Species-specific Differences of the Spectroscopic Properties of P700
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m304776200
ISSN1083-351X
AutoresHeike Witt, Enrica Bordignon, Donatella Carbonera, Jan P. Dekker, Navassard V. Karapetyan, Christian Teutloff, Andrew N. Webber, Wolfgang Lubitz, E. Schlodder,
Tópico(s)Photoreceptor and optogenetics research
ResumoWe applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae (Chlamydomonas reinhardtii CC2696), and higher plants (Spinacia oleracea). Remarkable species-specific differences of the optical properties of P700 were revealed monitoring the (3P700–P700) and (P700+·–P700) absorbance and CD difference spectra. The main bleaching band in the Qy region differs in peak position and line width for the various species. In cyanobacteria the absorbance of P700 extends more to the red compared with algae and higher plants which is favorable for energy transfer from red core antenna chlorophylls to P700 in cyanobacteria. The amino acids in the environment of P700 are highly conserved with two distinct deviations. In C. reinhardtii a Tyr is found at position PsaB659 instead of a Trp present in all other organisms, whereas in Synechocystis a Phe is found instead of a Trp at the homologous position PsaA679. We constructed several mutants in C. reinhardtii CC2696. Strikingly, no PS I could be detected in the mutant YW B659 indicating steric constraints unique to this organism. In the mutants WA A679 and YA B659 significant changes of the spectral features in the (3P700–P700), the (P700+·–P700) absorbance difference and in the (P700+·–P700) CD difference spectra are induced. The results indicate structural differences among PS I from higher plants, algae, and cyanobacteria and give further insight into specific protein-cofactor interactions contributing to the optical spectra. We applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae (Chlamydomonas reinhardtii CC2696), and higher plants (Spinacia oleracea). Remarkable species-specific differences of the optical properties of P700 were revealed monitoring the (3P700–P700) and (P700+·–P700) absorbance and CD difference spectra. The main bleaching band in the Qy region differs in peak position and line width for the various species. In cyanobacteria the absorbance of P700 extends more to the red compared with algae and higher plants which is favorable for energy transfer from red core antenna chlorophylls to P700 in cyanobacteria. The amino acids in the environment of P700 are highly conserved with two distinct deviations. In C. reinhardtii a Tyr is found at position PsaB659 instead of a Trp present in all other organisms, whereas in Synechocystis a Phe is found instead of a Trp at the homologous position PsaA679. We constructed several mutants in C. reinhardtii CC2696. Strikingly, no PS I could be detected in the mutant YW B659 indicating steric constraints unique to this organism. In the mutants WA A679 and YA B659 significant changes of the spectral features in the (3P700–P700), the (P700+·–P700) absorbance difference and in the (P700+·–P700) CD difference spectra are induced. The results indicate structural differences among PS I from higher plants, algae, and cyanobacteria and give further insight into specific protein-cofactor interactions contributing to the optical spectra. Photosystem I (PS I) 11 The abbreviations used are: PS I (II), photosystem I (II); ADMR, absorption-detected magnetic resonance; BA and BB, accessory chlorophylls ligated by subunit PsaA and PsaB, respectively; CAPS, 3-(cyclohexylaminol-1-propanesulfonic acid); Chl, chlorophyll; β-DM, n-dodecyl β-maltoside; ENDOR, electron nuclear double resonance; hfc, hyperfine coupling constant; LHC, light-harvesting complex; P700, primary electron donor of PS I; P700+·, cation radical of P700; 3P700, triplet state of P700; PA and PB, the two chlorophylls constituting P700 ligated by subunit PsaA and PsaB, respectively; T-S, triplet-minus-singlet; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TMPD, tetramethyl-p-phenylenediamine dihydrochloride; ZFS, zero