Features of Using 2,6-Dichlorophenolindophenol as An Electron Acceptor in Photosynthesis Studies

2,6-dichlorophenolindophenol (DCPIP) is a redox indicator widely used to study electron transfer reactions in biological systems, including in the process of photosynthesis. DCPIP exists in solution in two forms, “pink” and “blue,” which transform into each other during protonation/deprotonation. Upon reduction, the DCPIP is discolored. We investigated the pH-dependence of DCPIP reduction rate in the presence of the photosystem II (PSII) at two wavelengths: 522 nm (isobestic DCPIP point) and 600 nm (near the absorption maximum of the deprotonated “blue” form). It was shown that, in experiments with a change of the pH medium, measuring at a wavelength of 600 nm requires corrections related to changes in the ratio of the “blue” and “pink” forms of the acceptor as well as using the pK parameter of this acceptor, whose рK values vary in various sources, to calculate the DCPIP reduction rate. Measurements at the isobestic point (522 nm) allowed for avoiding these complexities. We also found that the maximum at the pH-dependence of the DCPIP reduction rate by PSII shifted by approximately 1 unit to the acidic region relative to the maximum of the acceptor pair 2,6-dichloro-p-benzoquinone–potassium ferricyanide reduction rate pH-dependence. This shift may be due to the lower availability of the QB site on the acceptor side of PSII for the charged deprotonated DCPIP form compared to the uncharged protonated form.


INTRODUCTION
The rate of oxygen evolution and the reduction rate of artificial electron acceptors are integral indicators of the functional activity of photosynthetic oxygenevolving objects [1]. In studies of membrane preparations of photosystem II (PSII) or thylakoid membranes, the release of oxygen is recorded polarographically using a Clarke electrode and artificial electron acceptors, such as derivatives of benzoquinone, ferricyanide, and other compounds. The reduction of colored artificial electron acceptors can also be detected photometrically. The latter method for assessing PSII activity is more universal since it is also applicable to drugs with a nonfunctional oxygen-evolving complex (OEC). In this case, artificial electron donors (diphenylcarbazide, Mn 2+ , donor system Mn 2+ + H 2 O 2 , etc.) are used. The redox indicator 2,6-dichlorophenolindophenol (DCPIP), which is discolored upon photoinduced reduction by photosystem II, is most often used as an electron acceptor [2][3][4].
The active centers of enzymes, including protein complexes of the electron transport chain of photosynthesis, include ionogenic groups. The pH-depen-dences of the enzymatic activity are obtained for the study of the properties of ionogenic groups. At the same time, it is important that the used method of assessing the activity is applicable over the entire investigated range of pH values. An important feature of DCPIP is the ability to exist in two forms, "pink" and "blue," which transform into each other during protonation/deprotonation when the pH of the medium changes [5]. This feature must be taken into account in the studies of the pH dependences of the reactions of electron transport of photosynthetic objects using photometry.
In this study, we investigated the features of measuring the rate of electron transport in PSII using DCPIP at various pH values of the medium. It was found that, when determining the rate of reduction of DCPIP in PSII at a wavelength of 600 nm, ambiguous results can be obtained when used in calculations of various pK values of the electron acceptor known from the literature. This ambiguity can be eliminated by using the 522 nm wavelength (DCPIP isobestic point) for activity measurements. In addition, it was shown that the inhibition of photoinduced reduction of DCPIP in the alkaline region can be mainly due to its RESEARCH ARTICLE deprotonation, which hinders the binding to the Q B site on the acceptor side of PSII.

MATERIALS AND METHODS
PSII membrane preparations of an active oxygenevolving complex (BBY particles) were isolated from spinach Spinacia oleracea L. according to the published method [6] with minor changes [7]. Their spectral and functional characteristics corresponded to those previously measured [8]. According to the literature data, the acceptor side of such PSII preparations completely preserves plastoquinones Q A , Q B and several plastoquinones of the pool [9]. The resulting preparations were stored at -80°С in buffer A containing 15 mM NaCl, 400 mM sucrose, 50 mM 2-(Nmorpholino)ethanesulfonic acid (Mes), pH 6.5. The total concentration of chlorophylls a and b was determined in an 80% acetone solution according to the known method [10]; it was 10 μg/mL in all experiments. Before measurements, the preparations were thawed in the dark at a temperature of -5°C for 1 h.
