Binuclear Manganese(III) Complexes as Electron Donors in Dl/D2/Cytochrome b 559 Preparations Isolated from Spinach Photosystem II Membrane Fragments S. I. Allakhverdiev3’bc, M. S. Karacan*3, G. Somerb, N. Karacanb, E. M. Khanc, S. Y. Ranec, S. Padhyec, V. V. Klimov3 and G. Rengerd a Institute of Soil Science and Photosynthesis, RAS, Pushchino, Moscow Region, 142292 Russia b Department of Chemistry, University of Gazi, Ankara, 06500, Turkey c Department of Chemistry, University of Poona, 411007, India d Institut für Biophysikalische und Physikalische Chemie, Technische Universität, D-10623 Berlin, Bundesrepublik Deutschland Dedicated to Professor Achim Trebst on the occasion o f his 65th birthday Z. Naturforsch. 49c, 587-592 (1994); received June 24/July 19, 1994 PS II Reaction Center, Manganese, Pheophytin Photoaccumulation, NADP+ Reduction The capability of different manganese complexes to act as PS II electron donors in D 1/D 2/ cytochrome b 559 complexes has been analyzed by measuring actinic light-induced absorption changes at 680 nm (650 nm) and 340 nm, reflecting the photoaccumulation of Pheophytin' (Pheo- ) and the reduction of NADP+, respectively. The data obtained reveal: a) the donor capacity of synthetic binuclear Mn(III)2 complexes containing aromatic ligands significantly exceeds that for MnCl2 in both cases, i.e. Pheo- photoaccumulation and NADP+ reduction; b) manganese complexes can serve as suitable electron donors for light-induced NADP+ reduction catalyzed by D 1/D 2/cytochrome £>559 complexes and ferredoxin plus ferredoxin- NADP+ reductase under anaerobic conditions and c) the specific turnover rate of the system leading to NADP+ reduction is extremely small. The implications of these findings are briefly discussed. Introduction be incorporated into a heterodimer of polypep­ The essential steps of photosynthetic water tides D l and D 2 of PS II in an analogous way as cleavage take place within an integral membrane the corresponding groups are incorporated into complex associated with extrinsic regulatory poly­ the heterodimer of the l- and M-subunit of the peptides. This operational unit is referred to as reaction centers of purple bacteria (Michel and photosystem II (PS II) complex. The primary reac­ Deisenhofer, 1988; Trebst, 1986). This idea is tions of PS II comprise light absorption, excitation strongly supported by the isolation of PS II com­ energy transfer to the photoactive pigment (a spe­ plexes that i) contain only D1 and D2 together cial chlorophyll a designated as P680) and elec­ with cytochrome b 559 (= Cyt b 559, two subunits) tron transfer from its excited singlet state to pheo­ and at least one smaller polypeptide, and ii) retain phytin a (Pheo) acting as primary acceptor and the ability to perform the primary charge separa­ subsequent stabilization of the primary charge tion (Nanba and Satoh, 1987; Barber et al., 1987). separation by rapid electron transfer from Pheo- Although deprived of QA and the oxygen-evolving to a special plastoquinone molecule QA (Renger, complex (OEC), the D 1/D 2/cytochrome £559 1992). Based on similarities of the functional and complexes were found to catalyze the electron structural organization of the primary processes, transport from an artificial electron donor (di- components P680, Pheo and QA were inferred to phenylcarbazide) to an exogenous electron accep­ tor like silicomolybdate (Chapman et al., 1988; Takahashi et al., 1989). This observation implies Reprint requests to Prof. Dr. Gernot Renger, Technische that an electron transfer can take place from Universität Berlin, Max-Volmer-Institut für Biophysika­ Pheo- to exogenous acceptors and that either this lische und Physikalische Chemie, Straße des 17. Juni 135, D-10623 Berlin, Bundesrepublik Deutschland. reaction or the electron donation are able com­ Telefax: 49-30-31421122. peting with the rapid internal charge recombina- 0939-5075/94/0900-0587 $ 06.00 © 1994 Verlag der Zeitschrift für Naturforschung. All rights reserved. 588 S. I. Allakhverdiev et al. ■ Binuclear Manganese(III) Complexes as Electron Donors tion between Pheo“ and P680+. As the redox the light-induced photoaccumulation of pheo- potential of Pheo/Pheo- is rather low (Em 7 = phytin~ (= Pheo'). Likewise, at 340 nm the photo­ -6 1 0 mV, see Klimov et al., 1979), it appeared reduction of NADP+ was measured. Optical worthwhile to check if D 1/D 2/cytochrome b 559 pathlength: 1 cm; measuring light beam \ = preparation are also able to reduce NADP+ 340 nm, 650 nm or 680 nm; I ~ 0.15 W-m~2; (Em 7 = -320 mV). Recent reports claim that this actinic light: A. > 600 nm, I ~ 100 W m-2. All can be the case (Allakhverdiev and Klimov, 1990, measurements were performed at 20 °C. 1992; A rnon and Barber, 1990; Klimov et al., The binuclear complexes 1986). In the present study manganese complexes [Mn(III)Mn(III)(HNQOX)4(OAc)2] and were tested as electron