field-splitting.11 The abbreviations used are: PS I (II), photosystem I (II); ADMR, absorption-detected magnetic resonance; BA and BB, accessory chlorophylls ligated by subunit PsaA and PsaB, respectively; CAPS, 3-(cyclohexylaminol-1-propanesulfonic acid); Chl, chlorophyll; β-DM, n-dodecyl β-maltoside; ENDOR, electron nuclear double resonance; hfc, hyperfine coupling constant; LHC, light-harvesting complex; P700, primary electron donor of PS I; P700+·, cation radical of P700; 3P700, triplet state of P700; PA and PB, the two chlorophylls constituting P700 ligated by subunit PsaA and PsaB, respectively; T-S, triplet-minus-singlet; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TMPD, tetramethyl-p-phenylenediamine dihydrochloride; ZFS, zero field-splitting. is a multisubunit pigment-protein complex located in the thylakoid membranes of cyanobacteria, algae, and plants that mediates light-induced electron transfer from plastocyanin or cytochrome c6 on the luminal side to ferredoxin on the stromal side (for a review see Refs. 1Brettel K. Biochim. Biophys. Acta. 1997; 1318 (references therein): 322-373Crossref Scopus (433) Google Scholar and 2Heathcote P. guest ed) Biochim. Biophys. Acta. 2001; 1507 (references therein)Crossref Scopus (8) Google Scholar). In plants and algae, PS I is composed of the core complex and the light-harvesting complex LHCI. LHC I complexes do not exist in cyanobacteria. The PS I core complexes of all organisms consist of two large subunits, PsaA and PsaB, and at least eight smaller subunits (3Scheller H.V. Jensen P.E. Haldrup A. Lunde C. Knoetzel J. Biochim. Biophys. Acta. 2001; 1507: 41-60Crossref PubMed Scopus (169) Google Scholar). However, some differences between the cyanobacterial and chloroplast PS I core complexes exist. (i) In cyanobacteria, two additional small subunits, PsaM and PsaX, are found not present in algae and plants, which instead contain another four additional subunits, PsaH, PsaG, PsaN, and PsaO, not found in cyanobacteria (4Manna P. Chitnis P.R. Singhal G.S. Renger G. Sopory S.K. Irrgang Govindjee K.D. Function and Molecular Genetics of Photosystem I in Concepts in Photobiology: Photosynthesis and Photomorphogenesis. Narosa Publishing House, New Delhi, India1999Google Scholar, 5Fromme P. Schlodder E. Jansson S. Green B. Parson W.W. Light-harvesting Antennas in Photosynthesis. Kluwer Academic Publishers Group, Dordrecht, Netherlands2003: 253-279Google Scholar). (ii) Trimer formation of PS I is common for cyanobacteria but is not observed in algae and plants. (iii) Another difference can be found regarding the type of electron carrier proteins used to transfer electrons to the oxidized primary donor P700+· and their interaction with PS I (6Baymann F. Brugna M. Mühlenhoff U. Nitschke W. Biochim. Biophys. Acta. 2001; 1507: 291-310Crossref PubMed Scopus (82) Google Scholar). In Thermosynechococcus elongatus, cytochrome c6 is the only electron carrier, whereas in Chlamydomonas reinhardtii cytochrome c6 or plastocyanin can be used, mainly depending on the nutrient conditions (7Rochaix J.-D. Plant Physiol. 2001; 127: 1394-1398Crossref PubMed Scopus (39) Google Scholar).Although only the three-dimensional structure of PS I from T. elongatus is known (8Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2029) Google Scholar), it is generally assumed that the PS I core complexes, and especially the cofactor arrangement in the reaction center, are similar in all organisms despite the differences described above. The two large subunits, PsaA and PsaB, each consisting of 11 transmembrane helices, form the heterodimeric catalytic core and coordinate most of the cofactors involved in the electron transfer process except for the terminal electron acceptors FA and FB (two [4Fe-4S] iron-sulfur clusters) that are bound by one of the extrinsic subunits on the stromal side, PsaC. Besides the reaction center, this heterodimer harbors the core antenna system, together with some of the smaller integral subunits, consisting of about 100 chlorophylls and 20 β-carotenes. The primary sequences of these large