OTHER INSTRUMENTAL METHODS 145
a rapidly advancing fi eld with applications in many areas of science and tech-
nology. Flash photolysis allows one to follow a reaction using fast (nanosecond
to microsecond) laser excitation pulses to cause absorption in the species of
interest. Following the excitation, one must use fast electronic devices to
measure the light emission or absorption by the species of interest. A neces-
sary criterium for the use of fl ash photolysis methods is that the molecule
under study must show a detectable change upon laser excitation.
An example of experimental data collected using fl ash photolysis, along
with conclusions reached as a result of the experiments, involves electron
transfer between heme a and heme a 3 in cytochrome c oxidase, CcO.^49 The
experimental setup relies on the fact that reverse electron fl ow between the
spatially close heme a and a 3 sites can be initiated by fl ash photolysis of carbon
monoxide (CO) from reduced heme a 3 (heme contains a Fe(II) ion) under
conditions when heme a is initially oxidized (heme contains a Fe(III) ion). See
Section 7.8 for more information about CcO and Figure 7.41 for a view of the
heme sites within the protein. The researchers followed the reaction using
transient absorption spectroscopy, with femtosecond (fs, 10 − 15 s) time resolu-
tion and a time window extending to 4 nanoseconds (ns, 10 − 9 s). Experimen-
tally, transient absorption pump probe spectroscopy was performed with a
55 - fs pump pulse centered at 590 nm and a < 30 - fs white light continuum probe
pulse. The probe continuum was generated by using the fundamental beam of
the laser system, centered at ∼ 615 nm. Absorbance changes after CO photoly-
sis were recorded for fully reduced (FR) CcO enzyme — heme a 3 and heme a
both having Fe(II) ions — and for mixed - valent (MV) enzyme — heme a 3 having
Fe(II) and heme a having Fe(III). The experimental results showed that there
were signifi cant spectral changes after photolysis for the MV state, whereas
for the FR state the spectrum hardly changed. This implied signifi cant differ-
ences in the behavior of the hemes for the different redox states. The ultravio-
let – visible absorption spectra of CcO were monitored for spectral evolution
at the so - called Soret ( ∼ 430 – 440 nm) and the α ( ∼ 590 – 605 nm) bands for CcO.
Comparison of picosecond (ps, 10 − 12 s) heme a 3 – CO photodissociation showed
signifi cant spectral interaction between the hemes. The most important fi nding
from the experiments were twofold: (1) The electron equilibration between
heme a 3 and heme a occurred in 1.2 ± 0.1 ns, and (2) electron equilibration
corresponded to aΔG^0 of 45 – 55 meV. The data suggest very fast equilibration
between the two hemes and demonstrate a low driving force (smallΔG^0 ) for
the redox reaction. Figure 5 of reference 49 summarizes the fi ndings for the
MV CcO complex: (1) Flash photolysis removes CO from heme a 3 , transfer-
ring it to Cu B ; (2) the effective midpoint potential of heme a 3 rises after
removal of CO, bringing it closer to heme a ’ s midpoint potential; (3) electron
redistribution based on this new equilibrium (with small energy difference
between the two hemes) occurs within 1.2 ns; and (4) CO is released from Cu B
and migrates out of the catalytic site within 3 μ s, a previously measured time
scale, then believed to be the fastest electron transfer reaction for CcO. The
previous experiments were carried out using microsecond instrumentation,^50