Inhibitory and Non-Inhibitory NH3 Binding at the Water-Oxidizing

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Inhibitory and non-inhibitory NH3-binding at the water-oxidizing manganese complex of photosystem II suggests possible sites and rearrangement mode of substrate-water molecules Nils Schuth, Zhiyong Liang, Matthias Schonborn, Andre Kussicke, Ricardo Assuncao, Ivelina Zaharieva, Yvonne Zilliges, and Holger Dau Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00743 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 7, 2017

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Inhibitory and non-inhibitory NH3-binding at the water-oxidizing manganese complex of photosystem II suggests possible sites and rearrangement mode of substrate-water molecules Nils Schuth,# Zhiyong Liang,# Matthias Schönborn, André Kussicke, Ricardo Assunção, Ivelina Zaharieva, Yvonne Zilliges, Holger Dau* #

These authors contributed equally.

Freie Universität Berlin, Department of Physics, 14195 Berlin, Germany

*corresponding author: [email protected]

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Abstract. Identity and rearrangements of substrate-water molecules in photosystem II (PSII) water oxidation is of high mechanistic interest and is addressed herein by comprehensive analysis of NH4+/NH3 binding. Time-resolved detection of O2-formation and recombination fluorescence as well as FTIR difference spectroscopy on plant-PSII membrane particles reveals: (1) Partial inhibition in NH4Cl buffer with a pH-independent binding constant of about 25 mM, which does not result from decelerated O2-formation, but from complete blockage of a major PSII fraction (ca. 60%) after reaching the Mn(IV)4 (=S3) state. (2) The non-inhibited PSII fraction advances through the reaction cycle, but modified nuclear rearrangements are suggested by FTIR difference spectroscopy. (3) The partial inhibition is explainable by anti-cooperative (mutually exclusive) NH3 binding to one inhibitory and one non-inhibitory site; these two sites may correspond to two water molecules terminally bound to the 'dangling' Mn ion. (4) Unexpectedly strong modifications of the FTIR difference spectra suggest that in the non-inhibited PSII, ammonia binding obliterates the need for some of the nuclear rearrangements occurring in the S2-S3 transition as well as their reversal in the O2-formation transition, in line with the carousel mechanism (Askerka et al, 2015, Biochemistry 54, 5783). (5) We observe the same partial inhibition of PSII by NH4Cl also for thylakoid membranes prepared from mesophilic and thermophilic cyanobacteria, suggesting that the above results are valid for plant and cyanobacterial PSII.


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Introduction Photosynthetic water oxidation by cyanobacterial and plant photosystem II (PSII) is a key process in global bioenergetics: it has shaped the atmosphere, by O2 production, and the biosphere, by its essential role in primary biomass production.1 The PSII protein-cofactor complex facilitates light-driven water oxidation at its (electron) donor side and plastoquinone reduction at its acceptor side.2, 3 Today the mechanism of the water oxidation reaction (WOR; or oxygen evolution reaction, OER) in PSII can be discussed at the atomic level at unprecedented detail, based on an impressive body of early and recent research results2, 4-13 and in conjunction with the structural information provided by PSII protein crystallography.14-19 Nonetheless, key aspects have remained insufficiently understood. This applies specifically to the coupling of the water oxidation chemistry to movements of (i) protons, which inter alia are reaction products, and of (ii) water molecules (and or water species), of which two (per produced O2 molecule) are the substrates of the water oxidation reaction. As opposed to protons, water molecules are visible in structures obtained by X-ray crystallography, but without any obvious tag for identification of the two specific water molecules that represent the WOR substrates. Identification and tracking of the two substrate-water molecules and in the course of the reaction cycle doubtlessly will be crucial for definitive elucidation of the mechanism of photosynthetic water oxidation. We note in passing that non-biological water-oxidizing Mn(Ca) oxides exhibit surprising structural and functional similarities to the biological catalyst in PSII so that mechanistic understanding of biological water oxidation can support also the knowledge-guided development of inorganic catalysts for production of non-fossil (solar) fuels.20-24

Figure 1. Redox-active tyrosine (YZ) and Mn4Ca cluster of photosystem II. The graphic corresponds to the dark-stable S1-state of PSII.16, 19 Four Mn ions (Mn1 to Mn4; violet spheres), and a single Ca ion (green) are interconnected by five bridging oxygens (O4, O5 and others; red). Terminal water ligands of Mn4 (W1, W2) and of the Ca ion (W3, W4) are part of the displayed water-protein H-bond network. Selected amino acid residues are shown, which are part of the D1, D2 or CP43 subunits of


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PSII. The residues coordinating the five metal ions are not shown, with exception of D1-Glu333, which also contributes to binding of the indicated chloride ion.

Atomistic key players of the PSII water oxidation reactions are shown in Figure 1.16, 18 The absorption of a photon by the PSII pigments is followed by ultrafast electron transfer from the donor to the acceptor side of PSII.2, 3 Thereby a highly oxidizing chlorophyll cation radical, P680+ (not shown in Fig. 1), is formed. Within less than 100 ns, P680+ oxidizes a specific tyrosine residue denoted as TyrZ or YZ.13 The oxidation of YZ is not coupled to release of a proton, but to a shift of the phenolic proton within a strong H-bond to His190,13, 25 and likely also further rearrangements within the protein-water environment of the Mn4Ca-oxo core cluster.26, 27 The oxidation of YZ represents the starting point for the various reaction steps in water oxidation. They are mostly discussed in terms of the classical S-state cycle model proposed already in 1970 by Kok and coworkers,28 which later has been extended to account for intermediate states formed by proton removal from the catalytic site.5, 27, 29, 30 Kok's basic Sstate cycle model represents the foundation for the experiments and discussion of results in our present study. We employ sequences of saturating flashes of visible light provided either by Xenon flash lamps (for time-resolved O2 polarography) or nanosecond laser flashes (5 ns, 532 nm; for variable PSII fluorescence yield and delayed, recombination fluorescence; FTIR difference spectra) to drive the catalytic metal complex stepwise through its reaction cycle. The first flashes applied to dark-adapted PSII induce predominantly the S1→S2 (1st flash), S2→S3 (2nd), S3→S4=>S0 + O2 (3rd), and S0→S1 (4th) transitions of the Kok cycle, where the subscript indicates the number of oxidizing equivalents accumulated at the catalytic site before O2-formation in the S4=>S0 transition induced by the 3rd flash. The efficiency of each transition is less than unity and equals 1-m, where m is the so-called miss parameter introduced by Kok and coworkers. The Kok model predicts correctly a damped oscillatory pattern of the O2-evolution yield with maxima at the 3th, 7th and 11th flash for small values of m. Earlier31-34 and more recent35, 36 spectroscopic results have provided strong evidence that up to the S3 state, three oxidizing equivalents are accumulated by means of oxidation of Mn ions; partial oxidation of water in early S-states is not observed (but earlier spectroscopic37, 38 and a recent crystallographic18 study disagree regarding the S3-state). Experimental and computational investigations suggests that the S2→S3 transition involves transformation of a five-coordinate Mn(III) ion into a six-coordinate Mn(IV),12, 32-35, 39-41 but this was not seen in a recent crystallographic study.17 We conclude that the nuclear rearrangements in the S2-S3 transition are insufficiently understood, especially regarding their relation to the substrate water molecules, which in the subsequent transitions undergo the O2-formation chemistry; this knowledge gap hinders mechanistic understanding of photosynthetic water oxidation seriously. In investigation of enzymatic catalysis, the inhibition by binding of substrate analogues can be employed for interrupting the catalytic cycle by stabilization of intermediate states,


