The P and F transient intermediates of the reaction cycle are characterised by a distinct visible absorption maximum at 607 nm of the P state, and at 580 nm of the F state [50,51]. Meanwhile, their Soret band positions are the same 428 nm [52].
Although the major principles of how oxygen reduction occurs are understood, the precise structures (including sites involved in de/protonation) and chemical properties of the transient P and F intermediates are still under question [53-55]. Oxygen enters and binds to the distal pocket of haem a3 when the BNC is reduced [56]. The name of
35 P (peroxy) was given since it was originally proposed to have a peroxide structure [50]. However resonance Raman spectroscopy data now show that the O-O bond is already broken in the P state from a band at 803 cm-1 attributed to the Fe4+=O2- stretch of an oxyferryl structure of haem a3 [57-59]. This was also supported by mass spectrometry
data of H218O that is released into the bulk solution when 18O2 reacts with CcO to form
the PM state [60]. The conversion from FR to A to P is fast (10 µs and ~200 µs [55])
and, although it requires 4 reducing equivalents, it takes place in a single measurable step. Hence the electrons must be donated by the BNC as described above. One of these electrons is suggested to come from Y244 that would form a radical [38,48,49].
Visible absorbance spectra of the P and F states, rule out the possibility that the fourth electron comes from the haem a3 macrocycle, which would result in a porphyrin
π-cation radical (haem.+ a
3 (Fe4+=O2-)), as occurs in some peroxidases. This is
concluded because the Soret band (400-430 nm) of a porphyrin radical associated ferryl intermediate, is expected to have the same peak position as the ferric state, and a smaller extinction coefficient as is observed, for example, in horseradish peroxidase [61,62]. However the Soret band of the P and F states are red shifted and equivalent in intensity to the ferric state. This behaviour is consistent with the ferryl haem only in both species, with any radical in the P state being located instead on an amino acid, as is observed, for example, in compound I (an electronic equivalent of the P state) of cytochrome c peroxidase [63].
1.9.1 Experimentally induced P and F states
P and F are catalytic transient intermediates that can be experimentally induced in a manner that last for several minutes to permit analysis. These artificially generated forms have been given names that refer to the conditions used to induce them
(PM/PR/F/ F
.
(F dot)) [54,55]. Figure 1-12 summarises how the various forms of P and F
Figure 1-12. Schematic showing how the various P and F states differ in their protonation state and electron status, together with the position of their visible absorption band. See main text for how each state is experimentally generated.
1.9.1.1 P
MThe PM state can be formed with two different approaches. In the first, fully oxidised
CcO is reacted with CO/O2 at alkaline pH. CO donates two reducing equivalents and
two protons to reduce only the BNC where it is released as CO2. This forms a mixed-
valence state that binds and reacts with O2 to form A that spontaneously decays to
form the PM state (Figure 1-13) [64,65]. In the second approach, fully oxidised CcO is
reacted with stoichiometric levels of H2O2 at alkaline pH [52]. H2O2 reacts with the BNC
to form PM and H2O is released. In both reactions, CuA and haem a remain oxidised.
Hence PM will be an oxyferryl species with an associated radical, equivalent to the P
state with a visible absorption peak at 607 nm.
Figure 1-13. Reaction mechanisms of fully oxidised CcO with CO/O2 or stoichiometric levels of H2O2, at alkaline pH to form PM state.
The protonation state of tyrosine within the BNC is not definitively established.
P
M607 nm
P
R607 nm
F 580 nm
F(dot) 580 nm
1e- 1e - +H+ +H+Cu
A2+a
3+Cu
B2+a
33+Cu
A2+a
3+ CO + H 2O CO 2a
34+=O
2- O 2TyrO
—TyrO
—TyrO
.
Cu
B1+(2H
+)a
32+Cu
A2+a
3+Cu
B 2+(H
2O)
Fully oxidised Mixed valance P M oxyferryl state H 2O2 H 2O37
1.9.1.2 P
RThe PR state is a transient state that forms when the same CO/O2 reaction occurs
with the fully reduced enzyme at alkaline pH. Therefore it can only be observed at low temperatures (-90 oC) [66]. Here the BNC contains one more electron than the PM state
that is provided haem a. However, the visible spectrum of PR is the same as PM with a
peak at 607 nm [66]. This transient PR state forms the F state with the uptake of a
proton.
