Rutherford Aw

The influence of the quinone-iron electron acceptor complex on the reaction centre photochemistry of Photosystem II

In a number of PSII preparations, no EPR detectable triplet signal is observed when QA is singly reduced. This is in contrast with observations from the purple bacterial reaction centre where the triplet is easily seen at liquid helium temperature.

A CHLOROPHYLL TILTED 30 RELATIVE TO THE MEMBRANE IN THE PHOTOSYSTEM II REACTION CENTRE

The orientation properties of the reaction centre triplet in Photosystem II (PS II) were determined. A high triplet yield was generated in PS II membranes by double reduction of the primary quinone electron acceptor Q A . It is deduced that the triplet state is localised on a chlorophyll, the tetrapyrrolic plane of which is tilted at 30° to the membrane. A similar orientation was found in D1 / D2 / cyt b -559 particles demonstrating that the triplet is confined to the reaction centre. Optical work in the literature has been interpreted as indicating that the triplet is localised on a monomeric chlorophyll and that, in the singlet state, P680 consists of this molecule weakly coupled to a second chlorophyll. The weakness of the coupling, compared to the coupling in the special pair of purple bacteria, allows P680 to be considered as a monomer. Taking the optical data into account, we propose that P680 is a chlorophyll molecule oriented at 30° to the membrane. This result is discussed in terms of the structural analogy between PS II and the reaction centre of purple bacteria. A model is favored in which P680 is a chlorophyll, structurally analogous to one of the monomeric bacteriochlorophylls of the bacterial reaction centre. In addition, the orientation data indicate that this chlorophyll is rotated by 45° in its ring plane compared to the monomeric bacteriochlorophylls in the reaction centre of Rhodopseudomonas viridis .

Rapid formation of the stable tyrosyl radical in photosystem II.

Two symmetrically positioned redox active tyrosine residues are present in the photosystem II (PSII) reaction center. One of them, TyrZ, is oxidized in the ns–μs time scale by P680+ and reduced rapidly (μs to ms) by electrons from the Mn complex. The other one, TyrD, is stable in its oxidized form and seems to play no direct role in enzyme function. Here, we have studied electron donation from these tyrosines to the chlorophyll cation (P680+) in Mn-depleted PSII from plants and cyanobacteria. In particular, a mutant lacking TyrZ was used to investigate electron donation from TyrD. By using EPR and time-resolved absorption spectroscopy, we show that reduced TyrD is capable of donating an electron to P680+ with t1/2 ≈ 190 ns at pH 8.5 in approximately half of the centers. This rate is ≈105 times faster than was previously thought and similar to the TyrZ donation rate in Mn-depleted wild-type PSII (pH 8.5). Some earlier arguments put forward to rationalize the supposedly slow electron donation from TyrD (compared with that from TyrZ) can be reassessed. At pH 6.5, TyrZ (t1/2 = 2–10 μs) donates much faster to P680+ than does TyrD (t1/2 > 150 μs). These different rates may reflect the different fates of the proton released from the respective tyrosines upon oxidation. The rapid rate of electron donation from TyrD requires at least partial localization of P680+ on the chlorophyll (PD2) that is located on the D2 side of the reaction center.

The influence of the double reduction of QA on the fluorescence decay kinetics of Photosystem II

Abstract The acceptor QA of PS II was doubly reduced by treatment of PS II-enriched membranes (200–300 chlorophylls per PS II-reaction centre) with dithionite and benzyl viologen. After double reduction of QA, two major differences appeared in the fluorescence decay kinetics (at 4′C), as compared to the situation with all QA singly reduced: (1) a dominant fast phase (lifetime approx. 200 ps) was observed, similar to that in samples with QA oxidised: (2) a slow phase with a lifetime of approx. 7 ns was observed, which disappeared upon reoxidation of the sample. The fluorescence yield was approximately half of that of samples with singly reduced QA. The fast phase is interpreted as being indicative of a high efficiency of charge separation due to the absence of a negatively charged QA. This is explained by the double protonation of doubly reduced QA giving rise to the electrically neutral quinol. Similar observations were made in a core complex preparation (60–80 chlorophylls per reaction centre). This preparation involves a detergent solubilisation step and data from both EPR and fluorescence indicated that it was more susceptible to double reduction of QA by dithionite (as compared to PS II membranes). The possibility that this is a general phenomenon in detergent solubilised PS II preparations is discussed.