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ideally even providing insight in transition state properties. In photosynthetic water oxidation, the substrate molecules are water and thus identical to the solvent. Complete exchange of the all water molecules against similar molecules is impossible in experiments on PSII. Yet ammonia (NH3) can be employed as a substrate analogue because the NH3 molecule likely binds competitively to high-valent manganese ions with an affinity that exceeds the binding affinity of the replaced water molecules by several orders of magnitude (half-effect NH4+ concentration around 25 mM versus H2O concentration of 55 mol/L, as detailed later). Recently Cox and coworkers concluded, based on highly advanced EPR approaches in combination with computational chemistry, that ammonia replaces a manganese-bound water molecule denoted as W1, which is terminally coordinated to Mn4 of the Mn4Ca complex of PSII (Fig. 1).42 Supported by their mass-spectroscopy results and earlier findings,43 they assume non-inhibitory ammonia binding and arrive at the important conclusion that W1 is not a substrate water molecule, but possibly W2. Using a similar protocol for ammonia treatment, recent crystallographic data was interpreted as evidence for ammonia binding at the W2 site and it was proposed that W2 is not a substrate water, but possibly W1.17 These (conflicting) conclusions regarding the identity of the substrate water molecules depend critically on (i) the assumption of non-inhibitory ammonia binding and (ii) the presence of a single ammonia binding sites that is visible in the respective experiment. Both assumptions are not easily reconciled with the large body of experimental results on ammonia binding to PSII as they have been reported since 1975. Motivated by early studies on inhibition of Hill reactions by ammonium salts,44, 45 Velthuys and Delrieu investigated the influence of 50 mM NH4Cl (at pH 7.8) using selected light-flash sequences and detection of either the delayed chlorophyll fluorescence46 or the flash-yield of oxygen evolution.47 They detected ammonia binding in the S2 and S3 state with halftimes of 0.5 s and 10 s and saw that the ammonium salt caused only partial inhibition of O2formation yield (about 50%) as well as reduced the S-state transition probability (increased miss factor) in the non-inhibited PSII. Sandusky and Yocum analyzed the inhibitory effect of chloride and ammonium salts on the rate of oxygen evolution for PSII illuminated with continuous light of saturating intensity (RO2CW).48-50 Based on concentration dependencies of the RO2CW inhibition, they concluded that there is one site of competitive chloride-amine binding and one ammonia-specific binding site. Based on analysis of two EPR signals assignable to the S2-state of PSII, the largely featureless g4 signal and the intricate g2 multiline signal, Brudvig and coworkers proposed two ammonia binding sites, with a third binding state reached by simultaneous binding to both sites;51, 52 Andreasson and coworkers extended this two-site model by taking into account the conformations of the Mn4Ca cluster corresponding to the g2 and g4 signal, both associated with two ammonia binding sites, resulting, all in all, in six ammonia-bound states.53 The modification of the g2 EPR signal suggested coordination to a manganese ion, as confirmed by pulsed EPR by Britt and coworkers.54 Boussac, Rutherford and Styring


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combined light-flash illumination with low-temperature EPR spectroscopy and measured light-saturation curves of oxygen evolution for continuous illumination (RO2CW).43 They proposed non-inhibitory ammonia binding in the S2-state and very slow inhibitory binding in the S3-state, the non-inhibitory binding clearly associated with the ammonia-modified g2 multiline signal. Their analysis of light-saturation curves of oxygen evolution for continuous illumination (RO2CW) revealed about 50% inhibition by 100 mM NH4Cl (at pH 7.6) for subsaturating light intensities, but no inhibition at extremely high light intensities (several fold higher than needed for RO2CW saturation without NH4Cl). In an extension of investigations by Chu and coworkers, who discovered a parallel influence of NH4Cl on EPR and FTIR difference spectra,55-57 Noguchi and coworkers measured S1→S2 difference spectra and found that the magnitude of these spectral changes correlates with the extent of inhibition of RO2CW.58 They also compared the pH dependence of RO2CW at saturating light intensities with NaCl and NH4Cl (100 mM), and detected a fully pHindependent NH4Cl-inhibition of about 40%.58 The latter finding is in conflict with the previously common assumption that the concentration of the free base, NH3, rather than that of the cation, NH4+, is decisive for the inhibitory and other effects of ammonium salts on PSII water oxidation (for the relevant pH values below 8.5, the NH4+/NH3 pKa of 9.2 implies a roughly tenfold increase of the NH3 concentration per pH unit). Yet the pH influence of the NH4+/NH3 inhibition (or binding) has been investigated rarely (or not at all) in an exhaustive way that includes determination of binding constants at various pH. Therefore, in the following Results section, we will refer to 'ammonium' effects and present binding constants that relate to the concentration of the ammonium cation. In summary, the complex phenomenology of the ammonium influence on PSII water oxidation has been intensely investigated in the past 45 years. Nonetheless, there have remained major knowledge gaps, which directly relate to hypotheses on the identity of the two substrate-water molecules in PSII water oxidation. These knowledge gaps include the relation between inhibitory and non-inhibitory ammonia binding, the question of NH3 versus NH4+ binding, the assignment of spectral changes observed by EPR and FTIR (for the S1/S2 state) to either inhibitory or non-inhibitory binding, and lacking spectroscopic signatures for the ammonium influence on higher S-state transitions. We address these knowledge gaps by a combination of time-resolved O2 polarography, analysis of recombination fluorescence (delayed fluorescence) and variable (prompt) PSII fluorescence after laser-flash excitation, as well as FTIR experiments that go beyond the S1→S2 transition and thereby provide unexpected insights regarding the S2→S3 transition.

