144 INSTRUMENTAL METHODS
along with a second process known as “ proton pumping, ” results in the genera-
tion of a transmembrane proton gradient driven by the electron transfer (ET)
reaction. The reference 48 researchers intended to study proton access to the
catalytic site, at heme a 3 and Cu B (see Figures 7.39 , 7.40 , 7.41 , and 7.42 ), that
presumably limits the rate of electron transfer to heme a 3 during anaerobic
conditions. To do this, two forms of oxidized CcO were studied: (1) the so -
called “ resting ” form ( O in Figure 7.42) and (2) activated oxidized CcO pro-
duced immediately following reoxidation of the fully reduced enzyme with
O 2 — the so - callled OH metastable state. The reduction kinetics of oxidized CcO
(O ) by a hexaruthenium complex (Ru) were measured under anaerobic condi-
tions (Ar gas) using a stopped - fl ow apparatus. An anaerobic solution of ( O )
was mixed in a 1 : 1 ratio with the mixture of Ru and sodium dithionite (Ru -
DT) — fi nal concentrations of 2.5 mM Ru and 5 mM DT. The researchers also
compared the kinetics of electron transfer (ET) to heme a 3 of CcO in the ( O )
and ( OH ) states at pH 9.0 and 8.0. For measurement of ( OH ) anaerobic, the
procedure mixed, in the stopped - fl ow apparatus, fully reduced CcO (in 5 mM
Ru and 20 mM dithionite, DT) and air - saturated buffers maintaining the
desired pH. In the approximately 2 - ms dead time of the stopped - fl ow appara-
tus, CcO was oxidized with any excess O 2 present consumed by the dithionite,
both Cu A and heme a components of CcO are reduced by the excess reducing
agent, all within the dead time of the apparatus, so that only re - reduction of
heme a 3 was available to be monitored. This fact was demonstrated experi-
mentally by monitoring the UV – visible absorbances of the mixtures at 1 ms
(within the dead time of the stopped - fl ow apparatus) and at 1.9 s, at which time
maxima at 446 and 604 nm correspond to the fully reduced CcO. The large
increase in the Soret region absorbance band at 446 nm, which takes place in
the interval between 1 ms and 1.9 s, is characteristic of heme a 3 ’ s reduction
exclusively. These experiments isolate the kinetics of electron transfer to heme
a 3. The researchers found that ET to heme a 3 was controlled by the state of
ionization of a single amino acid residue with a p Ka of 6.5 ± 0.2. This p Ka was
attributed to glu60, located on the entrance to the so - called matrix side “ K ”
channel (see Section 7.8 and Figure 7.41 ), a proton channel distinguished by
important lysine (K) residues. Thus glu60 was thought to control proton entry
into the channel and therefore into the catalytic site by coupled proton trans-
fer. Additionally, a second factor was thought to be the rate of proton diffusion
in the channel. The researchers also concluded, by experiments with both ( O )
and ( OH ) states of CcO, that rates of ET to heme a 3 in the resting enzyme ( O )
and in CcO activated by reaction of the fully reduced enzyme ( OH ) with O 2
were the same. This implied that the catalytic sites of the two forms of the
enzyme, ( O ) and ( OH ), are essentially identical.
3.7.2.2 Flash Photolysis. Time - resolved spectroscopy techniques are a
powerful means of studying materials, giving information about the nature of
the excitations, energy transfer, molecular motion, and molecular environ-
ment, information that is not available from steady - state measurements. It is