FTIR spectroelectrochemistry combined with a light-induced difference technique: Application to the iron-quinone electron acceptor in photosystem II
Abstract
Photosystem II (PSII) in plants and cyanobacteria performs light-driven water oxidation to obtain electrons necessary for CO2 fixation. In PSII, a series of electron transfer reactions take place from the Mn4CaO5 cluster, the catalytic site of water oxidation, to a plastoquinone molecule via several redox cofactors. Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been extensively used to investigate the structures and reactions of the redox cofactors in PSII. Recently, FTIR spectroelectrochemistry combined with the light-induced difference technique was applied to study the mechanism of electron-transfer regulation in PSII involving the quinone electron acceptors, QA and QB, and the non-heme iron that bridges them. In this mini-review, this combined FTIR method is introduced, and obtained results about the redox reactions of the non-heme iron and QB, involving the long-range interaction of the Mn4CaO5 cluster with the electron-acceptor side, are summarized.
1.Introduction
Photosystem II (PSII), one of the major protein complexes that work in oxygenic photosynthesis, has a unique function of light-induced water oxidation [67]. Electrons from water are ultimately used for CO2 reduction and released protons generate a proton gradient across the thylakoid membrane to synthesize ATP. In addition, molecular oxygen, a byproduct of water oxidation, makes an oxygenic atmosphere, which is essential for sustenance of life on the earth. In photosystem II, light absorption triggers charge separation between the chlorophyll dimer (P680) and the pheophytin (Pheo) electron acceptor to produce P680+Pheo− [51]. On the electron-acceptor side, an electron on Pheo− is transferred to the primary quionone electron acceptor (QA) and then to the secondary quinone electron acceptor (QB) [16,38,48], while on the electron donor side, the electron hole on P680+ is transferred to the redox-active tyrosine (YZ) then to the Mn4CaO5 cluster, at which water oxidation takes place via a cycle of five intermediates called
Fig. 1.
The electron transfer reaction between QA and QB is controlled by the gap of their redox potentials (
Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been extensively used to investigate the structures and reactions of the redox cofactors in PSII [5,11,13,14,39–43]. In this method, photoreactions are initiated by illumination of continuous-wave light or flashes from a pulse laser, and the structural changes of the cofactors and surrounding protein moieties are studied by detecting small infrared absorption changes. On the other hand, an FTIR spectroelectrochemical method has been used to investigate redox reactions of biomolecules and proteins [3,6,7,20,34,35,37]. In these studies, the redox reactions were triggered by changing the electrode potential and FTIR difference spectra were measured. Spectroelectrochemistry utilizing UV-Vis and fluorescence spectroscopies has been used to determine the
2.Instrumental setup of FTIR spectroelectrochemistry combined with light-induced FTIR difference spectroscopy
Figure 2A shows a schematic view of an optically transparent thin-layer electrode (OTTLE) cell used in our FTIR spectroelectrochemical measurements. This cell is similar to the ones previously reported [7,37]. Because strong infrared absorption of water interferes with the bands of proteins, a very thin-layer metal mesh, for example 6 µm in thickness, is used as a working electrode. When an Au working electrode is used, the surface of the electrode is modified with thiols to prevent irreversible adsorption and denaturation of proteins [62]. A commercially obtained Ag/AgCl/3 M KCl reference electrode (Cypress Systems Inc., 66-EE009; +208 mV vs. SHE) with a diameter of 2 mm and a Pt black wire as a counter electrode are arranged in the Teflon body (Fig. 2A).
Fig. 2.
Because redox cofactors located inside a protein are difficult to interact with an electrode directly, redox mediators are necessary for electrochemical measurements. Several mediators are used to cover the potential regions of target cofactors. For example, for the measurement of the non-heme iron in PSII, Ru(NH3)6Cl2 (
For photosensitive proteins like PSII, there is a merit to combine the spectroelectrochemistry and a light-induced difference technique for studies of redox cofactors. The
For light-induced FTIR difference measurements of electrochemically controlled samples, the OTTLE cell wired with a potentiostat is set in the sample room of FTIR spectrophotometer (Fig. 2B), and light illumination is performed, for example by flashes from a Q-switched Nd:YAG laser (532 nm; ∼7 ns width), which are synchronized to FTIR scans by triggers from the spectrophotometer [39]. The temperature of the OTTLE cell is controlled by circulating cold water in a copper holder. Details of the methodology of light-induced FTIR difference spectroscopy and application to PSII have been described in previous review articles [5,11,13,14,39–43].