Materials and Methods Preparation of plant-PSII membranes. High-activity PSII membrane particles were prepared from spinach leaves, as described elsewhere,59 and subsequently stored at -80°C. Before use, the frozen PSII suspension was thawed on ice (for 60 min, in the dark) and resuspended


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in Buffer A (150 mM NaCl, 25 mM MES (pKa 6.15), 25 mM HEPES (pKa 7.55), 25 mM HEPPS (pKa 8.0), 1 M glycine betaine, 5 mM MgCl2, 5 mM CaCl2). The pH of buffer A was adjusted with a high-molarity NaOH solution. The protein sample was then centrifuged at 50000 g for 12 min, the supernatant discarded and resulting pellet resuspended again in Buffer A.

Cyanobacterial thylakoid membranes. Cyanobacterial strains, namely Thermosynechococcus elongatus BP-1 (wild-type) and Synechococcus sp. PCC 7002 (wildtype, ΔpsbA1 and ΔpsbA2 mutants; see Fig. S1 for details of the mutagenesis approach), were grown photoautotrophically in BG11 medium60 and in A+ medium61 at 48°C and 38°C, respectively, supplemented with CO2-enriched air (1 % v/v) and continuous white light of 80 μE·m−2·s−1. Cells from midlog growth phase (OD750 1.5) were harvested by centrifugation at 6000 x g (4°C), washed twice with and finally concentrated in ice-cold MCM buffer (20 mM MES-NaOH, pH 6, 20 mM CaCl2, 10 mM MgCl2) before cryopreservation at -80°C. After gentle thawing, thylakoid membranes from T. elongatus and Synechococcus sp. PCC 7002 were isolated as described in ref.62 (using betaine instead of glycerol as a cryoprotectant) and in ref.63 (identical to the protocol for Synechocystis sp. PCC 6803), respectively. The pelleted thylakoid membranes were gently resuspended in ice-cold glycine betainecontaining MCM buffer (for T. elongatus: 20 mM MES-NaOH, pH 6.0, 20 mM CaCl2, 10 mM MgCl2, 0.5 M glycine betaine; for Synechococcus sp. PCC 7002: 50 mM MES-NaOH (pH 6.0), 10% (v/v) glycerol, 1.2 M glycine betaine, 20 mM CaCl2, and 5 mM MgCl2) to a chlorophyll concentration of 1–1.5 mg mL−1 before being snap-frozen in liquid nitrogen and stored at −80°C (acetone-based chlorophyll assay64). All steps of the PSII preparations, handling and storage of the samples were done under dim green safety light or complete darkness. Time-resolved oxygen polarography. Time-resolved oxygen evolution measurements were conducted with an in-house built centrifugable bare platinum/silver electrode as described elsewhere.65 Buffers were prepared on the day of the respective measurement. We note that slow warming up of previously frozen buffers resulted in clear reduction of the inhibitory ammonium influence on PSII, likely because of ammonium evaporation; therefore, this protocol was avoided. The biological sample was (if not stated otherwise) stored on ice in Buffer A. The PSII sample (in Buffer A), additional Buffer A and an ammonium buffer of equal pH with 150 mM NH4Cl, 25 mM MES, 25 mM HEPES, 25 mM HEPPS, 1 M glycine betaine, 5 mM MgCl2 and 5 mM CaCl2 (Buffer B) were pipetted onto the electrode right before the centrifugation (12000 g, 12 min, 4 °C) for a total volume of 375 µl. The amounts of buffers A and B were such that a chlorophyll concentration of 270 µg/ml as well as the required ammonium concentration was achieved. The sample was flashed once 10 minutes before starting the measurement and the individual oxygen evolution transients were measured for 14-20 flashes (flash spacing of 900 ms, electrode potential of -0.95 V at the central Pt electrode vs. Ag ring electrode), which was turned on 15 s prior to the first flash. The temperature was adjusted using appropriately mounted Peltier elements and monitored during the measurements by a miniature sensor element immersed in the PSII sample solution; all measurements shown herein were conducted at 10°C.