Electron transport rate in PSII preparations was recorded photometrically using a Specord UV-VIS spectrophotometer (Carl Zeiss Jena, Germany) in cuvettes with 0.4 cm optical path length. LEDs with a maxima of 450 nm (when measuring the reduction of DCPIP) and 650 nm (when measuring the reduction of potassium ferricyanide (PFC)), providing a saturating light intensity of 1800 μE m -2 s -1 were used as exciting light sources. Photo-induced changes in optical density were recorded at wavelengths of 522 and 600 nm (DCPIP) as well as 420 nm (2,6-dichloro-pbenzoquinone (DСBQ) + PFC). The cut-off excitation glass filters were installed in front of the photo-multiplier tube of the spectrophotometer: yellow ZhS-18 (at a recording wavelength of 522 nm), orange OS-14 (at a recording wavelength of 600 nm), and blue SS-15 (at a recording wavelength of 420 nm).
pH measurements were carried out in buffer solutions containing 15 mM NaCl, 400 mM sucrose, 50 mM citric acid (for a pH range of 4.0-5.0), 50 mM Mes (for a pH range of 5.5-6.5), 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (for pH range 7.0-8.0). The required pH value of the buffer solution was obtained by adding a concentrated NaOH solution.
RESULTS AND DISCUSSION DCPIP can exist as a solution in two forms, depending on the pH of the medium: protonated (DH, "pink" form) and deprotonated (D -, "blue" form) with an absorption maxima at 515 and 605 nm, respectively. When these forms are reduced, H 2 Dand H 2 DH products are formed, which practically do not absorb light in the visible region [5,11]. Structural formulas of various DCPIP forms are shown in Fig. 1a. Intense absorption in the visible region, physiologically acceptable redox potential (E m in the range from +130 mV to +217 mV [12,13]), and the discoloration during reduction are the reasons for the widespread use of DCPIP for the registration of electron transport in various photosynthetic objects.
The absorption spectra of DCPIP in media with different pH values are shown in Fig. 1b. At pH < 4, DCPIP almost entirely exists in the "pink" DH form, while it exists in the "blue" Dform at pH > 7 [11]. In the pH range of 4 to 7, both forms are present in various ratios, depending on the pH and dissociation con-  stant. With an increase in pH, a regular transformation of the absorption spectrum is observed, which is a superposition of the spectra of two forms: a shift of the maximum to longer wavelengths and an increase in optical density at the maximum. The spectra intersect at the 522 nm isobestic point. At this wavelength, the molar extinction coefficients of the "pink" and "blue" forms coincide (ε 522 = 8600 M -1 cm -1 [14]).
When DCPIP is used as an electron acceptor for studying electron transport in thylakoid membranes, the effect of pH on the measurement results is insignificant since the optimal pH value is 7.5. At this pH, DCPIP is represented in a completely deprotonated Dform. A different situation occurs in studies of PSII membrane preparations (BBY particles) when the pH optimum for measuring the activity is in a rather wide range of 6.0-7.0. In this case, the solution will contain both forms of DCPIP (DH and D -) in different ratios. A similar pattern will be observed when obtaining the pH dependences of electron transport in photosynthetic objects.
Most often, a wavelength of 600 nm near the absorption maximum of the "blue" form is used for photometric measurement of the rate of photoinduced reduction of DCPIP by photosynthetic objects [15]. However, at this wavelength, the molar extinction coefficients of the "pink" and "blue" forms differ by almost an order of magnitude. In order to elucidate the effect of the pH-dependent transition of DCPIP between the DH and Dforms on the measured values of the electron transport rate, we investigated the dependences of the reduction rate of this electron acceptor by PSII membrane preparations on the pH of the medium at two wavelengths: 600 nm and 522 nm (isobestic point). Typical kinetic curves of the photoinduced change in optical density upon the reduction of DCPIP with BBY PSII particles are shown in Fig. 2. During illumination (30 s), an almost linear decrease in optical density was observed at both wavelengths and at all pH values of the studied medium (4.0-8.0). For the calculation of the rate of decrease in optical density, the kinetic curves were approximated by a straight line using the OriginPro 2015 software. The activity of the preparations was calculated based on the slopes of the obtained straight lines.
