Photosynthesis Research 38: 355-361, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.
Regular paper
A mass spectrometric analysis of the water-splitting reaction Klaus P. Bader 1, Gernot Renger a & Georg H. Schmid 1,*
1Universitdt Bielefeld, Fakultiit fiir Biologic, Lehrstuhl fiir Zellphysiologie, Postfach 10 01 31, 33501 Bielefeld, Germany; 2Max-Volmer Institut f~r Biophysikalische Chemic, Technische Universitdt Berlin, Germany; *Author for correspondence Received 26 May 1993; accepted in revised form 8 September 1993
Key words:
chloroplast, flash-induced oxidation, mass spectrometry, water-splitting
Abstract
Earlier mass spectrometric measurements, in which oxygen evolution was measured following short saturating light flashes, indicated that with a time resolution of about 30 s no form of bound water and/or an oxidation product exists up to the redox state S3 of the oxygen evolving center (R. Radmer and O. Ollinger, 1986, FEBS Lett 195: 285-289; K.P. Bader, P. Thibault and G.H. Schmid, 1987, Biochim Biophys Acta 893: 564-571). In the present study, isotope exchange experiments with H2180 were performed under different experimental conditions. We found: a) the isotope exchange pattern is virtually the same at both pH 6.0 and 7.8, although marked structural changes of the PS II donor side are inferred to take place within this pH-range (Renger G., Messinger J. and Wacker U., 1992, Research in Photosynthesis, II: 329-332); b) injection of H2~80 at about 0 °C gives rise to mass ratios of the evolved oxygen which markedly deviate from the theoretically expected values of complete isotope scrambling; and c) rapid injection of Hz180 into samples with high population of S 1and S2 and subsequent illumination with three and two flashes, respectively, spaced by a dark time of only 1.5 ms lead to similar 180-labeling of the evolved oxygen. Based on the published data on the interaction with redox active amines, possible pathways of substrate exchange in the water oxidase are discussed.
Abbreviations: a - atom fraction of 180; PS II - Photosystem II; Si - redox states of the water oxidase; Y z - redox active tyrosine ofpolypeptide D 1
Introduction
Photosynthetic oxidation of two water molecules to molecular oxygen and the coupled release of four protons into the thylakoid lumen take place within a manganese-containing fimctional unit referred to as the water oxidase. This unit, which is a part of the membrane integral Photosystem II (PS II) complex, is regulated by extrinsic proteins and other cofactors like Ca 2+ and C1-. Based on the fundamental work of Pierre Joliot and Bessel Kok and their coworkers (for a review, see Joliot and Kok 1975), the overall process was shown to consist of a sequence of univalent electron transfer steps until after the
accumulation of four oxidizing redox equivalents, molecular oxygen is released. This highly endergonic reaction sequence referred to now as the Kok cycle (Kok et al. 1970) is energetically driven by the oxidized form of the PS II reaction center chlorophyll a, P680 +, as the oxidant, formed during the primary step of light induced charge separation within the PS II reaction center complex (for a review, see Renger 1992). The water oxidase is functionally connected with P680 + via the component Yz, identified by site directed mutagenesis in Synechocystis sp. PCC 6803 as a tyrosine (Tyr- 161) ofpolypeptide D1 (Debus et al. 1988, Metz et al. 1989).
