Observación número 13/AOP/M04/2006:

pKa of tyrosine in the ground state is ~10, which is lowered to pKa 8-9 in the cross-

linked structure which must favour proton donation in CcO [222,233]. In the radical state the pKa is -2 and so it must be deprotonated [217]. The protonation state of

ground state tyrosine in fully oxidised CcO is still not resolved. The measured IR absorbance spectrum of protonated TyrHis(trimethyl), displays a large band at 1547 cm-1 of protonated TyrHis(trimethyl) (Figure 5-13C) (also observed in a related protonated Tyr-His compound (2-imidazole-1-yl-4-methylphenol) [108]. This could be attributed to the negative at 1547(-) cm-1 of oxidised CcO. Hence it would suggest tyrosine is protonated in oxidised CcO, and becomes deprotonated during the transition to PM. However the 1547 cm-1 is insensitive to H/D exchange or to tyrosine- and

171 cm-1 was assigned to a haem a3 vibrational mode [186,187,197]. The measured IR

absorbance spectrum of deprotonated TyrHis(trimethyl) displays a large band at 1301 cm-1 (Figure 5-13A) that is not present in the protonated spectrum (Figure 5-13C). Therefore the equivalent 1317(-) cm-1 observed in oxidised CcO may be of deprotonated tyrosine that forms the deprotonated tyrosine radical in the PM state.

Hence, overall, the data reported here suggest tyrosine in the PM minus oxidised IR

difference spectrum is deprotonated in the oxidised state. This is further supported by Gorbikova, et al. [214,244] that have used D channel mutants of P. denitrificans CcO and have shown the equivalent 1317 cm-1 (~1308 cm-1) band (also attributed to a deprotonated tyrosine) is present in the PR, F, and O states, and not in the FR state or

PM state. Thus it was proposed that tyrosine was protonated in the FR state, a radical

in the PM state and deprotonated in the subsequent PR, F, and O states of the natural

cycle. It was suggested that tyrosine would be reprotonated in the O→E catalytic step [214].

5.5 Conclusion

In conclusion, a Tyr-His model compound has been synthesised to mimic the conserved structural feature in CcO. This structure was adapted based on the structures of TMP and TBP to give a stable electrochemically induced radical species. CV was used to characterise its electrochemical properties and the pKa values (6.8

±0.4 of imidazole) and (9.2 ±0.2 of phenol) of TyrHis(trimethyl) were determined by titration of their UV-visible spectra. IR absolute spectra of fully protonated, neutral and fully deprotonated Tyr-His(trimethyl) have been tentatively assigned by comparison to IR bands predicted by calculation using Gaussian. Finally, the optimised electrochemical conditions to induce a radical on TMP, TBP and TyrHis(trimethyl) were combined with ATR-FTIR spectroscopy to record reduced minus oxidised (radical) IR difference spectra. Based on literature assignments, the bands at 1592 cm-1 (TMP) and 1572 cm-1 (TBP), have been assigned to v8a(C-C) and the bands at 1511 cm-1 (TMP) and 1507 cm-1 (TBP) to v7a(C-O.)of a phenoxyl radical. By comparison to TMP and TBP, the bands at 1550 cm-1 and 1510 cm-1 of TyrHis(trimethyl), were also assigned to v8a(C-C) and v7a(C-O.) of a phenoxyl radical, respectively. The peak/trough at 1301(+)/1314(-) cm-1 was only observed for TyrHis(trimethyl) and has been assigned to an imidazole ring stretch. Subsequently, the PM minus oxidised IR spectrum of bovine

CcO was recorded, and the presence of the PM state was confirmed by in situ

recording of visible spectra, that are well characterised for the PM state. In doing so

tentative assignments of possible tyrosine radical bands, in the PM intermediate, have

been made by comparison to the model compounds (TMP, TBP and TyrHis(trimethyl). An IR band at 1572 cm-1 or 1555 cm-1 has been assigned to v8a(C-C), and at 1519 cm-

1

to v7a(C-O.)of a phenoxyl radical of tyrosine in the PM state. 1336 cm-1 has been

assigned to a histidine ring stretch of the cross-linked Tyr His radical in the PM state.

The PM minus oxidised IR difference spectrum is also presented in the low frequency

range for the first time, and a band at 808 cm-1 has been shown to be 18O–sensitive, and has therefore been assigned to Fe4+=O2- stretch in the PM state.

173

5.6 Future work

Assignment of the reduced minus radical IR spectrum of TyrHis(trimethyl) was achieved by comparison to TMP and TBP. Definitive assignment of these bands, will require repeating the experiment with an isotopically labelled structure of TyrHis(trimethyl), for example 13C, where the two bands will be expected to shift.

It was clear that the reaction of TyrHis(trimethyl) was irreversible when the measurement was carried out in the IR spectroscopy setup, and is possibly because of the higher concentrations (10 mM) used of this compound, compared to the dilute conditions of the cyclic voltammetry measurements (0.2 mM). In order to overcome the irreversibility, the structure could be optimised further. It is possible that if a butyl group is placed at both 2C and 4C sites of the phenol ring the presence of the bulkier group may sterically stabilise/protect the radical, and possibly improve the reaction reversibility.

The electrochemistry-ATR-FTIR setup shown in the methods relied on diffusion of the species to and from the electrode surface and the ATR-prism. This factor may be another reason for the irreversible reaction observed for TyrHis(trimethyl). This is because the radical induced may have been short-lived resulting in a low yield. In contrast, in cyclic voltammetry the radical is always measured at the electrode surface. To overcome the limitation of time required for diffusion, an alternative ATR setup could be used with a boron doped diamond (BDD) ATR prism [245]. Here, a thin layer of boron doped diamond conducting material is deposited onto a silicon ATR prism. The BDD is optically transparent in the frequency range expected for a phenol radical, and so the IR beam would travel through the electrode material, and produce an evanescent wave that penetrates into the electrolyte on the top surface. In this way, the compound can be oxidised and reduced at the same site as the IR measurement. More importantly, it will allow IR spectra of the compound to be taken at the same time as the electrochemical reaction. However, preliminary cyclic voltammetry measurements with BDD did not detect any oxidation or reduction peaks for this compound. This could be due to the solvent conditions or the surface groups of the electrode material. In order to use this setup, different solvent conditions could be tested, for example acetonitrile containing tetraethylammonium hydroxide to deprotonate the compound and tetrabutylammonium hexafluorophosphate as supporting electrolyte. These conditions have been used for TBP and TMP with a platinum electrode [224] but they may also be compatible with a BDD electrode.

6 Possible functions of the H channel in yeast