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Prompt and delayed chlorophyll fluorescence transients. A PSII stock solution was mixed in an optical one-way cuvette at room temperature with Buffer A and B (plus 20 µM DCBQ, 2,6-dichloro-1,4-benzoquinone, as exogenous electron acceptor) such that a final chlorophyll concentration of 10 µg/ml as well as the desired ammonium concentration was reached. Using a thermostatted cuvette holder at 10°C, the sample was flashed once and 12 minutes later exposed to a sequence of 32 saturating laser flashes of nanosecond duration (532 nm, 2 mJ/cm2, half-width of about 5 ns). Time-courses of the variable yield of the prompt chlorophyll fluorescence were measured employing a commercial instrument (FL3000, Photon System Instruments, Brno, Czech Republic), which was adapted for application of saturating ns-laser flashes to the PSII sample suspension. The delayed fluorescence (DF) transients after excitation with ns-laser flashes were measured using an in-house built setup with a gated photomultiplier (PM) shielded against stray light by two long-pass filters (cutoff wavelength 600 and 632 nm); the data acquisition involved logarithmic averaging. For details on the DF measurements and data analysis, see refs.66-68 Time courses of the delayed fluorescence excited by the third applied flash (S3->S4->S0 transition) were simulated (leastsquares fit) using tri-exponential functions (plus constant term) after a correction taking into account (i) an artifact signal from glass and/or PM cathode material and (ii) the influence of QA- reoxidation on the DF signal, as described elsewhere.67, 68 Fourier-transform infrared spectroscopy (FTIR). Thawed PSII membrane particles were suspended in an aqueous buffer solution containing 1 M glycine betaine, 25 mM MES, 25 mM HEPES, 25 mM HEPPS, 5 mM MgCl2, 5 mM CaCl2 and additional NaCl or NH4Cl, followed by centrifugation for 12 min at 50000 g. Then the supernatant was discarded and the procedure repeated a second time. To the resulting pellet, PPBQ (phenyl-p-benzoquinone) dissolved in DMSO (dimethyl sulfoxide) was added as exogenous electron acceptor. Therefore we transferred the PSII pellet to a CaF2 plate using a spatula before adding PPBQ (in DMSO) with a pipette and thorough mixing with a spatula. The final concentration of PPBQ was close to 30 mM. Subsequently, vacuum grease was placed at the outer margin of the CaF2 surface and another CaF2 plate was placed on top of the first plate. The second plate was rotated and gently pressed onto the first plate such that a homogeneous sample film sealed by grease was obtained. The thickness of the PSII film corresponded to an IR absorption at 1655 cm-1 of about 1 OD. The PSII samples sandwiched between CaF2 plates were exposed to illumination by a single laser flash (to optimize S-state synchronization) before being mounted within the temperature-controlled sample chamber (in-house construction) of the FTIR spectrometer, where they remained for 4 h in complete darkness before start of FTIR data collection. FTIR spectra were collected at 10°C in a low humidity atmosphere ( 100 ms). Figure 3 shows, for the first flash applied to dark-adapted PSII, that QA- re-oxidation kinetics are not affected by NH4Cl addition (see Fig. S4 for the corresponding 2nd and 3rd flash data), verifying the absence of NH4+/NH3 influences on the PSII acceptor side.


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Figure 3. Ammonium influence on acceptor and donor side of PSII membrane particles assessed by detection of the variable PSII fluorescence yield (prompt fluorescence, PF; in A) and recombination fluorescence transients (delayed chlorophyll fluorescence, DF; in B-F). The PSII membrane particles were excited with sequences of 24 laser flashes spaced by 900 ms in a NaCl/NH4Cl buffer at pH 7.5. (A) PF time courses, which reflect the kinetics of the quinone electron transfer at the PSII acceptor side, detected after the 1st flash


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applied to dark-adapted PSII. (B) Flash pattern of the DF intensity at 2 ms after the laser flash. (C) Differences of the DF intensities of subsequent flashes (vertical offset for clarity). (D,E) Increase in DF at 2 ms after the 2nd and 24th flash with KM values of 30+/-6 mM and 22+/-2 mM, respectively. (F) DF at 10 µs after the 1st laser flash (KM of 120+/-50 mM).

3) Ammonium influence on delayed (recombination) fluorescence Time courses of the delayed PSII fluorescence (DF) emitted after laser-flash excitation were recorded and analyzed as described elsewhere.66, 67 In uninhibited PSII, a clear period-offour oscillation pattern is typically observed for the DF intensity record within 0.5 to 5 ms after the laser flash, which is closely related to the flash pattern of oxygen evolution. The typical pattern of the 2 ms fluorescence observed in the absence of ammonium (black data points in Fig. 2B) is increasingly modified upon exposure to ammonium. Period-of-four oscillations are still discernible but superimposed by a continuous rise in the fluorescence intensity that is indicative of PSII with a defective donor side. This rise is explainable by accumulation of the oxidized form of the redox-active tyrosine residue, which in PSII with a fully functional OEC is rapidly and completely re-reduced within milliseconds after laserflash excitation. To reduce the influence of this rise, the difference in the DF level for two subsequent flashes was calculated. In the resulting flash pattern (Fig. 3C) an oscillatory pattern is discernible also at higher flash numbers, but with a reduced amplitude suggesting that roughly half of the PSII undergoes S-state transitions also after the third flash and thus is not inhibited by ammonium exposure. The inhibition KM-values determined for this rise after the 2nd flash and after the 24th flash are 30+/-6 mM and 22+/-2 mM (Fig. 3D and E), which suggests, taking into account the limited precision, identity to the KM of 21+/-5 mM determined for inhibition of flash-induced O2-evolution (Fig. 2C). An ammonium influence is detectable already at 10 µs after the 1st laser flash and thus clearly before onset of the S1-S2 transition (Fig. 3F). This observation supports NH4+/NH3 binding already in the dark-stable S1-state, albeit possibly with reduced affinity. A binding constant (KM) of 120+/-50 mM is formally obtained by curve-fitting of the data of Fig. 3F based the standard equation for description of non-cooperative ligand binding (see inset Fig. S2). We note that, as opposed to the other Km values determined in our study, the value of 120 mM is highly uncertain as indicated by the large 1σ-confidence interval (67% likelihood) of +/-50 mM (2σ interval for 95% likelihood of +/-100 mM). The DF data facilitates the following conclusions: (1) Not only in the time-resolved O2evolution experiment, but also for the changed experimental conditions of the DF experiment (film of highly concentrated PSII on bare platinum electrode versus highly diluted PSII particles in solution; with and without artificial electron acceptor), partial inhibition of advancement in the S-state cycle is observed; the KM of this partial inhibition is close to 25 mM in both experiments. (2) The rise of the DF intensity after the 2nd flash demonstrates that this transition is affected by ammonium exposure, even though the


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extent of electron transfer from the OEC to the tyrosine residue is unchanged (as shown later). (3) The clear ammonium influence on the DF intensity detected 10 µs after the first flash suggests that either ammonium (or ammonia) binds already in the S1-state to the OEC or the binding occurs within clearly less than 10 µs after the laser flash initiating the S1=>S2 transition.