356 The four step univalent oxidative pathway within the water oxidase can be summarized by the following equation: y°XSzi +miH20--~YzSi+l+niH++Si302
(1)
where Si symbolizes the redox state of the water oxidase with i = 0, 1, 2 or 3 representing the number of accumulated oxidizing redox equivalents, ni describes the noninteger stoichiometry (see Rappaport and Lavergne 1991) of net H+-release and mi is the net uptake o f m water molecules coupled with oneelectron abstraction from the water oxidase in redox state S., (with 2 m. = 2), ~.. = Kronecker symbol (~3 = l'for i = 3, ott~erwise zJero). Equation (1) also tacitly implies that redox state S4is rapidly converted into SOwith the concomitant release of molecular oxygen. Thus, it cannot be detected as a kinetically separable component, i.e. i + 1 = 4 has to be read in Eq. (1) as zero. The kinetics of the elementary steps in the overall reaction sequence of PS II have been resolved in great detail by various spectroscopic techniques (for reviews, see Babcock 1987, Renger 1987a, Rutherford et al. 1992). In marked contrast, key mechanistic questions still remain to be answered (for a list see Renger 1987b). Likewise, the structure of the water oxidase and the nature of its protein matrix are not yet resolved (for a detailed review, see Debus 1992). Among the many open questions, two problems are of central relevance: a) the entry of water into the redox cycle (symbolized by the unknown mi in Eq. (1)) and b) the mode of oxygenoxygen bond formation. Isotope exchange experiments with H2~80revealed that with a time resolution of the order of half a minute no buried form of substrate water can be found up to the redox state S3 (Radmer and Ollinger 1986, Bader et al. 1987). This led to the widely accepted conclusion that the process b) takes place only at the redox level S4 (for a review, see Rutherford et al. 1992). However, as emphasized in a previous study (Renger 1987b) this conclusion should not be considered as straightforward because sufficiently fast equilibria ofredox and exchange reactions would mask the formation of state(s) with electronic configuration(s) and nuclear geometry corresponding with a peroxide at a redox level below S4 (Renger 1987b, Renger and Wydrzynski 1991). This idea might be supported by thermodynamic calculations which favor a
sequence of two 2-electron oxidation steps compared with a concerted 4-electron abstraction (Krishtalik 1990) and by experimental findings of peroxide formation at a disturbed manganese cluster of the water oxidase (Ananyev et al. 1992 and references therein). The present report is an attempt to address the question of the entry of water into the redox cycle and the mode of O-O bond formation by 180/160 isotope exchange experiments at different pH, temperature and regime of flash excitation. Materials and methods
Chloroplast membranes were prepared from N. tabacum leaves; they were isolated in 0.05 M Tris buffer containing 0.4 M sucrose, 0.28% pectinase, 0.2% bovine serum albumine and 0.01 M NaC1, pH 7.4, as described by Homaun and Schmid (1967). Chloroplast membranes were transferred from this buffer to 0.06 M Tricine and 0.03 M KC1. For our experiments, aliquots of the chloroplast membrane suspension, corresponding to 80/~g chlorophyll, in a total volume of 2 ml of 0.06 M Tricine and 0.03 M KC1 were used. Mass spectrometric experiments were performed as previously described by Bader et al. (1987, 1992). Measurements were carried out in a home-made cell directly connected to the ion source of the stable isotope ratio mass spectrometer 'Delta' from Finnigan MAT (Bremen, Germany). The construction principle and the geometry of the cell are decisive for the response and sensitivity of the mass spectrometric set-up. The cell consists of a stainless steel piece in which, on a support (a porous Teflon block), a Teflon membrane of 12.6 cm 2 surface and a thickness o f - 1 0 #m is put on. The cell can be covered with an air-tight Plexiglas lid. Usually the membrane surface is covered with 3 ml of assay solution containing the photosynthetic material (thylakoids or thylakoid particle preparations) in the buffer system with the respective isotope composition. The thickness of the total liquid layer on the membrane is, under our geometric conditions, 1.7 mm (Bader et al. 1987, 1992). Between the surface of the liquid phase and the Plexiglas lid, a gas phase space of 14 ml is available from which, due to the volume/surface ratio, a practically instant gas exchange with gas mixtures of any isotope composition is possible (Bader et al. 1992). The