3.FTIR spectroelectrochemical study on the non-heme iron
The non-heme iron connects QA and QB by a molecular bridge, QA-His214(D2)-Fe-His215(D1)-QB (Fig. 1) [18,21,63]. In addition to these His ligands, other two His residues, D1-His272 and D2-His268, and a bicarbonate ion function as ligands to the non-heme iron [18,21,63]. Under physiological conditions, the non-heme iron is not involved in the electron transfer reaction from QA to QB [16,38,48], because of its high
Figure 3 shows flash-induced FTIR difference spectra of the O2-evolving (a, black line) and Mn-depleted (c, red line) PSII membranes of spinach at pH 6.5 measured in an electrolytic solution at +600 mV (vs. SHE) [28]. Bands at 1339, 1258, 1229, 1109, and 1101 cm−1, which were observed in both spectra, are typical of the Fe2+/Fe3+ difference signals [4,22,44,60,61]. The 1339(+)/1229(−) cm−1 bands were attributed to the symmetric CO stretching vibrations of the bicarbonate ligand [22], while the positive band at 1258 cm−1 and a part of the 1229 cm−1 negative band were assigned to the CO stretching vibration of a Tyr side chain (either D1-Tyr246 or D2-Tyr244) structurally coupled to the non-heme iron using [4-13C]Tyr labeling [60]. In addition, the 1109(+)/1101(−) peaks were assigned to the CN stretching vibrations of the imidazole ring of the His ligands [4]. In the difference spectrum of intact PSII, signals were also observed at 1439, 1419, 1404 and 1365 cm−1, which arise from the transition from the dark-stable
Fig. 3.
Fig. 4.
From the intensities of the major signals around 1240 cm−1 in the thus-obtained Fe2+/Fe3+ spectra at a series of electrode potentials between +350 and +600 mV (Fig. 4A), the molar ratios of Fe3+ and Fe2+ were estimated and analyzed in a semilogarithmic Nernst plot (Fig. 4B) [28]. A linear relationship with slopes of 65 and 62 mV was obtained for the intact and Mn-depleted PSII samples, respectively; These slopes are similar to the theoretical value of 56 mV at 10°C (measuring temperature). The data from both PSII samples well followed the theoretical one-electron Nernstian curves (Fig. 4C), and the
Fig. 5.
The Fe2+/Fe3+ difference spectra also showed some changes in the COOH and His CN regions upon Mn depletion (Fig. 5). A negative peak at 1750 cm−1 was observed in the region of the C=O stretching vibration of COOH in intact PSII, whereas this band was not detected in Mn-depleted PSII (Fig. 5A). It was suggested that the
4.FTIR spectroelectrochemical study on QB
In contrast to many reports of the measurement of
Fig. 6.
Fig. 7.
This marker peak of QB at 1745 cm−1 was used to examine the redox state of QB at a series of the electrode potentials (Fig. 7A) [27]. Note that in this method, the QB signal is obtained as a light-induced change of QB, typically one electron reduction of QB, after reaching equilibrium of electrochemical reactions, and hence electrochemically-induced changes are not directly detected in FTIR spectra. The semilogarithmic Nernst plots of the relative intensities of this peak against the electrode potential (Fig. 7B) showed virtually linear relationships with slopes of 30 ± 2 and 39 ± 1 mV for intact and Mn-depleted PSII, respectively. These slopes are closer to 28 mV, the theoretical value of a two-electron reaction at 10°C (measurement temperature), than 56 mV, that of a one-electron reaction. The apparent redox potentials (
Fig. 8.
By determining the
5.Concluding remarks
The combined method of thin-layer electrochemistry and light-induced FTIR difference spectroscopy was applied to study the redox properties of the non-heme iron and the terminal quinone QB on the electron-acceptor side of PSII. In particular, the FTIR signal specific to QB reduction enabled the first direct measurement of the
Although the mechanism of electron transfer regulation in PSII was clarified by determination of the
Acknowledgements
This study was supported by JSPS KAKENHI (25410009 to Y.K., 24000018, 24107003, and 25291033 to T.N.).
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