4) Ammonium influence on flash-induced FTIR difference spectra


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Figure 4. Ammonium binding constant and flash-pattern modifications from FTIR difference spectra. S-state difference spectra of PSII membrane particles at various NaCl and NH4Cl concentrations were detected for excitation with sequences of saturating laser flashes of visible light (532 nm). (A) S2-S1 difference spectrum at four NH4Cl concentrations (0 mM, black line; 30 mM, blue; 60 mM, green; 120 mM, red). The spectra were shifted and scaled according to the spectrum without added ammonium (0 mM) and the double-difference amplitude at 1436 versus 1422 cm-1. The blue arrows mark an increase at 1380 cm-1, for increasing NH4Cl concentrations, and a concomitant decrease at 1363 cm-1. (B) Difference of the S2-S1 amplitudes at 1380 and 1363 cm-1 (blue arrows in panel A) for increasing NaCl (black) and NH4Cl (red) concentrations. The red line corresponds to a simulation with an ammonium binding constant (KM) of 16+/-6 mM. (C) Flash pattern from FTIR difference spectra at 1342 cm-1 at 120 mM NaCl (black) and 120 mM NH4Cl (red). (D) Flash pattern of the difference in absorption at 1100 and 1110 cm-1 (A1100- A1110). The magnitude of changes in optical-density units is indicated by the length of the black bars. All FTIR spectra were collect at 10°C and pH 7.5. (In A and B, the buffer solution contained MES only, whereas the FTIR data shown in panels C, D, and Figure 5 were collected using a ternary buffer system containing MES, HEPES, and HEPPS. For further buffer constituents and concentrations, see Materials and Methods section.)

We investigated the influence of NH4Cl on the mid-infrared difference spectra recorded for application of a laser-flash sequence using PPBQ as an artificial electron acceptor. Ferricyanide is routinely used as artificial electron acceptor in FTIR investigations on cyanobacterial PSII core complexes (see, e.g., ref.72, 73). In extensive preliminary experiments, we found that for the herein used PSII membrane particles from spinach, the employment of ferricyanide at sufficiently high concentration (sufficient to ensure complete re-oxidation of the primary quinone acceptor in the time period between flashes) resulted (i) in partial dark-oxidation of the non-heme iron at the PSII acceptor side and (ii) insufficiently well synchronized S-state cycling (miss parameter, m, significantly exceeding 10%). Specifically the latter point is highly problematic in the context of the present investigation. In contrast, the use of PPBQ reproducibly resulted in well-synchronized Sstate cycling (m of about 8%). and thus is used herein. The changes in the FTIR S-state difference spectra caused by NH4Cl were compared to changes caused by addition of NaCl. Only a very minor NaCl influence on the spectra was detected (not shown). First we focus on the most prominent ammonium effect observed in the first-flash difference spectrum (S1-S2 transition), where ammonium binding causes an intensity loss around 1363 cm-1 and a concomitant intensity gain around 1380 cm-1, possibly indicating upshift of the vibrational frequency assignable to a specific, but presently still unidentified carboxylate55-57 (Fig. 4A). Comparable changes are not observed for addition of sodium chloride. The ammonium affinity (binding constant) is determined to be 16+/-6 mM (1σ error, Fig. 4B) and thus is, within the accuracy limits of the data, close to the ammonium affinity determined for inhibition of the time-resolved O2-signal (Fig. 2). Figure 4 illustrates a further important aspect of the FTIR data. In NaCl buffer, a clear periodof-four pattern (Fig. 4C, assignable to reactions of the water-oxidation cycle) or period-of-


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Biochemistry

two pattern (Fig. 4D, acceptor side reactions) is observable in the intensities of the bands of the S-state difference spectra. With increasing flash number, a moderate damping of the oscillation amplitudes is observable. Yet in the presence of NH4Cl, the behavior is changed. For the 1st and 2nd flash, the oscillation amplitude is comparable to that observed in the presence of NaCl. At higher flash numbers, oscillations are still observed, but their amplitude is clearly reduced. This observation implies that addition of saturating ammonium concentration does inhibit advancement in the S-state cycle in a fraction of PSII, but another significant fraction of PSII remains uninhibited and continues to advance through the S-state cycle synchronously. In Figure 5, FTIR difference spectra obtained for application of a sequence of ns-laser flashes are shown for PSII in a buffer supplemented either by 100 mM NaCl or 100 mM NH4Cl (both at pH 7.5). In these experiments, we used PPBQ as an electron acceptor, which results in an intricate redox chemistry at the PSII acceptor side involving oxidation of the non-heme iron on the first flash and reduction on the second flash. Consequently, a binary oscillation of the features in the difference spectra around 1100, 1230, and 1250 cm-1 is observed (marked by brown bars in Fig. 5A). Similar spectral changes assignable to the oxidation of Fe2+ to Fe3+ have been described before for the PSII membrane particles74-76 and cyanobacterial PSII.77, 78 In the present work, we use the magnitude of these acceptor-side redox features to assess the number of electrons transferred from the donor to the acceptor side of PSII.


Biochemistry

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Figure 5. FTIR difference spectra at pH 7.5 (10°C) with either 120 mM NaCl (black lines) or 120 mM NH4Cl (red or grey lines). On the 3rd, 4th and 5th flash, the NH4Cl spectra were rescaled to match the magnitude of the acceptor side features in NaCl spectra (red lines in C, D, and E). Prominent spectral changes caused by ammonium exposure are marked by colored areas. Changes occurring on both 1st and 5th flash are assignable to non-inhibited PSII; they are marked in blue and red (blue, decreased


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Biochemistry

magnitude; red, increase); small spectral shifts on both 1st and 5th flash are indicated by two blue arrows. Grey indicates inverse spectral changes in the 2nd and 3rd flash spectra (mirror symmetry of the grey areas), which always represent a decrease in the magnitude of the respective spectral feature (or its disappearance on the 3rd flash); orange indicates spectral changes without mirror symmetry. Green indicates spectral changes on the 2nd (1564 cm-1) or 3rd flash (1382 cm-1) that are reversed on the 4th flash. For comparison of ammonium exposure and genetic modifications, similar spectral changes in genetic PSII variants are marked by blue-letter labels (site-specific mutagenesis by Debus and coworkers, see main text for references). We emphasize that marked spectral changes are not directly assignable to vibrational modes of the mutated residues itself. It is likely that the genetic modifications affect the H-bonded water cluster at the active site (Figure 1) and thereby indirectly vibrational frequency and oscillator strength of further residues (of still unknown identity).