subunits are highly conserved and the same holds for the primary photochemistry of the reaction center of PS I, although there is a discussion going on that in some species both branches participate in the primary charge separation whereas in other species only the A-branch seems to be active (9Guergova-Kuras M. Boudreaux B. Joliot A. Joliot P. Redding K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4437-4442Crossref PubMed Scopus (267) Google Scholar, 10Xu W. Chitnis P. Valieva A. van der Est A. Brettel K. Guergova-Kuras M. Pushkar J. Zech S.G. Stehlik D. Shen G. Zybailov B. Golbeck J.H. J. Biol. Chem. 2003; 278: 27876-27887Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).After absorption of light by an antenna pigment, the excitation energy is transferred to the primary electron donor P700, a chlorophyll a dimer. P700 being in the lowest excited singlet state donates an electron to the primary electron acceptor Ao, a chlorophyll a monomer. Charge stabilization is achieved by subsequent electron transfer to secondary acceptors, the phylloquinone A1 and Fx, a [4Fe-4S] iron-sulfur cluster and finally to FA and FB. The x-ray crystallographic analysis of cyanobacterial PS I (8Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2029) Google Scholar) has revealed that the primary electron donor P700 of photosystem I is a dimer composed of one chlorophyll a and one chlorophyll a′ that is the 132 epimer of Chl a. The coordinating ligand of Chl a′ (PA) is provided by a His from PsaA (His-A676) (numbering according to Chlamydomonas throughout the text), whereas the one of Chl a (PB) is provided by PsaB (His-B656). Based on ENDOR spectra of P700+·, which exhibit nearly identical hyperfine coupling constants of the methyl protons of all investigated species, it has been concluded that the positive charge is mainly localized on PB (11Käss H. Die Struktur des primären Donators P700 in Photosystem I. Ph.D. thesis, Technical University, Berlin1995Google Scholar, 12Krabben L. Schlodder E. Jordan R. Carbonera D. Giacometti G. Lee L. Webber A.N. Lubitz W. Biochemistry. 2000; 39: 13012-13025Crossref PubMed Scopus (82) Google Scholar, 13Käss H. Fromme P. Witt H.T. Lubitz W. J. Phys. Chem. B. 2001; 105 (references therein): 1225-1239Crossref Scopus (90) Google Scholar, 14Webber A.N. Lubitz W. Biochim. Biophys. Acta. 2001; 1507: 61-79Crossref PubMed Scopus (127) Google Scholar).Despite the remarkable similarities described above, optical spectroscopy of PS I complexes from different species indicates considerable differences. For example, although the flash-induced (P700+·–P700) absorbance difference spectra of T. elongatus and Spinacia oleracea (spinach) are rather similar (15Hiyama T. Ke B. Biochim. Biophys. Acta. 1972; 267: 160-171Crossref PubMed Scopus (453) Google Scholar, 16Pålsson L.-O. Flemming C. Gobets B. van Grondelle R. Dekker J.P. Schlodder E. Biophys. J. 1998; 74: 2611-2622Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), those recorded for Anabaena variabilis (15Hiyama T. Ke B. Biochim. Biophys. Acta. 1972; 267: 160-171Crossref PubMed Scopus (453) Google Scholar) and C. reinhardtii (17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar, 18Redding K. MacMillan F. Leibl W. Brettel K. Hanley J. Rutherford A.W. Breton J. Rochaix J.-D. EMBO J. 1998; 17: 50-60Crossref PubMed Scopus (82) Google Scholar) look different.To investigate the reasons for species-specific differences, we were looking for differences in the primary sequences. In all organisms sequenced so far, the amino acid residues around P700 are highly conserved. Based on the 2.5-Å structure of PS I from T. elongatus, we searched for all amino acids located within a distance of 10 Å with respect to the magnesium atoms of PA and PB. The sequence comparison shows that the respective amino acids are identical for T. elongatus and spinach. Two differences exist between C. reinhardtii and T. elongatus. A Ser is found at position A654 instead of an Ala in T. elongatus. The exchange of this Ala against Ser using the program Swiss PdbViewer indicates that the Ser