A high-field EPR tour of radicals in photosystems I and II

High magnetic-field electron paramagnetic resonance (HFEPR) has been extensively applied to the study of photosynthetic reaction centers. HFEPR experiments have provided accurate measurements ofg-values of radicals in photosystems I and II. Theg-values not only reflect the structure of the radical, but also its immediate environment. Hence, valuable information about radicalprotein interactions can be obtained fromg-values. Data on tyrosine-, quinone-, pheophytin- and chlorophyll-based radicals are reviewed. Experiments dealing with spin pairs in photosystem I and II are examined. The spectrometer with which numerous experiments on photosystems have been carried out is described, as well as the 10 T magnet around which the spectrometer is based.

Electron spin echo envelope modulation spectroscopy in photosystem I.

The applications of electron spin echo envelope modulation (ESEEM) spectroscopy to study paramagnetic centers in photosystem I (PSI) are reviewed with special attention to the novel spectroscopic techniques applied and the structural information obtained. We briefly summarize the physical principles and experimental techniques of ESEEM, the spectral shapes and the methods for their analysis. In PSI, ESEEM spectroscopy has been used to the study of the cation radical form of the primary electron donor chlorophyll species, P(700)(+), and the phyllosemiquinone anion radical, A(1)(-), that acts as a low-potential electron carrier. For P(700)(+), ESEEM has contributed to a debate concerning whether the cation is localized on a one or two chlorophyll molecules. This debate is treated in detail and relevant data from other methods, particularly electron nuclear double resonance (ENDOR), are also discussed. It is concluded that the ESEEM and ENDOR data can be explained in terms of five distinct nitrogen couplings, four from the tetrapyrrole ring and a fifth from an axial ligand. Thus the ENDOR and ESEEM data can be fully accounted for based on the spin density being localized on a single chlorophyll molecule. This does not eliminate the possibility that some of the unpaired spin is shared with the other chlorophyll of P(700)(+); so far, however, no unambiguous evidence has been obtained from these electron paramagnetic resonance methods. The ESEEM of the phyllosemiquinone radical A(1)(-) provided the first evidence for a tryptophan molecule pi-stacked over the semiquinone and for a weaker interaction from an additional nitrogen nucleus. Recent site-directed mutagenesis studies verified the presence of the tryptophan close to A(1), while the recent crystal structure showed that the tryptophan was indeed pi-stacked and that a weak potential H-bond from an amide backbone to one of the (semi)quinone carbonyls is probably the origin of the to the second nitrogen coupling seen in the ESEEM. ESEEM has already played an important role in the structural characterization on PSI and since it specifically probes the radical forms of the chromophores and their protein environment, the information obtained is complimentary to the crystallography. ESEEM then will continue to provide structural information that is often unavailable using other methods.

Location of the calcium binding site in Photosystem II: A Mn2+ substitution study

The whereabouts of the Ca2+ site in Photosystem II (PSII) was investigated by experiments in which Mn2+ was substituted for Ca2+. When stoichiometric amounts of Mn2+ ions were added to Ca2+-depleted PSII, the Mn2+ was not detected by EPR. The titration of Ca2+ back into Ca2+-depleted/Mn2+-containing PSII resulted in the simultaneous release of the Mn2+ and the loss of the two EPR signals which are characteristic of the Ca2+-depleted enzyme (i.e., the stable, modified S2 multiline signal arising from the intrinsic Mn cluster and the split S3 signal from an organic radical interacting with the Mn cluster). These results indicate that the Mn2+ occupies the functional Ca2+ site. The S2 and S3 EPR signal characteristic of this kind of Ca2+-depleted preparation were unaffected by the binding of the Mn2+ Since, from earlier results, it seems likely that the modification and stability of S2 multiline signal in these PSII preparations is due to binding of chelator to or close to the Mn cluster, the present results indicate that the Ca2+ site (at least when occupied by Mn2+) does not overlap with the chelator binding site. Since Mn2+ binding does not effect the S2 EPR signal from the Mn cluster, it can be concluded that the Mn2+ is not involved in detectable magnetic interactions with the cluster. This result indicates that the Mn2+-occupied Ca2+ binding site is outside the first co-ordination sphere of the Mn cluster. The relaxation properties of TyrD. were enhanced by the presence of the Mn2+ when the Mn cluster was in the S1 state.

