3.4.1 An Unexpected Result: The Light-induced ZnSe FTIR Difference Signal
At liquid helium temperatures, we ultimately discovered, quite unexpectedly, that the ZnSe windows used in the cryostat responded to illumination at 10 K and gave a pronounced FTIR difference signal. The ZnSe signal has an illumination-dependent kinetic behavior – green il- luminations at 10 K would induce a transient signal that decays over∼10 minutes (as well as
a stable component), while 200 K green illumination combined with 10 K near-infrared illu- mination induced a stable ZnSe signal. These are shown in Figure 3.2.The 200 K green + 10 K NIR illumination regime was initially designed to induce stable oxidation of TyrZ via the transfer of an electron hole from the WOC in the S2state, while a 10 K green illumination was expected to induce a transient TyrZ◦ signal. The ZnSe signal was thus, for some time, assumed to correspond to that of TyrZ oxidation.
FIGURE 3.2: FTIR difference signals from 10 K Illumination on the cryostat, using NIR light (A) and green light (B). The NIR illumination was for 1 minute, and the difference spectrum was taken immedi-
ately after illumination.
Subsequent experiments were designed to investigate the temperature dependence of the signal. 10 K NIR illumination without pre-illumination at 80 K or above would induce the stable ZnSe signal only if longer illuminations were undertaken (2 minutes or more), and the signal could notbe induced at 80 K, either by NIR or green light. Furthermore, we found that the ZnSe signal could be induced by light up to wavelengths as long as 820 nm, which was the long wavelength limit of the Ti:Sapphire laser in its current configuration. These spurious signals interfered with all PSII experiments performed at 10 K (examples shown in Figure 3.3), thus necessitating the pre-illumination of samples using NIR light to saturate the ZnSe signal.
FIGURE 3.3: Stable light-induced signals in dark-adaptedT.vulcanusand spinach PSII membranes, in- duced by green light (A) and 6 minutes of NIR illumination (B) at 10 K. The labeled peaks in (A) are characteristic of the ZnSe signal. NIR illumination also induced P700+/P700 signals (labeled peaks in
(B)), due to substantial PSI contamination in spinach PSII membrane samples.30
3.4.2 TyrD Oxidation at Cryogenic Temperatures
TyrD oxidation was measured in pH 8.5 spinach PSII membrane samples, at 77 K and 10 K in FTIR, and 7.5 K in EPR. Spinach PSII membranes were used for this set of experiments, as our spinach PSII core samples were largely inactive following high pH treatment, and T.vulcanus samples were only available in small quantities. EPR measurements indicate that TyrD is largely reduced in pH 8.5 membrane samples prepared in total darkness. Subsequent illumination at 7.5 K produces the TyrD◦ radical signal (see Figure 3.4). This EPR result confirms that TyrD◦ is photo-generated under the given sample and illumination conditions, in subsequent FTIR exper- iments.
FIGURE 3.4: EPR measurements of the TyrD radical, before and after 3s white illumination at 7.5 K, in PSII membranes poised at pH 8.5. EPR parameters: Microwave frequency 9.42 GHz, microwave power
5µW, modulation amplitude 2 G.
One of the more challenging aspects of FTIR studies at cryogenic temperature, is that illumina- tion at these temperatures always results in electron donation from a range of secondary donors, making it very difficult to extract the signal of one specific redox species out of these differ- ence spectra.31,32 Although it is not possible to fully prevent oxidation in multiple secondary donors, the preferred secondary donor species can still be manipulated to an extent using chemi- cal treatments. For this purpose, we generated the (green) light-minus-dark spectra of 4 different samples, denoted A – D, to perform spectral subtractions, so as to obtain a “clean” TyrD◦/TyrD FTIR difference signal.
Prior to 10 K illuminations, the samples were pre-illuminated for 6 minutes with NIR light so as to pre-oxidise P700, and saturate the ZnSe signals. This allowed for better spectral subtractions. The treatments used in each sample are detailed in Table 1. The treatments used in each sample are detailed in Table 3.1.
