Fe4S] (S3) signal
overlaps at g ~2.02, with that from oxidatively-deactivated cytosolic aconitase in which the labile Fe atom is
a
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[90]
2+
lost from the active, EPR-silent [4Fe4S] cluster to form an [3Fe4S] cluster . However, the very different
temperature dependences of S3 and aconitase allow deconvolution by recording at 2 temperatures (e.g.,
[80]
12 and 40 K) . Another signal in that region with a g ~2.015 turning point and flanking resonances at
g ~2.03 and 1.98 has been assigned to a stable, dipolar-coupled ubisemiquinone pair in the vicinity of S3 in
[91]
Complex II .
Although the redox cofactors of Complex III (cytochrome bc complex; CoQH -cyctochrome c reductase)
1
2
have been studied extensively in vitro, the EPR spectra of tissue and cells do not provide much information
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due to their low intensities. The only signal routinely assignable to complex III is the reduced [2Fe2S] Rieske
cluster with a distinct sharp g resonance at 2.03, a derivative feature at 1.90 (generally not resolved from
1
the composite ‘g = 1.92’ signal), and a very broad g feature at g ~1.78 that is observable in heart and muscle
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samples that do not exhibit overlapping Mn(II) signals [79,92-94] . The other EPR signals observable from isolated
complex III are resonances that overlap in the region g ~3.8-3.3 and are due to g of cytochromes c , b , and
eff
L
1
1
b H [92,95] . These signals are sometimes detected but are difficult to characterize and quantify because they are
weak, part of a very broad signal envelope, and their resonance positions and line widths can be sensitive to
the specific environment.
The final redox-active MRC component, Complex IV (cytochrome c oxidase), exhibits resonances at g ~3.0
eff
and g ~2.2 due to low-spin heme a, and, under some conditions, resonances at g ~2.18 and 2.0 due to the
eff
dinuclear S = / Cu center [96,97] . Additional complex IV signals at g ~12 and 2.95 can be difficult to detect
1
A
eff
2
in some tissues and cells due to low signal levels and overlap with the heme a g ~3.0 resonance, respectively,
eff
and are associated with the heme a3-Cu coupled center [98-100] .
B
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Additional signals that may be observed in the spectrum arise from the [3Fe4S] cluster of aconitase
[90]
described above ; a rhombic high-spin ferriheme signal from catalase with g ~6.45 and g ~5.33 [81,101] , and a
y
x
signal at g ~4.2, with a characteristic splitting at the crossover, due to transferrin Fe 3+[102-104] . A characteristic,
1
5
2+
5
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and sometimes intense six-line I = / hyperfine-split signal due the S = / , M = ± / manifold of Mn is
S
2
2
2
observed in some tissues, particularly liver, and may be distorted due to the rapid-passage relaxation effects
[79]
at the low temperatures needed to observe the other signals .
TISSUE SAMPLE PREPARATION FOR EPR
The goals of EPR of tissue for the characterization of MD are (1) to provide a snapshot of the redox status of
metabolism in actively metabolizing tissue; and (2) to report on instantaneous and chronic exposure to ROS.
It is important, then, that tissue is excised and frozen before either the exhaustion of reducing equivalents
or of a terminal electron acceptor (usually oxygen) alters the local and global redox potentials, and before
further non-physiological ROS-mediated damage occurs. Traditional tissue mounting techniques cannot be
used: EPR spectra of formalin-fixed brain tissue, for example [105] , are devoid of almost all of the characteristic
signals observed in freshly frozen brain [106,107] . Similarly, human tissue-bank muscle samples exhibited intense
free-radical signals and signals due to Fe but no signals ascribable to metabolic components were observed.
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