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the split pheophytin signal titration phenomenon it was necessary to investigate the QA/QA" mid-point potential and also the behaviour of the E, in +/- bicarbonate systems. All the titrations were carried out at pH 7.0? other pHs mentioned refer to the pH at which the different PS2 complexes were prepared.

A redox titration of the g=1.9 Qx‘Fe2+ EPR signal observed in pH 7.5 OGP PS2 + 50 mM HC03‘ showed that the signal titrates in a single step (Fig.3.32). An n=l Nernst fit gave a curve with an E„7 of -32 mV. In the several titrations performed on the bicarbonate dependent signal the mid-point potential was found to vary between +5 and - 32 mV. This variability probably originated from errors inherent in the titration method, possibly due to insufficient equilibration after dithionite additions.

A titration of the g=1.8 Qx"Fe2+ EPR signal in pH 6.0 OGP PS2 (Fig. 3.33) gave an Eb7 of -102 mV. This was lower than that for the Qa/Qa' couple obtained by titrating the

g=1.9 signal. However, the significance of this result is questionable because the Qx'Fe2+ EPR signal obtained in this titration was a mixture of both the g=1.8 and 1.9 forms. The partial conversion back to the native bicarbonate situation may have occurred because of C02 contamination in the N2 used to keep the titration anaerobic or during the prolonged incubation in 1 M Tris-HCl pH 8.8 required for inactivation of the OEC. It proved difficult to maintain

110. 99.0 BB.O 77.0 66.0 c => >* c. a c. 55.0 o 44.0 cn < 33.0 22.0 11.0 40.0 100. Potential mV.

Figure 3.32 Determination of the mid-point potential of Qa/Qa‘ in pH 7.5 + 50 mM bicarbonate OGP PS2. A theoretical

one electron Nernst curve is plotted for the rise of the g=1.9 native QA iron-semiquinone EPR signal as the potential is lowered. The E,7 was calculated to be -32 mV but this was found to vary between +5 and -32 mV in the several titrations performed.

110. gg.o BB.O 77.0 66.0 c a> = > N -*-i > CD l. a rH £_ 55.0 a u ■rt L CO < 44.0 33.0 11.0 — *—e---1 p obo 1 ■60.0 -20.0 40.0 100. 140. -200. -260. -320. -360. 440. Potential mV.

Figure 3.33 Redox titration of the g=1.8 QK iron- semiquinone EPR signal in pH 6.0 OGP PS2 to determine the mid-point potential of the primary quinone. The theoretical one electron Nernst curve fitted to the data gives a value of -102 mV for the QA/QA‘ couple in this bicarbonate depleted system.

the g=1.8 signal in 100% of reaction centres at pH 7.0, in the pH 6.0 OGP PS2, without the addition of formate. The g=1.8 QA‘Fe2+ signal in pH 6.0 OGP PS2 + 50 mM HCOa' was found to titrate in a single step but with a higher E, of -5 mV

(Fig.3.34).

The narrow splitting of the pheophytin signal observed in the g=1.8 QA"Fe2+ type of samples presented a further problem (the split signal in formate treated samples had a similar lineshape and width as that observed in Fig.3.27a). Contaminating signals were found in the g=2 region in all the titrations which obscured the true size of the narrow split signal and prevented any accurate measurements. No conclusions about the effects of bicarbonate depletion on the split pheophytin signal titrations could be drawn. The wider bicarbonate dependent split signal was unaffected by this problem. The contaminating radicals displayed various widths and sizes, depending on the potential, so were thought to arise from the redox mediators. Mediators are normally chosen because they are thought to undergo a single step two electron reduction but it is possible that the semi-reduced states can become stabilised, perhaps by association with proteins.

In an effort to ensure adequate redox mediation in the -100 to -250 mV range anthraquinone-1,5-disulphonate (E^ - 170) was initially included in the titrations. Some of the spectra obtained are shown in Fig.3.35. Fig.3.35a is the g=1.9 QA'Fe2+ signal in a pH 7.5 OGP PS2 + 50 mM HC03" dark sample poised at -106 mV. Fig.3.35b is an identical sample

110. gg.o 68.0 77.0 66.0 >. c. a c. 55.0 a a ■el C. cn < 44.0 33.0 22.0 11.0 Potential mV.

Figure 3.34 Redox titration of the g=1.8 QA iron- semiquinone EPR signal in pH 6.0 OGP PS2 + 50 mM formate to determine the mid-point potential of the primary quinone. The theoretical one electron Nernst curve fitted to the data gives a value of -5 mV for the QA/QA' couple.

1-9 1-8 1-7 16 1-5

mT

400 450

350

Figure 3.35 QA iron-semiquinone EPR signals obtained from a redox titration of this signal in pH 7.5 + 50 mM HC03' OGP PS2 where the mediator anthraquinone-1,5-disulphonate was included. (a) dark spectrum from a sample poised at -106 mV, (b) as (a) but poised at -337 mV, and (c) is a 200 K illuminated spectrum from a non-reduced dark adapted control sample lacking the mediator. Chi concentration 2.5 mg/ml. EPR conditions: temperature 5 K, microwave power 25 mW, modulation amplitude 1.25 mT.

at -337 mV but in addition to the g=1.9 signal the spectrum also includes a large signal peak at g=1.65 and a trough at g=1.55. This signal was only observed in samples in the potential range -250 to -350 mV but w a s . absent if the mediator was excluded.

Anthraquinones are known inhibitors of PS2 electron transport [Ottmeier et a l .. 1988] and probably act at the Qb binding site. EPR signals at g=1.65 have previously

[Corrie et a l .. 1991] and here (section 3.1) been shown to be associated with an interaction between the QA and QB iron-semiquinones. Therefore the new signal probably represents an interaction between the Qx iron-semiquinone and the semi-reduced anthraquinone bound either in the QB binding site on D1 or nearby. The deviation of the mediator from the quoted mid-point potential to a lower potential could be an effect of the binding environment. It is concluded that quinone mediators of this sort should be avoided in EPR titrations of the PS2 quinones. Stable potentials were obtained with the range of mediators described in section 2.6.

Fig.3.35c is a non-(dithionite)reduced 200 K illuminated control, a g=1.9 QA~Fe2+ signal in pH 7.5 OGP PS2 + 50 mM HC03". The g=1.9 QA'Fe2+ signal arising from chemical reduction at 283 K (Fig. 3.35a) was different to that obtained by photoreduction at 200 K (Fig.3.35c). The low field peak was significantly broader in the chemically reduced samples. This may be due to reaction centres assuming a preferred conformation upon QA reduction at 283

K. At cryogenic temperatures this reorganization could be