3.5.2 Cryogenic TyrD ◦ FTIR Signals
Through multiple spectral subtractions, including two different subtraction pathways to enable the identification of spectral subtraction artifacts, we have isolated a difference signal that should correspond to the TyrD◦/TyrD signal at 80 K and 10 K; EPR control experiments have confirmed that TyrD oxidation does occur under the given sample conditions. The intensity of the 1653 cm−1feature appears to be consistently stronger in the first subtraction pathway. We suspect
that it is because Cyt b559is partially reduced in sampleC, since it contains 10 mM ascorbate. Although the Cyt b+559/Cyt b559 difference signal has not been fully characterised in PSII, the double-difference between the high and low potential forms of Cyt b559has a strong differential feature at 1661(+)/1655(-).42If a fraction of Cyt b559acts as a donor inC, then this differential feature could cause a spurious enhancement of the 1653(+) cm−1 band.
The TyrD◦/TyrD signals at 80 K and 10 K are similar to each other, but are quite different from the room temperature TyrD◦/TyrD signal. This is somewhat unexpected, as the EPR signal of the TyrD◦radical at 80 K is the same as that at room temperature, whereas the 10 K TyrD◦ rad- ical is highly electropositive, and has been postulated to be in a different protonation state.18,43 Furthermore, large temperature-dependent changes in PSII cofactor difference signals is not a general occurrence, as the Q−A/QA signal obtained at 10 K is only slightly changed from the
room temperature signal.33 In the 80 K signal, the ∼1737(+) cm−1, 1717(+) cm−1, 1703(-) cm−1,∼1697(+) cm−1, 1676(+) cm−1, 1653(+) cm−1, 1643(-) cm−1, 1631(+) cm−1, 1559(+) cm−1, 1543(+) cm−1, 1282(-) cm−1,∼1251 cm−1and 1226 cm−1bands appear to be related to room temperature bands, albeit with shifted frequencies (up to +12 cm−1 for the 1543(+) cm−1 feature, if it is indeed related to the 1532(+) cm−1 band) and also different band shapes.15,28,44 However, the bands at 1662(-) cm−1, 1621(+) cm−1,∼1478 cm−1and almost all of the features from 1000 cm−1up to 1400 cm−1(except 1282(-) cm−1,∼1251 cm−1and 1226 cm−1), do not appear to be related to room temperature features. The characteristic 1503(+) cm−1 feature in the room temperature signal is also changed at 80 K, and appears to be split into two peaks at
1511(+) cm−1and 1501(+) cm−1.
In the room temperature TyrD◦/TyrD difference signal, the modes that are shifted in frequency upon selective tyrosine labeling are 1503(+) cm−1, 1280(-) cm−1, 1250 cm−1 and 1220 cm−1, with the (1280(-) cm−1and 1220 cm−1) bands only appearing at high pH.15The 1503(+) cm−1 feature is assigned to theν(C-O) mode, while 1280(-) cm−1, 1250(-) cm−1and 1200 cm−1have been assigned to vibrations involving the phenolic C-O bond of TyrD, possibly aδ(COH) mode. If the 1511(+), 1501(+), 1282(-), ∼1255(-) and 1226(-) modes correspond to the same room
temperature difference features, then the 1282(-),∼1255(-) and 1226(-) modes are collectively
upshifted compared to their room temperature counterparts. One possible cause is an increase in hydrogen bonding strength to TyrD (all negative features belong to TyrD, while positive features belong to TyrD◦), since bending modes upshift with hydrogen bonding.45 The splitting of the 1503(+) cm−1feature into 1511(+) and 1501(+) bands is also consistent with a change in hydro- gen bonding to the phenolic CO group. The 1511 cm−1mode is remarkably similar in frequency to theν(C-O) mode of TyrZ◦, and it is thought that the higherν(CO) frequency is indicative of stronger hydrogen bonding in TyrZ to its His partner (D1-His190), compared to TyrD.9 This change in theν(CO) mode is not induced by high pH treatments at room temperature, but at 80 K and below, TyrD◦may be prevented from relaxing to the room temperature conformation, as some proton transfers are likely to be inhibited at cryogenic temperatures. The assignments of these features to TyrD◦/TyrD remain tentative, until experiments using isotopically labeled tyrosine can be performed to positively identify these signals.
The strong absorptions from 1600 cm−1 and above do not shift in frequency with isotopic la- beling, and most likely arise from peptide C=O and carboxylate asymmetric stretching modes or water bending modes (His189 only contributes weakly in this region).28 These difference features may be caused either by the electrostatic interaction between TyrD◦ and its environ- ment, or changes in the hydrogen bonding network responsible for proton egress from TyrD. The D2-His189Gln mutation strongly alters these bands, and induces features at 1659(-) cm−1 and 1623(+) cm−1, amongst other observed differences to the wildtype. Interestingly, the 80 K difference signal has similar features at 1662(-) cm−1 and 1621(+) cm−1. It is known from
which would almost certainly affect proton egress from the TyrD pocket.46 It is then possible that cryogenic temperatures may inhibit proton transfer from TyrD in a manner similar to the D2-His189Gln mutation.