could be located close to PA between the vinyl and methyl side chains. Although the polarity of the binding pocket of PA might be slightly higher for C. reinhardtii, we do not expect that this amino acid residue has a significant influence on the optical properties of P700. A striking difference is a Tyr at position B659 in C. reinhardtii, whereas a Trp is conserved in all other organisms. For Synechocystis three differences are found, namely Gly instead of Ala-A675, Phe instead of Leu-A622, and a Phe is present at position A679 which is the homologous position to B659 on PsaA where Trp is always found in the other organisms. We did not investigate further the differences found for Synechocystis due to the mutagenesis system chosen (see below). Unfortunately, the amino acid sequence of Spirulina platensis is not available so far. The amino acid residues at positions B659 and A679 are the most interesting ones due to their close vicinity to P700 and the accessory Chls (within 4 Å). An additional interesting aspect of these residues is their location one helix turn toward the stromal side with respect to the coordinating ligands, forming a roof-like structure orthogonal to P700 (Fig. 1). In C. reinhardtii, the Tyr-B659 might be able to form a hydrogen bond to the keto group of the phytyl side chain of PB. This would be analogous to the proposal based on the crystal structure that the conserved Tyr-A731 might be able to form a hydrogen bond with the phytyl ester carbonyl oxygen of PA. As the single amino acid deviations mentioned above are the only obvious differences between the different species, site-directed mutagenesis has been applied to clarify whether these differences are responsible for the spectroscopic peculiarities of the species. For T. elongatus, it has not been achieved to perform site-directed mutagenesis whereas such a system is established for C. reinhardtii and this organism has been preferred as a model organism for the analysis of photosynthesis using (sitedirected) mutagenesis (12Krabben L. Schlodder E. Jordan R. Carbonera D. Giacometti G. Lee L. Webber A.N. Lubitz W. Biochemistry. 2000; 39: 13012-13025Crossref PubMed Scopus (82) Google Scholar, 17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar, 18Redding K. MacMillan F. Leibl W. Brettel K. Hanley J. Rutherford A.W. Breton J. Rochaix J.-D. EMBO J. 1998; 17: 50-60Crossref PubMed Scopus (82) Google Scholar, 19Hippler M. Redding K. Rochaix J.-D. Biochim. Biophys. Acta. 1998; 1367: 1-62Crossref PubMed Scopus (56) Google Scholar). C. reinhardtii is able to grow heterotrophically on media with acetate as the carbon source (20Harris E. Chlamydomonas Sourcebook. Academic Press, San Diego1989: 25-64Google Scholar) and is easily amenable to genetic manipulation with the advent of chloroplast genome transformation and selectable markers. Site-directed mutagenesis in turn is a powerful tool to identify the role of individual amino acids and to study the relationships between structure and function of photosynthetic reaction centers.To get reliable information on species-specific differences, we measured and compared triplet-minus-singlet (T-S) difference, (P700+·–P700) absorbance difference, and CD difference spectra of PS I core complexes from various species all purified using β-dodecyl maltoside as detergent. To study the influence of the amino acid side chains described above on the spectroscopic properties of P700 and to gain further insight into protein-cofactor interactions, we mutated Trp-A679 to Ala, Tyr, and His, Tyr-B659 to Trp, Leu, Ala, and His, and Tyr-A731 to His in C. reinhardtii. The effects of these mutations were analyzed using steady state and transient absorbance difference spectroscopy. Additionally, we applied redox titrations and electron nuclear double resonance (ENDOR) spectroscopy.MATERIALS AND METHODSStrains, Chloroplast Transformation, and Growth Conditions—As the recipient of the donor plasmids, strain C. reinhardtii CC2696 was used which in contrast to wild type carries the DS-521 nuclear mutation leading to a deficiency in the