357 thylakoid particle preparation is sedimented on the membrane and the chlorophyll concentration of the preparation is chosen as to give a monolayer on the membrane. The Teflon membrane of the measuring cell separates the normal 'atmospheric' assay condition (buffer system in equilibrium with air, for instance) from the high vacuum condition of the ion source. The system as we use it may be open or closed and represents a unidirectional gas flow towards the ion source. Oxygen evolved by the photosynthetic membrane, or by the particle preparation derived from it, is mixed with the constant O2-flow which passes the membrane and is instantaneously in the detection system. The functional principle of this measuring cell is practically the same as that used in the large surface 'Three Electrode System' described by Schmid and Thibault (1979) and discussed by Schulder et al. (1990) in context with the determination of the time constants for photosynthetic oxygen evolution. The addition of H2180 (Figs. 1 to 4) is brought about by the addition of 1 ml of 97% H2180 to 2 ml of assay mixture. The amount of this addition to the only 1 mm thick layer of 2 ml assay medium warrants immediate mixing of the bulk water phase in the system. The stable isotope ratio mass spectrometer 'Delta' from Finnigan MAT (Bremen, Germany) is a magnetic sector field instrument which measures isotope ratios in the mass range of 2-50 with an internal precision of 0.1%. This instrument was substantially modified for our experiment as described by Bader et al. (1987). Calibration of the setup and calculation of the isotope distribution was carried out by two procedures: First, the average of at least 10 determinations of the signals at m/e = 32, arde = 34 and rrde = 36 for 'normal' air was correlated with the well-known natural atomic abundance of 99.7587% oxygen-16 and 0.2039% oxygen-18, and second, various concentrations of hydrogen peroxide were exogenously added to yield definite signals in the detection system upon decomposition by the addition of catalase. Corrections for the isotope dilution were made according to the equation given by Peltier and Thibault (1985). Signals for 1602, 160180 and 1802 were simultaneously detected in Faraday cups and recorded on a SE 130-03 BBC (Brown Boveri Corp.) Metrawatt 3-Channel recorder. Flash illumination was performed via a Stroboscope 1539 A of General
Radio which yields flashes of 5/Js duration. H2180 was obtained from CEA-Oris, Bureau des Isotopes Stables, Gif-sur-Yvette, France. Results and discussion
Hashimoto et al. (1986) have shown that in horseradish peroxidase the isotope exchange of the oxygen bound to the heine iron exhibits a marked pH dependence. Likewise, the Fe-O distance becomes significantly reduced at high pH (Chang et al. 1993). These phenomena were ascribed to the breakage of a hydrogen bond from the oxygen due to deprotonation of an amino acid residue. Based on these findings, the possibility was considered that the 160/180 exchange in the water oxidase could be also pH-dependent through an analogous effect. Therefore, mass spectrometric experiments were performed at two different pH-values where the oxygen evolution capacity becomes only marginally affected (Renger 1977) while marked structural changes are inferred to take place at the PS II donor side (Renger et al. 1992, and references therein). The results obtained by mass spectrometry at pH 6.0 and pH 7.8 are shown in Fig. 1. An evaluation of the data reveals that the extent of isotope exchange remains virtually unaffected. Similar results were obtained in samples populated in S2 and S3 by one and two preflashes, respectively, before the rapid injection m/e =36
1~ 3 Floshes {z~t=l.5rns) "f
Addition of
Dork Adopt 6otionPHH~ 8~ I"
~ 30s
2 pMo[es 02
,a
-, 3Floshes {z~t=1.5ms}
pH7.8 Additioofn 18 H20
oles 02
Dork Adoptotion
I-
30s
Fig. 1. Photosynthetic oxygen evolution by thylakoids from Nicotiana tabacum var. John William's Broadleaf in a mass spectrometric assay detected as the oxidation of H2180. The signals were induced by illumination of the thylakoid suspension at pH-values of 6 or 7.8 with 3 short (5/is) saturating light flashes spaced 1.5 ms apart.