The similarity of the 10 K and 80 K signals suggests that there are no temperature-related changes in the bonding of TyrD◦, but there are still some small differences that can be ob- served in the double-difference spectrum (shown in Figure 3.7). These differences seem to be caused by small frequency shifts of only a few wavenumbers, as the differential features are quite small and sharp. This is consistent with electrostatic effects, which are expected to shift infrared modes by several wavenumbers, depending on the magnitude of the modes’ dipole moment and its alignment relative to the charge.47 Furthermore, C=O stretching modes (which are highly sensitive to electrostatic effects) tend to absorb in the∼1600-1700 cm−1region, where the most prominent features of the 10 K-minus-80 K double-difference spectrum are observed. Thus, the FTIR result is generally in-keeping with the increased TyrD◦ electropositivity observed in EPR.18
The origin of this increased TyrD◦ electropositivity at 10 K remains uncertain. While proton transfer from TyrD is most likely, being further retarded at 10 K compared to 80 K, it is uncer- tain as to where the proton is trapped. One possible explanation is that the mobile water that serves as a proton carrier for TyrD plays some role in PCET even at high pH.48,49If this water ac- cepts a proton from TyrD, but is completely immobilised at 10 K, this could cause the observed increase in TyrD◦ electropositivity. Warming the sample to 80 K could be sufficient to enable thermal relaxation, which in turn relieves electrostatic strain in the system. This may be tested in future experiments using deuterated samples, as the 10 K-minus-80 K double-difference, es- pecially in the 1600-1700 cm−1 region, is likely to contain features derived from water modes if this were the case.
We note that these results do not agree with the proposal by O’Malley et al., that an imida- zolium intermediate is formed upon TyrD oxidation at 10 K.43 Based on FTIR studies on 4- methylimidazole radicals, the imidazolium form would have strong absorptions in the C=O
region that are not present for the imidazole, amongst other differences.50No positive features of the appropriate magnitude (∆A ∼10−4) were observed in the 10 K-minus-80 K double- difference spectrum.
3.5.3 TyrZ Oxidation at Liquid Helium Temperatures
Based on EPR studies, TyrZ undergoes photo-oxidation at liquid helium temperatures, with the resulting TyrZ◦ showing illumination wavelength-dependent kinetics. Visible light illumina- tion would induce a TyrZ◦ radical that decayed in minutes, while NIR illumination on samples poised in S2 or S3 would result in a stable TyrZ◦ radical. Unfortunately, both of these illumi- nation regimes induced a ZnSe FTIR difference signal with very similar decay kinetics, which hindered analysis of the TyrZ◦/TyrZ difference signal. We were only able to isolate a PSII- related signal from samples continuously illuminated with green light. The features of QA/QA– could not be eliminated through signal subtraction in this instance, as there was insufficient time to obtain a “clean” QA/QA– fromT.vulcanusPSII cores, after we had identified the ZnSe signal effects interfering with observation of PSII-related signals.
Nevertheless, it is uncertain as to why NIR illumination on samples poised in S2 did not give rise to a signal that can be associated with PSII, despite parallel EPR experiments showing that a PSII signal is induced using this illumination protocol, albeit with differences in illumination time owing to the optical thickness of the EPR sample compared to the FTIR sample. TyrZ oxidation in this case is thought to involve direct NIR excitation of the WOC, leading to electron transfer from TyrZ to the WOC.51,52Considering that, some possible explanations for this null result are:
1. The NIR-induced difference signal is inherently weak, and was completely obscured by the ZnSe signal.
2. The NIR illumination used in the FTIR case was not long or strong enough to induce TyrZ photo-oxidation.
3. NIR illumination at 10 K does not cause an electron hole to transfer from the WOC in S2 to TyrZ. Instead, some change not detectable using FTIR spectroscopy has occurred.
In the first case, the issue is easily resolved by using non-photoactive IR windows, since FTIR is able to identify absorption changes as small as 10−5absorption units. As for the second case, it is plausible that the illumination used in these experiments was not sufficiently strong, since optical absorption at the WOC is∼1000 times lower than that of the surrounding chlorophylls
in PSII.53 The third case has a precedent. Onodaet al. have shown that the NIR-induced S2 multi-line to g4.1 conversion produces no observable FTIR difference signals.54 While there is no evidence indicating that the NIR-induced EPR signal is due to a WOC spin-state conversion, the formation of a TyrZ◦ radical is expected to cause structural changes measurable by FTIR difference spectroscopy.
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