1st flash (S1→S2). The first flash initiates the S1→S2 transition and although largely similar, the FTIR difference spectra show reproducible modifications caused by ammonium exposure. Examples are the already mentioned differences near 1363 cm-1 and 1380 cm-1 as well as reproducible ammonia effects detectable at 1402, 1461, 1529, 1615, 1651, and 1699 cm-1 (marked by blue areas and arrows in Fig. 4A). In the S1→S2 difference spectrum in the presence of NH4Cl, we also see a negative shoulder at 1390 cm-1 (even more clearly visible in Fig. S5), which has been assigned to ammonium binding to a carboxylate group resulting in inhibition of PSII oxygen evolution,58 as opposed to ammonia binding (to a manganese ion) without inhibition of PSII and associated with a characteristic positive peak at 1380 cm-1.55-57 The negative shoulder at 1390 cm-1 is absent and the 1380 cm-1 peak is enhanced in our 5th flash difference spectrum, in line with assignment of the negative shoulder to inhibited PSII (thus not visible on the 5th flash) and the positive peak at 1380 cm-1 to non-inhibited PSII (thus enhanced on the 5th flash). 2nd flash (S2→S3). The differences between ammonium exposed and control PSII are clearly more pronounced for the 2nd flash initiating the S2→S3 transition at the PSII donor side. The yield of electron transfer to the PSII acceptor side remains essentially unaffected as indicated by the unchanged magnitude of the spectral features assignable to the PSII acceptor side reactions. We note that the reduction process at the acceptor side does not necessarily imply that the S2→S3 transition is accomplished at the donor side of PSII; also alternative electron donors might be involved, e.g. oxidation of the cytochrome b559 or specific carotenoids within the PSII core complex.79 Yet essentially identical difference at wavenumbers below 1200 cm-1 and above 1690 cm-1 spectra regarding both line shapes and amplitudes (see Figure 5B) provide evidence against oxidation of alternative electron donors. Moreover, also between 1200 cm-1 and 1690 cm-1 there are regions where the signal amplitude is only slightly affected. Thus we conclude that, most likely, ammonium exposure does not inhibit manganese oxidation in the S2→S3 transition. Nonetheless, in the S2→S3 difference spectrum many spectral features are strongly modified (grey, orange and green areas in Fig. 5B) suggesting that either the donor redox chemistry itself is modified or


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the response to manganese oxidation of the H-bonding network formed by the metal-bound and nearby water molecules is significantly different in the presence of ammonium. 3rd flash (S3→S0 + O2). For the 3rd flash inducing the oxygen-evolution transition (S3→S0 + O2), the magnitude of the acceptor side features is significantly reduced implying that in only about half of the PSII an electron is transferred from the donor to the acceptor side (compare grey and black line in Fig. 5C). This low yield of the oxygen-evolution transition indicated by the FTIR spectra confirms the low O2-formation yield detected polarographically (Fig. 1). The 3rd flash difference spectra were up-scaled to facilitate comparison between control-PSII and ammonium-PSII. There are spectral regions where the difference spectra of control-PSII and up-scaled ammonium-PSII differ only slightly, in particular from 1260 to 1350 cm-1, where the 3rd flash spectrum resembles the inverted 2nd flash spectrum closely (2nd/3rd-flash mirror image region). There are minor ammonium effect in the mirror-image region, e.g. shifts in the peaks at 1341 and 1320 cm-1 by 2-5 cm-1. In other spectral regions, there is a clearly stronger ammonium influence on the 3rd flash spectrum. Several positive and negative peaks present in the 2nd flash and 3rd flash spectra of control PSII are significantly reduced or fully absent in ammonia-PSII, again mostly in a mirror-image-like fashion (grey areas in Fig. 5B and 5C). However, there are also exceptions from 2nd/3rd-flash mirror image behavior. For example, the negative peak at 1564 cm-1 in the 2nd flash spectrum, which is matched by a positive peak in the 4th flash spectrum (dominated by S0→S1 transition), is diminished or even absent in ammonium-PSII. Similarly, the positive peak at 1382 cm-1 in the 3rd flash spectrum and the corresponding negative peak in the 4th flash spectrum are affected in ammonium-PSII. In the amide-I region, two negative peaks at 1668 and 1647 cm-1 are diminished in ammonium treated PSII (orange areas), possibly corresponding to an ammonium influence on the corresponding positive peaks in the 5th flash spectrum assignable to the S1→S2 transition of ammonium-affected, but not inhibited PSII. 4th flash (S0→S1). An influence of ammonium on the up-scaled 4th flash spectrum, which reflects predominantly the S0→S1 transition, is detectable: peaks at 1383, 1564 and 1689 cm-1 are disappearing. Yet the ammonium influence on the FTIR spectra is small in comparison to 2nd flash and 3rd flash spectra. 5th and 6th flash. The up-scaled 5th flash spectrum shows the ammonium influence on the S1→S2 transition in non-inhibited, oxygen-evolving PSII. The spectral differences between control and ammonium-PSII resemble closely the differences seen in the 1st flash spectrum suggesting that also the latter are assignable to non-inhibited PSII. Similarly, the 6th flash spectrum resembles the 2nd flash spectrum regarding the ammonium influence (Fig. S6B).

Reversibility of ammonium effects. After collection of the FTIR difference spectra discussed above, a second set of difference spectra was collected. The time period between application of the first sequence of 12 laser flashes and the second sequence amounted to about 30 hours (in the dark, at 10°C). This long dark-adaptation period inter alia ensures


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reversal of all PSII to the dark-stable S1-state. The resulting difference spectra are shown in Figure S5. The amplitudes of the second set of difference spectra collected after 30 hours of dark-adaptation are found to be moderately reduced only, for both control-PSII (in highNaCl buffer) and ammonium-PSII. Moreover, essentially the same ammonium influence on the S-state difference spectra was detected as had been for the first flash sequence confirming (i) the absence of irreversible inhibition (damage) caused by long-term exposure to ammonium and (ii) the unbinding of ammonium/ammonia in the dark-stable S1-state.

FTIR results - summary. The central results coming from the FTIR investigation of the ammonium influence can be summarized as follows: (1) The yield of the S1→S2 transition as well as the yield of the S2→S3 transition is not affected by ammonium, whereas the yield of the S3→S0 transition is significantly reduced. There is a population of PSII (about 50%) that cannot advance beyond the S3 state and consequently cannot contribute to O2 evolution (ammonium-inhibited PSII), in line with the polarographic data. (2) The remaining, non-inhibited PSII population clearly is affected by ammonium with regard to its FTIR difference spectra, but these PSII still can advance through the complete Sstate cycle. All S-state transitions seem to be affected, but the S2→S3 and S3→S0 transitions display an especially strong ammonium influence, largely in a mirror-image-like manner. This points to an S3 state conformation of the OEC and its protein-water environment that is modified by non-inhibitory ammonia binding, either regarding the structure of the metaloxo complex or the H-bond network formed by water molecules and nearby amino acid residues.