Cab proteins and a deletion in psbA causing the loss of PS II. Therefore, this strain is well suited for the preparation and analysis of PS I core complexes.Chloroplast transformation and selection of the transformants on Tris acetate-phosphate plates containing 150 μg/ml spectinomycin were carried out as described previously (17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar).For enzyme purification, strains were grown heterotrophically in Tris acetate-phosphate and high salt medium/acetate medium (20Harris E. Chlamydomonas Sourcebook. Academic Press, San Diego1989: 25-64Google Scholar) at 26 °C under low light conditions and supplemented with spectinomycin for mutant strains.Site-directed Mutagenesis—Site-directed mutagenesis was performed according to the altered sites mutagenesis procedure (Promega, Heidelberg, Germany) on psaA-3 and psaB. Following mutagenesis and sequencing, a ClaI-PstI fragment of psaA-3 was subcloned into pKR154 (17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar, 21Leibl W. Brettel K. Nabedryk E. Breton J. Rochaix J.-D. Redding K. Garab, G Photosynthesis: Mechanisms and Effects. Vol. 1. Kluwer Academic Publishers Group, Dordrecht, Netherlands1998: 595-598Google Scholar) and reintroduced into C. reinhardtii CC2696. Plasmid pG528G, which was used for the reintroduction of psaB, has been described before (22Cui L. Bingham S.E. Kuhn M. Käss H. Lubitz W. Webber A.N. Biochemistry. 1995; 34: 1549-1558Crossref PubMed Scopus (29) Google Scholar). For mutations in psaB, the EcoRI-PstI fragment that encodes the psaB gene and part of rbcL was subcloned into pAlter-1 to perform site-directed mutagenesis.Preparation of PS I Core Complexes—Isolation of native and mutated PS I core complexes from C. reinhardtii CC2696 was performed according to Witt et al. (17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar) and from S. oleracea (spinach) as described by van der Lee et al. (23van der Lee J. Bald D. Kwa S.L.S. van Grondelle R. Roegmer M. Dekker J.P. Photosynth. Res. 1993; 35: 311-321Crossref PubMed Scopus (68) Google Scholar). The trimeric PS I core complexes from S. platensis have been prepared as described in Kruip et al. (24Kruip J. Karapetyan N.V. Terekhova I.V. Roegner M. J. Biol. Chem. 1999; 274: 18181-18188Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), from T. elongatus as described by Fromme and Witt (25Fromme P. Witt H.T. Biochim. Biophys. Acta. 1998; 1365: 175-184Crossref Scopus (112) Google Scholar) and from Synechocystis PCC 6803 (kind gift of M. Rögner) as described in Kruip et al. (26Kruip J. Boekema E.J. Bald D. Boonstra A.F. Roegner M. J. Biol. Chem. 1993; 268: 23353-23360Abstract Full Text PDF PubMed Google Scholar).The Chl/P700 ratio has been determined for all PS I core complexes from all the species used in this work. It was calculated from the maximum flash-induced absorbance decrease in the Qy region due to photo-oxidation of P700 by using the molar extinction difference coefficients given under "Results" and from the flash-induced absorbance increase at 826 nm due to the formation of P700+· using an extinction coefficient of 7500 m–1 cm–1. For all PS I core complexes from all the species, the ratio is 100 ± 15. For the PS I preparations from C. reinhardtii and spinach, this gives evidence that virtually no LHC I is present bearing in mind that the Chl a/P700 ratio for PS I holocomplexes is about 200–250.After purification the native and mutated PS I core complexes from C. reinhardtii CC2696 were characterized by SDS-PAGE and Western blot analyses in comparison to the PS I holocomplex isolated from C. reinhardtii CC125 where LHC I is still present. The same amount of native and mutated PS I from CC2696 and wild type PS I from CC125 was loaded in each lane of a gel, and Western blot analysis with antibodies directed against PsaF showed that PsaF is present at the same level in the purified PS I complexes (not shown). Western blot analysis performed with antibodies directed against PsaA, -C, and -D demonstrated the existence of these subunits. For the native and mutated