358 o f H2~80 (data not shown). These results show that within the time domain o f about 30 s no form o f bound water and/or an oxidation product exist up to the redox state S3 which is drastically affected by breaking o f a hydrogen bond within the physiological pH range. Interestingly, the C1--binding at PS II exhibits a marked decline above pH 7.5 (Lindberg and Andrrasson 1992). Accordingly, this bound chloride does not seem to be related to substrate water exchange. Another line of evidence that can be used to argue against the involvement o f a strongly bound form o f water comes from the observation that there seems to be little or no isotope effect associated with the O2-evolving step (Guy et al. 1993). The stability o f r e d o x states S 2 and S3 was found to be markedly enhanced at lower temperatures (Vass and Styring 1991, Styring and Rutherford 1992, Messinger et al. 1993). Likewise possible redox and/or ligand exchange equilibria could be retarded with decreasing temperature. To check this possibility, H2180/I-I2160 exchange experiments were performed at around 0 °C. In a procedure identical to that used in previous experiments (Bader et al. 1987), S2 and S3 were generated by one and two preflashes, respectively, and 1 ml aliquots o f H2180 cooled on ice were rapidly injected into the sample cuvette, also kept at about 0 °C. The results obtained are shown in Fig. 2. Two interesting phenomena emerge from the data: a) isotope exchange takes place within 30 s also at around 0 °C, and b) the mass ratio o f 160180 to I802 markedly deviates from the expected theoretical value for a homogenous mixture o f H2a60 and H2180. The latter phenomenon is very interesting because it suggests that at low temperatures the scrambling o f the water molecules is rather slow and clusters o f Hz~80 are bound and/or exchanged at the water oxidase. In order to investigate the kinetics of the effect, described above, the samples were illuminated with groups o f 10 flashes at different times after injection of H2180. The results obtained for masses 32, 34 and 36 are depicted in Fig. 3. (The initial signal for mass 32 is not shown, since after injection this mass cannot be determined precisely.) It shows that the theoretical ratio is achieved only after more than 30 min. If one assumes that at temperatures near the freezing point the mixing o f the water molecules at the microscopic level is rather slow, this result suggests that clusters o f water molecules are
role=36 ~ 2Flashes (At=300ms Addition of 18 H20 1 Preflash r~] DarkAdaptation ~,~~" I,, 30s Addition of 18 H20 2 Preflashes k Dark Adaptation J.
30s
,h 1 Flash pNoles 02
,I
Fig. 2. Photosynthetic 1802-evolutionby Nicotiana tabacum
thylakoids in a mass spectrometric experiment carried out at 0 °C. The initial reaction assay contained only normal water (i.e. H2160). Afterextensivedarkadaptationone or two preflashes were fired to populate the S2 and S. redox state, respectively. Immediatelyafter the preflashes, Hj80 was added and oxygen evolution at m/e=36 was measured by one or two analyzing flashes. The control experiment with addition of H2tsO to an assaymediumthathad not beenpreilluminatedyieldedabsolutely no 1802-evolution. m/e=32
10min.after HlgO2_Addition
7.
mle=3/,
m/e=36
_ ~
30mitt
TheoreficoI Isotope Distribution:/5.9684
43.8832
10.3684
Fig. 3. Isotopic distribution of oxygen that was evolved from Nicotiana tabacum thylakoids upon a train of 10 consecutively
firedflashesin an assay systemcomposedof a mixture ofH2160 and H2180correspondingto the atomic abundance of a = 0.322. The signals were recorded 10, 20 and 30 min after the addition of H2180 to the equilibrated and dark-adapted assay medium that contained only H2t60. The theoretical values that can be mathematicallyobtainedfor 1602,160180and 1802,respectively, are given in the bottom line of the figure. exchanged near the catalytic site o f the water oxidase. This idea could imply that the catalytic site o f the water oxidase becomes accessible to substrate water from the bulk phase only in the redox state S3
359 and/or S4. However, at least for S3this conclusion is difficult to rationalize if one takes into account results on the interaction of water soluble redox active amines with S2 and S3. The reduction of S3 with either NH2NH2 or NH2OH (both compounds are isoelectronic with H202) is slower than that of S2 by at least one order of magnitude (Franck and Schmid 1989, Messinger et al. 1991). This finding reveals that S3is by no means particularly susceptible to interaction with hydrophilic molecules from the bulk phase but appears to be much more 'protected' than S2. In this context it should be noted that early mass spectrometric experiments with the filamentous cyanobacterium Oscillatoria chalybea, demonstrating the existence ofmetastable $3, have