donors for photoaccumu­ [M n(III)-0-M n(III)(HNQ0X)2(0Ac)2(H20 )2] lation of Pheo- in the absence of exogenous elec­ symbolized by M-2 and M-3, respectively, con­ tron acceptors and for NADP+ reduction under tained 2-hydroxy-l.4-naphthoquinone monoxime anaerobic conditions in the presence of ferredoxin (HNQOX) as terminal and CH3COO~ (OAc) as (Fd) and Fd-NADP+ reductase. The results ob­ bridging ligands (in M-3 there exist in addition a tained indicate that synthetic binuclear complexes p-oxo-bridge). These complexes were synthesized are efficient electron donors in D 1/D 2/cyto­ according to previously described procedures chrome b 559 preparations. (Charles, 1963; W ieghardt et al., 1985) and charac­ terized by spectroscopic and electrochemical Materials and Methods methods (Khan, 1993). Ferredoxin and ferredoxin- D 1/D 2/cytochrome £559 preparations from NADP+ reductase were purchased from Sigma. spinach were obtained from digitonin/Triton The compositions of the sample suspensions X-100 PS II fractions (DT 20 preparations, see used in the different experiments are given in the Allakhverdiev et al., 1992; Klimov et al., 1982) figure legends. according to the method of Nanba and Satoh (1987) with some modifications as described in Results and Discussion Allakhverdiev et al. (1992). The pigment content When thylakoid and PS II membrane fragments of the isolated D 1/D 2/cytochrome £559 was are exposed to actinic illumination in the presence determined by HPLC. A ratio of about six chloro­ of the strong reductant dithionite (Na2S20 4), phylls per two pheophytins was found. Pheo- accumulates and gives rise to a marked A single-beam differential spectrophotometer quenching of the chlorophyll fluorescence with a phosphoroscope similar to that described (Allakhverdiev and Klimov, 1990, 1992; Allakh­ previously (Klimov et al., 1979, 1986; A llakhver­ verdiev et al., 1978, 1979, 1986; Shuvalov et al., diev and Klimov, 1990, 1992; Allakhverdiev et al., 1980; Renger and Kayed, 1987). Analogously, 1992) was used to monitor at 650 nm and 680 nm Pheo^ photoaccumulation can be achieved also in Fig. 1. Actinic light-induced absorption changes at 650 nm and 680 nm as a function of time in Dl/D2/cyto- chrome6559 complexes. The sample suspension contained: D 1/D 2/cyto­ chrome b 559 complexes (5 pg chloro­ phyll/ml), 35 mM NaCl. 5 m M MgCl2, 1 (.im methylviologen, 20 m M tris(hy- droxymethyl)aminoethane-HCl, pH = 8.0. Arrows indicate turning on (up) • 8-1 and off (down) the actinic light. Trace 1: control with no further addition; trace 2: addition of 1 mg Na 2S204/ml; trace 3: addition of 50 |.im MnCl2, traces 4a/b: addition of 5 |.im manganese com­ plex M-3. S. I. Allakhverdiev et al. ■ Binuclear Manganese(III) Complexes as Electron Donors 589 D 1/D 2/cytochrome b 559 preparations in the pres­ (10-12 manganese per P680). No further increase ence of methylviologen (MV) and Na2S20 4 of the signal amplitude is observed at tenfold M-3 (B arber et al., 1987; Nanba and Satoh, 1987; concentrations (data not shown). This effect Allakhverdiev et al., 1992; Shuvalov et al., 1989) clearly shows that compound M-3 acts as an ef­ and monitored by characteristic absorption ficient electron donor to D 1/D 2/cytochrome b 559 changes in the red region. Light-induced absorp­ preparations. In order to check that the photo- tion changes of D 1/D 2/cytochrome £559 prep­ bleaching at 682 nm mediated by compound M-3 arations are shown in Fig. 1. In the absence of really reflects Pheo' formation, comparative Na2S20 4/MV virtually no absorption change can measurements were performed at 650 nm be seen at 682 nm (trace 1 of Fig. 1), a wavelength (trace 4 b of Fig. 1) where the difference spectrum characteristic for Pheo“ formation (Allakhverdiev exhibits a positive band (Allakhverdiev et al, et a l, 1992; Klimov et al, 1979). Lack of light- 1992; Klimov et a l, 1978, 1979). The amplitude induced absorption changes is understandable be­ ratio AA650/AA682 of the data nicely fits with that cause the primary radical pair recombines via kin­ of the difference spectrum of Pheo- versus Pheo. etics in the time domain of tens of nanoseconds In a recent study (Allakhverdiev et al, 1994) (Crystall et a l, 1989; Danelius et al, 1987; Taka- evidence was presented that the binuclear manga­ hashi et a l, 1987). On the other hand, in the pres­ nese complexes M-2 and M-3 are much more ence of Na2S20 4/MV the actinic light causes a efficient than MnCl2 in electron donation to strong bleaching at 682 nm that biphasically re­ PS II and restoration of the water oxidase activity laxes in the subsequent dark period (trace 2 of in PS II preparations that are almost completely Fig. 1). This effect is understandable by electron deprived of their endogenous manganese by donation to P680+ competing with the fast recom­ N,N,N\N'-tetramethylendiamine (TEMED) treat­ bination reaction by Pheo-.