5) Minority fraction of PSII with slow oxygen evolution A closer inspection of the flash-induced O2-transients revealed an ammonium influence on the decay following the initial rise (Fig. 6). Under high-ammonium conditions, the decay was reproducibly slowed down (Fig. 6B). Slow decays of the O2-transients have been observed before for exchange of specific amino acid residues that interact with the water cluster interfacing the Mn4Ca(µ-O)5 core of the OEC (e.g. D1-D61, D2-K317, CP43-R357). In these PSII variants, the rate of oxygen evolution was slowed down strongly reaching values of 3060 ms.65, 80-82 Therefore we approached simulation and curve-fitting of all O2-transients employing a simple one-dimensional diffusion model that covers light-induced O2production (by PSII), O2-diffusion (within the PSII film and in the bulk solvent), and O2reduction (at the bare Pt electrode), as described elsewhere.65 Good simulations of the fast rise and slowed down decay were obtained by assuming the presence of two PSII populations: (i) a PSII with fast oxygen evolution (1.7-4.0 ms) and (ii) PSII with slow oxygen evolution (ca. 50 ms) in the presence of ammonium (Fig. S7A). The time constants of fast and slow oxygen evolution were found to be essentially independent of the ammonium concentration (Fig. S7B and C). For ammonium concentrations above 20 mM, the fraction of


Biochemistry

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1.0

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PSII with slow O2-evolution increased from about 8% at pH 6.5 to about 20% at pH 8 (Fig. 6D).

Figure 6. Minority fraction of PSII with slow oxygen evolution in the presence of ammonium. (A) Rise and (B) decay of normalized O2-transients at various ammonium concentrations (pH 7.5) illustrating unchanged rise time, but variation of the decay time. Very good simulations of the complete O2transients measured by time-resolved polarography at 10°C (see Fig. 2) were obtained for a majority population of 'fast PSII' with time constants for flash-induced oxygen evolution ranging from 2 ms (pH 6.5) to 4 ms (pH 8) (Fig. S7B) and a minority population of 'slow PSII' with an O2-evolution time constant close to 50 ms (Fig. S7C). Shown are the relative amplitudes of 'fast' (C) and 'slow' (D) PSII populations.


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6) Time-resolved O2-evolution for cyanobacteria with various variants of the D1 protein Recently, the ammonium binding has been investigated by advanced EPR spectroscopy combined with computational chemistry for PSII core complexes from Thermosynechococcus elongatus, a thermophilic cyanobacterium for which crystallographic models of the PSII structure have been obtained (for T. elongatus or the closely related T. vulcanus).14-18 The rate of substrate water exchange had been compared between control and ammonium-treated PSII by mass spectroscopic detection of the evolved dioxygen,42 which has not provided any indications for a significant inhibitory effect of ammonium treatment, as opposed to the results we obtained for PSII particles from spinach (Fig. 1). The contrasting results prompted us to compare ammonium binding in plant and cyanobacterial PSII.

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Biochemistry

80% 60% 40% 20% 0% 1

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Figure 7. Fraction of PSII from plants and various cyanobacteria that is not inhibited by 120 mM ammonium (at pH 7.5 and 10°C). To illustrate the extent of reproducibility, 6 replicas are shown for PSII from spinach, 4 replicas for a mesophilic cyanobacterium (Synechococcus sp. PCC 7002), 2x2 replicas for genetic variants of PCC 7002 lacking either the psbA1 or the psbA2 genes (multiple gene copies of the D1 protein of PSII), and 6 replicas for the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. After averaging the amplitudes of the O2-evolution transients of the 3rd to 6th flash, the resulting values for PSII in 120 mM NH4Cl buffer were divided by the corresponding value in the absence of ammonium (in 120 mM NaCl buffer), to obtain the percentage of non-inhibited PSII for each investigated species. The data was collected on PSII membrane particles (for spinach PSII) or thylakoid preparations (cyanobacterial PSII).

We investigated the flash-induced O2-formation for thylakoid membranes prepared from T. elongatus and a mesophilic cyanobacterium (Synechococcus PCC 7002), where we analyzed the wild-type PSII and genetic variants containing only two of three D1 isoforms. In this series of measurements, the level of inhibition in the presence of 120 mM NH4Cl (at pH 7) was consistently close to the level observed in PSII particles prepared from spinach (Fig. 7).


Biochemistry

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In conclusion, partial inhibition of O2-formation is observable for PSII from several organisms and also for various D1 isoforms of the cyanobacterial PSII.

Discussion (I) Characteristics of inhibitory and non-inhibitory NH4+/NH3 binding The influence of ammonium salts on PSII oxygen evolution was discovered by analysis of the (partial) inhibition of the rate of oxygen evolution measured under continuous illumination (see Introduction section). By time-resolved O2-polarography, we also observe inhibition of PSII oxygen evolution, with a binding constant (KM) of about 25 mM, at all investigated pH values (from pH 6.2 to 8.0), but typically only about 50-60% of the PSII are inhibited. We note that the KM value of 25 mM implies that the standard concentration of about 100 mM ammonium frequently used in spectroscopic experiments is sufficient for saturation of the here characterized effects. It has been proposed that ammonium inhibition results from slowed-down oxygen evolution,43 which might explain an apparent partial inhibition for detection of the rate of oxygen evolution during continuous illumination. Yet our time-resolved O2 polarography experiments reveal that the inhibition of O2-formation is predominantly an on/off effect, and not explainable by slowed down O2-formation. Depending on the ammonium concentration, an increasing number of PSII is fully inhibited, whereas the rate constant of O2-formation of the S3→S4→S0 transition, which ranges from 250 s-1 at pH 8 to 600 s-1 at pH 6.2, is independent of the ammonium concentration in the buffer solution. For genetic modifications of amino acid residues that are not coordinated to manganese ions, but affect the H-bonded water cluster in vicinity of the catalytic water cluster, the O2formation step was slowed down significantly,81-83 from time constant values of about 2 ms to about 50 milliseconds.65 Also under the influence of ammonium, we detect a minor fraction of PSII that is not fully inhibited, but slowed down to a level of the O2-formation time constant close to 50 ms. In the following discussion, we will focus on the main effects and thus will not consider the minority fraction of the slowly oxygen-evolving PSII in more detail. The FTIR experiments reveal that the S1→S2 transition induced by the 1st laser flash applied to dark-adapted PSII as well as the S2→S3 transition (2nd flash) are both not inhibited by ammonium, whereas the yield of the oxygen-evolution transition induced by the 3rd flash was reduced by about 50%; also all later flashes induced an S-state transition in only about 50% of the PSII. Importantly, in the non-inhibited PSII, which continue to advance through the S-state cycle largely synchronously, the ammonium influence on the 5th and 6th flash difference spectra resemble closely the ammonium influence on the 1st and 2nd flash spectra. Consequently, the detected spectral changes are assignable to the non-inhibited