shown that under particular conditions 02 given in the first flash is unlabeled even after several minutes in labeled water (Bader et al. 1983). It is not easy to rationalize that the Si-states should exhibit an entirely different order of interaction with the water molecules. Therefore, it appears to be more likely that the unusual isotope ratio at short times after injection of H2180 is caused by a limited number of water molecules within the protein matrix near the catalytic site of the water oxidase. The transport of substrate water through the protein matrix does not cause a complete disruption of (H2180)n-clusters. This conclusion implies that substrate water is probably transferred in clusters to the catalytic site rather than step by step as single molecules. Accordingly, the isotope ratio of the product dioxygen (i.e. x(160180):x(180=) where x is the molar fraction of 160180 and 1so2, respectively) corresponds only to the macroscopic stoichiometry of H2160:H2180 if complete scrambling of the isotope is achieved at the level of microscopic water clusters. Another problem of mechanistic relevance is the binding of substrate water to the catalytic site for which very little data is available. The observation of superhyperfine splitting of the S2 multiline EPR signal in samples incubated with H2170 indicates possible interaction of substrate water with the manganese in redox state S2 (Hansson et al. 1986). However, the comparatively long preincubation does not preclude an isotope exchange between 170 and 160 in $1, e.g. at the presumed #-oxo-bridges of dimeric manganese units (Yachandra et al. 1992). In the present study another approach was used to address the problem ofsubstrate water entry: H2180 was injected into a suspension of dark-adapted
thylakoids and after 30 s of equilibration the sample was illuminated with 3 flashes spaced by a dark time of 1.5 ms. An analogous experiment was performed where after a single flash H2180 was injected and two further flashes were fired. Typical traces are shown in Fig. 4. The evaluation of these experimental results reveals that in both cases virtually the same amount Of I802 was evolved provided that in the second case the decay of S2 between the first and the other two flashes is taken into account. These results can be explained in three different ways: i) the substrate water interacts with the water oxidase already in redox state S I (and probably also So), ii) the entry of substrate water into the oxidase is very fast in S2 and/or S3 states, or iii) there exists a pool of exchangeable water which interacts with the manganese cluster only in state S4. At present an unambiguous decision between these alternatives cannot be achieved. However, for different reasons, explanation i) seems to be more attractive. First, a time of 1.5 ms between the flashes is short compared with ligand exchange reactions at metal centers in higher redox states (for a discussion, see Sharp 1992). Accordingly, in S2 and S3states, a possible (substrate) water exchange at the manganese cluster should not be faster than in $1 provided that no special structural changes of the protein matrix take place which selectively affect the ligand substitution of the metal coordination
role 36
li{ 3 Flashes (zxt=1.5ms) Addition 18 of H2O
18
Moles 0 2
Dork Adaptation •
30s
Addition of 18 H20
~-[
~ 2 Flashes (At=1,5ms) 18 h~2.65pMoles 02
IPreflash, ~ Dark Adaptation'~
I\ I~
3os
"I
Fig. 4. Mass spectrometric determination of the oxygen-18 evolved by Nicotiana tabacum thylakoids in an assay system that contained H2180 (a = 0.322) and was illuminated with three flashes spaced 1.5 ms apart. The lower tracing represents the condition where H2180 had been added after 1 preflash and only two analyzing flashes were given.
360 sphere. Secondly, it is not easily understandable why S 1should be inert to isotope exchange because, in this case, the environment of the water oxidase has to be practically free of water molecules in equilibrium with the bulk phase. In the latter case a special mechanism is required which permits water access to the catalytic site of water oxidation in S4. The results of this study suggest that within the time resolution of the assay (approx. 30 s) the water oxidase can interact with the bulk aqueous phase in all redox states St. This phenomenon is not affected bypH where the water oxidase is inferred to undergo significant structural changes (Renger et al. 1992). The nature of the binding sites for water molecules inside the protein matrix and their mode of interaction with the catalytic site remains to be clarified.
Acknowledgements The financial support of the Deutsche Forschungsgemeinschaft to G. Renger (Re 354/10-2) and to K.P. Bader (Ba 1290/2-1) is gratefully acknowledged.
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