1.9.1.3 F
The F state can be formed when fully oxidised CcO is reacted with an excess of H2O2 at alkaline pH. Initially, the PM state is formed as described above. However, this
reacts with a second H2O2 that donates a third electron and a proton (and is released
as a superoxide O2- or hydrogen superoxide HO2
.
) to reduce the radical species in PM
and so form a stable F state.
The ratios of PM and F states generated by the H2O2 reaction in the steady state
are dependent on the relative amount of H2O2 reacted and the pH [67,68]. At low pH
the F state is favoured and at high pH with equal ratios of H2O2 and CcO, the PM state
is favoured [52].
1.9.1.4 F
.(F dot)
An F
.
state is formed by reaction of oxidised CcO with stoichiometric levels of H2O2at low pH [69]. This state is also formed by lowering the pH of a sample of CcO in the PM state that has been prepared at alkaline pH with either CO/O2 or stoichiometric
levels of H2O2 [69,70]. F
.
is isoelectronic with PM, but its visible spectrum is similar to
the F state with an absorption maximum at 580 nm instead of 607 nm. Since it is
isoelectronic with PM, the F
.
state must also have a radical amino acid. The shift from a
607 nm (PM and PR) to a 580 nm (F and Fdot) species is most likely the result of
protonation of a site within PM or PR, rather than an effect on the haem a3 spectrum of
an amino acid radical [54]. For example, the 607 nm peak is present in the PR and PM
1.9.2 Detection of a radical species in P
MEPR spectroscopy has in fact detected one or sometimes two radicals in CcO when it reacts with H2O2 [52-54,71]. The broad and narrow EPR signals detected were
dependent on the pH conditions used. At alkaline pH (8.0) the PM state exhibits a
narrow signal attributed to a tyrosine (Y244 [53]) or a porphyrin cation radical [54]. At
low pH (6-6.5) F
.
has a broad signal that was attributed to the migration of the radicalto a nearby tryptophan [54,70] or another Y129 [53,72]. Intriguingly the same broad signal was detected by Budiman, et al. [73] in P. denitrificans CcO but was independent of pH and, through mutagenesis (Y167F), it was assigned to Y167, a residue 10-13 Å away from the haem a3-Fe (equivalent to Y129 in bovine CcO). In all
cases, there has not always been a direct correlation between the amount of the radical species and specific intermediates of the reaction cycle. The radicals have been suggested to arise from a side reaction after H2O2 has reacted with the BNC, and
subsequent migration of the radical to other sites [54,55]. For example, the background
narrow EPR signal of a porphyrin radical was detected in PM, F
.
and also in the F
intermediate that should not contain a radical [54]. Non-specific side reactions can also
occur from the toxic by-products, superoxide O2- or hydrogen superoxide HO2
.
, of this
reaction.
In contrast, in the PM state generated by the less damaging reaction of oxidised
CcO with CO/O2 at alkaline pH, no EPR signal attributable to a tyrosine, tryptophan
radical or porphyrin cation radical was detected [52,54,70]. The absence of an EPR signal was proposed to be due to the spin coupling of the true cross-linked Y244(- H240) radical (S=1/2) with paramagnetic CuB2+ (S=1/2) because the CuB distance from
Tyrosine-OH group is 5.7 Å and CuB is a ligand of H240 [38,54,70,74,75].
Radioactive iodide labelling of the PM state (bovine CcO) prepared using the CO/O2
method followed by peptide cleavage and detection of labelled residues has located the radical site to be on Y244 [76]. Resonance Raman spectroscopy has detected a band at 1489 cm-1 that was tentatively assigned to a C-O. stretch of a tyrosine radical of the PM state of E. coli CcO [77] but its definitive assignment still requires isotope
labelling.
IR spectroscopy can also offer a means to detect a protein associated radical and this has been explored in Results Chapter 5 with the aid of model compounds that have provided reference spectra.
39