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PSII fraction. In conclusion, in the non-inhibited PSII all S-state transitions can proceed at reasonable efficiency, but each S-state transition is more (S2→S3 and S3→S0) or less (S0→S1 and S1→S2) strongly modified by the binding of ammonium or ammonia, at least regarding the corresponding S-state difference spectra. The herein described FTIR experiment provides—for the first time—a spectroscopic signature of the ammonium influence for each of the four light-induced transitions between semi-stable S-states. The ammonia-modified S2-state multiline signal most likely originates from an ammonia molecule bound to a manganese ion.42, 52 Boussac et al showed that this EPR signal exhibits a period-of-four flash pattern and thus is assignable to PSII with bound ammonia.43 Taking into account that in earlier and recent EPR investigation similar 'standard' conditions were used regarding ammonium concentration and pH as used in our experiments, we conclude that, most likely, the same, non-inhibitory ammonia-binding site gives rise to the modified multiline signal and the modified FTIR difference spectra.

(II) pH dependence – ammonia versus ammonium binding Recent results obtained by advanced EPR spectroscopy and computational chemistry strongly support ammonia binding in the first coordination sphere of a manganese ion,42, 8486 in agreement with earlier conclusions.52, 54 An ammonium ion (NH4+) stably bound to a metal cation represents a chemically highly unlikely option because the metal binding would lower the pKa of NH4+ drastically, resulting in immediate deprotonation. In aqueous solution, the formation of NH3 by deprotonation of NH4+ is described by a pKa of 9.2 so that, e.g., at pH 7.5 the ammonia concentration in the buffer solution is predicted to be about 10 times higher than at pH 6.5 (for the same concentration of added ammonium salt, which was in our study NH4Cl). Therefore, it might be assumed that for a given NH4Cl concentration, the binding of ammonia is strongly favored by alkaline pH. Here we find that the binding constant (KM) for inhibition as well as the fraction of inhibited PSII is essentially pH-independent (Fig. 2), as previously also found for the rate of oxygen evolution measured under continuous illumination.58 Also the characteristic modification in the S2-S1 FTIR difference spectra has previously been found to be similar at pH 5.5, 6.5 and 7.5.58 The lacking pH dependence has been interpreted as evidence against binding of ammonia to a manganese ion. But does the lacking pH dependence really exclude Mn-NH3 formation? To address this question, we consider four (simplified) scenarios for ammonia binding to one of the Mn ions in PSII: (A) pH-dependent KM and kon ('conventional' binding model'). Ammonia (NH3) from the bulk solution moves into the PSII protein, reaches the active site and binds to a Mn ion. The NH4Cl concentration dependence of the experimentally determined Km of ammonia binding to manganese (Km-value with respect to [NH4+]) thus is predicted to be proportional to the concentration of 'free' NH3 in the aqueous bulk phase. A roughly ten-fold change in [NH3] and thus Km per pH-unit is predicted for the here considered pH values (which are clearly


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below the NH4+/NH3 pKa). We note that, to a first approximation, the rate constant for NH3 binding, kon (with Km defined as (kon/koff)/[NH4]) is predicted to exhibit a similarly strong pH dependence, whereas the unbinding rate constant, koff, may be pH-independent. (Here and in the following, kon and koff are effective rate constants describing the probability of NH3/NH4+ binding with respect to the employed NH4Cl concentration in the aqueous buffer solution. The unit of kon is mol-1s-1; the unit of koff is s-1. The values of these effective rate constants may be pH dependent.) (B) pH-dependent KM, but pH-independent kon. Ammonium (NH4+) reaches the OEC. At the OEC, the NH4+ ion deprotonates coupled to binding as a NH3 ligand to a Mn ion of the OEC; subsequently the proton is released into aqueous bulk. In this case, the rate constant of MnNH3 formation (kon) could be pH-independent. Yet the NH3 unbinding (Mn-NH3 dissociation) likely will be coupled to NH3 protonation according to: Mn-NH3 + H2O + (H+)local => Mn-H2O + (NH4+)local The probability of having an 'additional proton' locally at the OEC site may be extremely small, but will depend on the proton concentration in the bulk solvent. Consequently, koff and thus KM are predicted to be pH-dependent. (Here the pH dependence of KM does not relate to the increased NH3 concentration at alkaline pH, but to accelerated Mn-NH3 dissociation at more acidic pH values.) We note that the low probability of having an 'additional proton' available at the OEC could render the unbinding of NH3 extremely slow. Consequently, establishment of a complete equilibrium may be exceedingly slow, especially at high pH values. This means that for experimental conditions with incomplete equilibration, a pH-independent kon could mask the pH dependence of the equilibrium KM. (C) pH-independent KM. Ammonium (NH4+) from the bulk solution reaches the OEC. At the active site, the NH4+ ion deprotonates and binds as a NH3 ligand to a Mn ion of the OEC. The proton from NH4+ deprotonation now is assumed to stay within the protein; it binds to a (nearby) protein-internal group. Although NH3 binds to a Mn ion, the outlined situation corresponds to NH4+ binding to PSII. Consequently, the Km value is predicted to be pHindependent within the herein investigated pH range (pH

Inhibitory and Non-Inhibitory NH3 Binding at the Water-Oxidizing

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