Illuminations of samples A to C were performed for 3 seconds, while illuminations on sample D were limited to 1 second to avoid electron donation from ChlZ, which occurs upon longer illuminations under highly oxidising conditions.34 The difference spectra obtained from these samples are shown in Figure 3.5.
Sample Treatment pH Redox Species formed by Illumination
A 10 mM Ferrocyanide 8.5 TyrD◦, Car+, QA–
B 5 mM ferricyanide, then washed 6 Car+, QA–
C 20 mM Ascorbate 6 QA–*
D 40 mM Ferricyanide, 0.6 mM SiMo 6 Car+
TABLE3.1: The spectra collected in order to obtain the TyrD◦/TyrD difference signal via spectral sub- tractions. *Cyt b559 would be expected to donate under these conditions, but the resultant difference
signal was very similar to QA–/QAobtained at 200 K.33
FIGURE 3.5: Light-minus-Dark difference spectra obtained from sample A, B, C and D at 80 K and 10 K. Illumination was performed with green light for 30 s, and each difference spectrum is an average from 3 samples, for a total scan time of 30 minutes (8085 scans). All 10 K difference spectra were corrected using the Rubberband Baseline correction function, to correct for the residual baseline slope that comes from illumination of the ZnSe windows. The difference spectrum of D was only collected at 80 K, because B-minus-C double-difference spectra were identical at 80 K and 10 K (not shown), implying that
Car+/Car did not change between 80 K and 10 K.
Two different spectral subtractions were used to obtain the TyrD◦/TyrD signal. These spectral subtractions were performed by scaling and matching the characteristic features of some given redox species, to remove all spectral contributions arising from that redox species and to isolate those of TyrD◦/TyrD. For instance, the most prominent Car+/Car features are the peaks at 1465 cm−1, 1440 cm−1 and 1147 cm−1, while the characteristic QA–/QAfeature is a peak at∼1480 cm−1. The two different subtraction pathways allow for TyrD◦/TyrD features to be separated from those that are merely artifacts arising from multiple spectral subtractions; features that truly arise from TyrD oxidation should be similar in both resultant spectra. In the first subtraction pathway, the carotenoid signals of sample B and sample A were matched, and B was subtracted from A. The remaining QA–/QAsignal would then be subtracted using the signals of sample C. In the second subtraction pathway, the QA–/QAsignals of sample B were matched with sample
A, with B subtracted from A again. This would over-subtract the carotenoid features, so the signals of sample D were (fractionally) added to compensate for the over-subtraction.
Both subtraction methods resulted in largely similar TyrD◦/TyrD difference signals (see Figure 3.6), albeit with a few notable differences. The intensity of the positive 1653 cm−1 peak tends to be stronger in Subtraction 1, while a 1480 cm−1positive feature in the 10 K spectrum only
appears in Subtraction 2. Some features also appear at slightly different frequencies, e.g. 1739 cm−1vs 1737 cm−1.
FIGURE3.6: TyrD◦/TyrD at 80 K and 10 K, obtained using two different spectral subtractions. Subtrac- tion 1 (Red): A – B – C, Subtraction 2 (Black): A – B + D. The peaks that are present in both spectra are
labeled in blue, while the features unique to each spectra are labeled in their respective colours.
A double subtraction between the 80 K and 10 K TyrD◦/TyrD signals revealed many small, sharp difference features, with larger changes occurring in the 1600 – 1700 cm−1 region (see Figure 3.7). The smaller features are not due to noise since they appear consistently in a number of double-difference spectra.
FIGURE 3.7: 10 K TyrD◦/TyrD minus 80 K TyrD◦/TyrD, with (a) obtained via A – B – C, and (b) obtained via A – B + D. The subtractions were optimised for the disappearance of peaks at 1653 cm−1 and 1559 cm−1, as these were the two most distinct features in low temperature TyrD◦/TyrD difference