The dependence of the activity of PSII BBY particles on the pH of the medium obtained at a wavelength of 522 nm is shown in Fig. 3a (curve 1). The curve is bell-shaped with a maximum at pH 5.5. The complete dependences of PSII activity on the pH were shown in many studies [16][17][18][19][20]. In this case, the measured parameter was the rate of oxygen evolution, recorded using various artificial electron acceptors. The pHdependence of the rate of DCPIP reduction as an indicator of PSII activity was previously investigated in only two studies [21,22]. A decrease in the activity of PSII preparations in the acidic region (at pH 5.0 or less) may be associated with the dissociation of three peripheral OEC proteins: PsbP, PsbQ, and PsbO [23].
In the alkaline region (at pH 6.0 and more), the cause of inhibition may be the extraction of Clions from OEC [19]. Difficulties associated with the presence of two forms of DCPIP (DH and D -) in the medium and their mutual transitions with varying pH arise when calculating the activity of PSII BBY particles (in units of μmol DCPIP (mg chlorophyll) -1 h -1 ) based on the rate of photoinduced decrease in optical density at a wavelength of 600 nm. At 600 nm, we used the apparent molar extinction coefficient of the acceptor for determining the activity, which was calculated according to the following formula: where ε 600 (DH) = 2700 M -1 cm -1 , ε 600 (D -) = 22000 M -1 cm -1 are the molar extinction coefficients of the "pink" and "blue" forms, and pK is the negative decimal logarithm of the DCPIP dissociation constant [14]. This formula can be obtained from the conditions for the additivity of the optical density and the equilibrium ratio of the concentrations of the two acceptor forms at varying pH. According to various authors, the pK value of DCPIP varies over a wide range from 5.57 to 5.9 [5,11,14]. The reason for the ambiguity in determining the pK value may be the instability of the protonated DCPIP form in an acidic medium [14]. The pH dependences of PSII activity calculated using pK values of 5.57, 5.7, and 5.9 are shown in Fig. 3a (curves 2, 3, and 4). When using different pK values in the calculations, the maximum of the pH dependence of the rate of DCPIP reduction remains in the range of 5.5-6.0, while the absolute value of the maximum activity varies significantly. Thus, with an increase in pK from 5.57 to 5.9, the maximum activity increases by 27.6%. Thus, the values of the activity of PSII preparations calculated based on the rate of DCPIP reduction derived from the decrease in optical density at 600 nm are largely sensitive to the pK value of the electron acceptor used in the calculations. At the same time, when using a wavelength of 522 nm, at which both forms of DCPIP have the same extinction coefficient, such difficulties do not arise. At this wavelength, the recorded photoinduced decrease in optical density will reflect the total rate of PSII reduction of both forms of the acceptor, regardless of their ratio determined by the pH of the medium. Therefore, there is no need to make corrections related to the pH dependence of the extinction coefficient. An additional advantage of using the  For the determination of the activity, the apparent molar extinction coefficients of DCPIP at 600 nm were used, calculated using the formula provided in the text, with pK 5.57 (curve 2), 5.7 (curve 3), and 5.9 (curve 4). (b) Dependences of the apparent extinction coefficient of DCPIP at 600 nm on pH, calculated using the formula provided in the text, with pK 5.57 (curve 1), 5.7 (curve 2), and 5.9 (curve 3). (c) The pH dependences of the activity (normalized to the maximum value) of PSII obtained using DCPIP as electronic acceptors (measurement wavelength was 522 nm, curve 1) and DCBQ + PFC pair (measurement wavelength was 420 nm, curve 2). The concentrations of DCPIP, DCBQ, and PFC were 80 μM, 200 μM, and 2 mM, respectively. The maximum absolute value of activity measured in the presence of the DCBQ + PFC pair (at pH 6.5) was 2100-2200 μmol PFC · (mg chlorophyll) -1 h -1 . 