PS I preparations from CC2696, a minor contamination by LHC I can be deduced both from the measured Chl a/b ratio of 15 ± 5 and from the Western blotting with antibodies directed against LHCI proteins. Chlorophyll concentrations were determined according to Porra et al. (27Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4580) Google Scholar).Transient Absorption Spectroscopy—Flash-induced absorbance difference spectra of (P700+·–P700) were measured at room temperature as described previously (17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar) with PS I complexes diluted to 10 μm Chl in 20 mm Tricine (pH 7.5), 25 mm MgCl2, 100 mm KCl, 0.02% β-DM, 5 mm ascorbate, and 10 μm phenazine methosulfate. The difference between the molar extinction coefficients of P700+· and P700 at the peak wavelength was calculated from the flash-induced absorption change of N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) according to Hiyama and Ke (15Hiyama T. Ke B. Biochim. Biophys. Acta. 1972; 267: 160-171Crossref PubMed Scopus (453) Google Scholar). TMPD is oxidized by the flash-induced P700+·. An extinction coefficient of 12,000 m–1 cm–1 has been determined for oxidized TMPD (pH 8.0).For the light-minus-dark absorbance difference spectra at 77 K, PS I core complexes were diluted to 10 μm Chl in 20 mm Tricine (pH 7.5), 25 mm MgCl2, 0.02% β-DM, 5 mm ascorbate, and 60% glycerol. Measurements were performed as described previously (17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar).To study the triplet state of P700, flash-induced T-S spectra have been recorded at 5 K. PS I core complexes were diluted to 10–15 μm Chl in 100 mm CAPS (pH 10), 10 mm MgCl2,10mm CaCl2, and 0.02% β-DM. Glycerol was added to a final concentration of 65% (v/v). 10–20 mm dithionite was added to this solution under argon. The samples were then illuminated at 270 K for 3 min with a 250-watt focused tungsten lamp filtered by water and additional heat-absorbing filters. This procedure leads to the pre-reduction of the secondary electron acceptor A1 whereby further electron transfer to A1 is blocked. The primary radical pair P700+·Ao-· formed under these conditions recombines with a high yield to the triplet state of P700. Measurements were performed according to Ref. 28Schlodder E. Paul A. Cetin M. PS2001 Proceedings of the 12th International Congress on Photosynthesis, Brisbane, Australia, August 18–23, 2001. CSIRO Publishing, Melbourne, Australia2001Google Scholar.Circular Dichroism—CD spectra were recorded on a Jasco J-720 spectrometer at room temperature as described previously (17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar). PS I complexes were diluted to 13 μm Chl in 20 mm Tricine (pH 7.5), 25 mm MgCl2, 100 mm KCl, 0.02% DM. For the measurements the solution was divided into two samples. Ferricyanide (1 mm) was added to one sample to oxidize P700, whereas 5 mm ascorbate and 10 μm phenazine methosulfate was added to the second sample to keep P700 in the reduced state. Spectra of the samples with P700 oxidized or reduced were measured alternating. Oxidized-minus-reduced difference spectra were obtained by subtracting the average reduced from the average oxidized spectrum.Redox Titration—To determine the oxidation midpoint potential of P700, the flash-induced absorbance change at 826 nm, associated with oxidation of P700, was measured as a function of the redox potential. Purified PS I complexes were diluted to 20–30 μm Chl in 20 mm Tricine (pH 7.5), 100 mm KCl, 25 mm MgCl2, 0.02% β-DM, and the redox potentials were adjusted by adding ferricyanide and ferrocyanide. After each experiment, the potential was measured using a combination Pt/Ag/AgCl electrode (Schott PT5900A) which was calibrated against the redox potential of a saturated solution of quinhydrone as a function of pH. A pH-meter (Knick PHM82) was used to read out the redox potential. All redox potentials are given relative to the standard hydrogen electrode (normal hydrogen electrode).ENDOR—ENDOR measurements were performed on a Bruker ESP 300E X-band EPR spectrometer with home-built ENDOR accessories (29Rautter J. Lendzian F. Lubitz W. Wang S. Allen J.P. Biochemistry. 