(A) 285 GHz and (B) 9
GHz EPR spectra of Tyr radicals formed in
the W191F (top, black), W191F/W51F (middle, magenta), and W191F/W51F/Y187F/Y229F/Y236F
(bottom, purple) variants of cytochrome c peroxidase
upon reaction with a 2-fold excess of hydrogen peroxide, using a mixing
time on ice of 10 s. (A) The 285 GHz spectra (shown in g scale) were recorded at 10 K, using a frequency modulation of 30
kHz; a field modulation of 5 G was used for the top and middle spectra,
while a field modulation of 10 G was used for the bottom spectra because
of the much lower radical yield. The top and middle spectra result
from averages of four scans, while 10 scans on average were used for
the bottom spectrum. (B) The 9 GHz spectra (top and middle) were recorded
at 40 K, with a modulation amplitude of 1 G, a microwave power of
0.08 mW, and a modulation frequency of 100 kHz. For the bottom spectrum,
a modulation amplitude of 3 G and a microwave power of 0.2 mW were
required because of the much lower radical yield.