CYTOCHROME c OX IDASE 439
The mixed valence state stores only half the electrons necessary to reduce
one molecule of O 2 to water and therefore the reaction stops at the PM state,
halfway through the catalytic cycle. Model compounds synthesized in this state
provide convenient conditions for enzyme mechanistic studies as will be
described in Section 7.8.4. The fully reduced and mixed valence pathways have
similarA states at the heme a 3 – Cu B catalytic site after addition of substrate
dioxygen. Evidence includes a UV – visible absorption band at 595 nm and a
resonance Raman band at 571 cm − 1 assigned to ν (Fe II – O). The pathways
diverge at that point to produce thePR (fully reduced) or PM (mixed valence)
states. The PM state is produced at a ∼ 4 – 5 times slower rate than PR. Both PR
andPM show indications of the ferryl – oxo intermediate at heme a 3 — resonance
Raman band at 803 cm − 1 assigned to ν (Fe IV =O) for PM. In addition, experimen-
tal evidence suggests that the P M state contains a tyrosine radical, YO • , and it
is postulated that the radical arises from tyr244, which is covalently bonded to
CuB ’ s his240 ligand (bovine enzyme numbering). The fully reduced pathway
next undergoes a pH - dependent step to state F that exhibits its ν (Fe IV =O)
resonance Raman band at 785 cm − 1. As shown in Figure 7.42 , it is thought that
an equilibrium mixture of two redox states at the heme a – Cu A sites exists in
stateF. An additional proton has been added to the catalytic site in state F ;
and although this is shown being added to the Cu B – OH moiety of state A ,
the location of this proton has not been established experimentally. The rates
of heme a→ Cu A electron transfer and the state PR → state F transition
are pH - dependent, and the step is accompanied by proton pumping
(translocation). The fourth electron enters to form the O state with its ferric
hydroxide intermediate characterized by time - resolved resonance Raman
spectroscopy to have aν (Fe – OH) stretching mode at 450 cm − 1. The F → O
transition is pH - dependent and accompanied by proton pumping. What
happens next in the cycle must be further protonation and release of H 2 O, but
the exact mechanism for these processes is not resolved at the time of this
writing.
If one can make overall conclusions about the cytochrome c oxidase cycle,
the reference 143 authors do so. They conclude that effi cient proton pumping
into the cytosolic space (P - side of the membrane) in CcO relies on (1) rapid
proton uptake through the D - pathway from the matrix (N - side of the mem-
brane) and (2) fi nely adjusted p Ka values of proton shuttling groups — for
example, glu286, D - ring propionate, and others within the enzyme complex.
Conclusions about the conformation or oxidation state of dioxygen at the
heme a 3 – Cu B catalytic site are more diffi cult. Probably the presence of the
peroxo ligand can be dismissed as a long - lived intermediate, although it
appears in many of the cytochrome c oxidase model complexes to be discussed
in Section 7.8.4. The dioxygen superoxo ligand intermediate has a greater
probability of existence, and the ferryl iron – oxo intermediate has exhibited its
spectroscopic signature in many experimental studies. Whether or not the
synthesis and investigation of cytochrome c oxidase model compounds can
help answer these questions is the topic of the next section.