1994; 33: 12077-12084Crossref PubMed Scopus (75) Google Scholar). ENDOR experiments were carried out on the cation radical P700+· of PS I complexes in frozen solution at 150 K as described earlier (17Witt H. Schlodder E. Teutloff C. Niklas J. Bordignon E. Carbonera D. Kohler S. Labahn A. Lubitz W. Biochemistry. 2002; 41: 8557-8569Crossref PubMed Scopus (72) Google Scholar). Samples contained between 3 and 5 mm Chl. P700+· was generated by continuous illumination at room temperature with two 150-watt halogen lamps equipped with a water filter (2-cm path length) and a 700-nm edge filter for 20 s followed by rapid freezing under illumination.ADMR Spectroscopy of the P700 Triplet State—Measurements were performed on thylakoid membranes that were diluted to 0.1 mm Chl in 100 mm CAPS (pH 10), 10 mm MgCl2, 10 mm CaCl2. Oxygen was removed using a glucose/glucose oxidase system, and 20 mm dithionite was added under nitrogen. Glycerol was added to a final concentration of 60% (v/v). The procedure for the pre-reduction of the secondary electron acceptor A1 was essentially the same as described for the flash-induced T-S spectra. Measurements were performed at 1.8 K. The absorption-detected magnetic resonance and the T-S microwave-induced spectra were recorded using the laboratory-built apparatus described previously (30Carbonera D. Collareta P. Giacometti G. Biochim. Biophys. Acta. 1997; 1322: 115-128Crossref Scopus (32) Google Scholar).RESULTSBy applying standard site-directed mutagenesis techniques, we constructed several mutants in PsaA and PsaB of PS I from C. reinhardtii CC2696 to investigate the influence of Trp-A679 and Tyr-B659 on the properties of P700. No PS I could be detected in mutants where Tyr-B659 was replaced by Trp or Leu. This is very surprising for the Trp mutant because all other species have a Trp at this position. The native and the mutated PS I core complexes WA A679, WH A679, WY A679, YA B659, and YH B659 from C. reinhardtii were purified and spectroscopically characterized together with PS I core complexes from four other species (T. elongatus, S. platensis, Synechocystis sp. PCC 6803, and S. oleracea) by steady state and transient absorption spectroscopy, circular dichroism, ADMR, ENDOR, and redox titrations.Mutant Phenotype—As the mutations were introduced into the Chlamydomonas strain CC2696, which in contrast to wild type contains the DS-521 nuclear mutation leading to a deficiency in the Cab proteins and a deletion in psbA causing the loss of PS II, PS I is the main chlorophyll-binding protein. It is therefore possible to detect consequences of the amino acid substitution on the amount of PS I by inspection of the phenotype. All mutants contain PS I amounts comparable with the native CC2696 except for YW B659 and YL B659 which show a yellow phenotype indicating the absence of PS I. The negative phenotype of YW B659 was confirmed by Western blots (not shown) using thylakoid membranes and antibodies directed against PsaA which did not show any detectable amount of PS I.Triplet-minus-Singlet (T-S) Absorbance Difference Spectra—To study the triplet state of P700, measurements were performed with PS I complexes under reducing conditions with the secondary acceptor A1 in the reduced state. Therefore, the electron transfer to A1 is blocked and the primary radical pair, P700+·A0-·, recombines to the triplet state of P700 with high yield. Fig. 2, A and B, shows the flash-induced T-S absorbance difference spectra of C. reinhardtii, T. elongatus, S. platensis, Synechocystis, and S. oleracea (spinach) detected at 5 K. These spectra reflect the absorbance difference between P700 in its triplet state and its singlet ground state. The spectra have been normalized between 660 and 726 nm to the same area assuming that the loss of oscillator strength upon triplet formation of P700 is the same in all species. The flash-induced absorbance difference spectra at 5 K attributed to 3P70
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