522 nm wavelength (compared to 600 nm) is the more intense absorption of the protonated DCPIP form at this wavelength, which allows more reliable measurements of the relatively low activity of PSII preparations in the acidic region. It should be noted that the form of the pH dependences of the activity of PSII BBY particles provided in published works [16][17][18][19][20][21] varies considerably. Both the position of the maximum and the width of the region with practically a maximal constant activity change. The reason for the differences can be both the PSII preparation itself, which is used in the measurement, and the electron acceptor(s). For the identification of the influence of the nature of the electron acceptor on the shape of the dependences of PSII activity on the pH of the medium, we measured the pH dependences with DCPIP and the acceptor pair DCBQ + PFC for the same PSII preparations. DCBQ belongs to the same group of PSII electron acceptors as DCPIP (quinone analogs are acceptors that bind to Q B -site) [1]. When DCBQ is used together with PFC, the latter plays the role of a noncompetitive secondary electron acceptor. PFC oxidizes DCBQ reduced by PSII and, thereby, maintains the concentration of oxidized quinone at a constant level [1]. In this case, the secondary electron acceptor PFC plays the role of a "drain" of electrons donated by PSII during the photooxidation of water. The reduction of PFC was recorded photometrically at its absorption maximum at 420 nm (ε 420 = 1020 M -1 cm -1 ). The dependences of the activity of PSII BBY particles (in % of the maximum value) on the pH of the medium for DCPIP (measured at 522 nm) and a pair of electron acceptors DCBQ + PFC are shown in Fig. 3b. The maximum of PSII activity when using DCPIP (at pH 5.5) is shifted by one unit to the acidic region compared to the maximum when using DCBQ and PFC. It should be noted that the authors of one of the studies [21] also found a shift in the maximum activity of PSII particles measured by the rate of DCPIP reduction to the acidic region as compared to the maximum activity measured by the rate of oxygen evolution in the presence of an electron acceptor phenyl-p-benzoquinone. It is important to note that the decrease in activity during the transition from the neutral to alkaline region for DCPIP is much more pronounced than for DCBQ + PFC. At pH 7.0, the rate of PFC reduction decreases by only 10% compared to the maximum rate at pH 6.5, and the rate of DCPIP reduction decreases by 45% in comparison with the maximum rate at pH 5.5. The reason for the differences in pH inhibition of PSII activity in the alkaline region when DCPIP and DCBQ + PFC are used as electron acceptors is the nature of the acceptors themselves. Probably, under otherwise equal conditions (the concentration of the electron acceptor, the level of inhibition of PSII at a certain pH value), the rates of electron transfer from PSII to the protonated and deprotonated forms of DCPIP differ. In this case, the accessibility of the binding site of the electron acceptor, the hydrophobic Q B site of PSII for an uncharged protonated form, should be higher than for a charged deprotonated form [15]. It should be noted that the ability to reduce DCPIP directly in the Q B site remains only a guess. It is possible that DCPIP is reduced from plastoquinol Q B H 2 , but plastoquinone Q B and its fully reduced form Q B H 2 relatively loosely bound to protein D1 of PSII and can be replaced in the Q B site both by electron transport inhibitors and exogenous electron acceptors [24]. In any case, the free Q B site of PSII or Q B H 2 bound to it are located deep in the hydrophobic region of the membrane, and their availability for DCPIP will depend on its charge, e.g., on its state (protonated or deprotonated). Since the deprotonated form of DCPIP predominates in the alkaline region in the medium, which accepts electrons from PSII less efficiently as compared to the protonated form, the decrease in the rate of DCPIP reduction with increasing pH occurs rather sharply.
Thus, when using DCPIP as an electron acceptor for studying electron transport in PSII, even for a slight variation in the pH of the medium, it is necessary to take into account not only the dependence of the enzyme activity on the pH but also an additional factor: a change in the ratio of the "pink" and "blue" forms, which are probably acceptors of different "strength."