Orientation selection in photosynthetic PS I multilayers: structural investigation of the charge separated state P700+A1− by high-field/high-frequency time-resolved EPR at 3.4 T/95 GHz

The radical-pair state of the primary electron donor and the secondary electron acceptor (P(700)(+z.rad;)A(1)(-z.rad;)) of the photosynthetic reaction center (RC) photosystem I (PS I) of Synechocystis PCC 6803 was studied by time-resolved electron paramagnetic resonance (TREPR) at high field/high frequency (3.4 T/95 GHz) using orientation selection in multilayers. The goal of the present article is to work out the basis for future studies, in which the improved resolution of such multilayers may be used to detect mutation-induced structural changes of PS I in membrane preparations. This approach is particularly interesting for systems that cannot be prepared as single crystals. However, in order to use such multilayers for structural investigations of protein complexes, it is necessary to know their orientation distribution. PS I was chosen as a test example because the wild type was recently crystallized and its X-ray structure determined to 2.5 A resolution [Nature 411 (2001) 909]. On the basis of our experimental results we determined the orientation distribution. Furthermore, a simulation model for the general case in which the orientation distribution is not axially symmetric about the C(2) symmetry axis of the RC is developed and discussed. Spectra simulations show that changes in the TREPR spectra of PS I are much more significant for these oriented multilayers than for disordered samples. In this way the use of oriented multilayers, in conjunction with multifrequency TREPR measurements on oriented as well as on disordered samples, is a promising approach for studies of structural changes of PS I systems that are induced by point mutations.

Reaction centre photochemistry in cyanide-treated photosystem II.

EPR was used to study the triplet state of chlorophyll generated by radical pair recombination in the photosystem II (PSII) reaction centre. The spin state of the non-haem Fe2+ was varied using the CN--binding method (Y. Sanakis, V. Petrouleas, B.A. Diner, Biochemistry 33 (1994) 9922-9928) and the redox state of the quinone acceptor (QA) was changed from semi-reduced to fully reduced (F.J.E. van Mieghem, W. Nitschke, P. Mathis, A.W. Rutherford, Biochim. Biophys. Acta 977 (1989) 207-214). It was found that the triplet was not detectable using continuous wave EPR when QA- was present irrespective of the spin-state of the Fe2+. It was also found that the triplet state became detectable by EPR when the semiquinone was removed (by reduction to the quinol) and that the triplet observed was not influenced by the spin state of the Fe2+. Since it is known from earlier work that the EPR detection of the triplet reflects a change in the triplet lifetime, it is concluded that the redox state of the quinone determines the triplet lifetime (at least in terms of its detectability by continuous wave EPR) and that the magnetic state of the iron, (through the weakly exchange-coupled QA- Fe2+ complex) is not a determining factor. In addition, we looked for polarisation transfer from the radical pair to QA- in PSII where the Fe2+ was low spin. Such polarisation is seen in bacterial reaction centres under comparable conditions. In PSII, however, we were unable to find evidence for such polarisation of the semiquinone. It is suggested that both the short triplet lifetime in the presence of QA- and the lack of polarised QA- might be explained in terms of the electron transfer mechanism for triplet quenching involving the semiquinone which was proposed previously (F.J.E. van Mieghem, K. Brettel, B. Hillmann, A. Kamlowski, A.W. Rutherford, E. Schlodder, Biochemistry 34 (1995) 4798-4813). It is suggested that this mechanism may occur in PSII (but not in purple bacterial reaction centres) due the triplet-bearing chlorophyll being adjacent to the pheophytin at low temperature as suggested from structural studies (F.J.E. van Mieghem, K. Satoh, A.W. Rutherford, Biochim. Biophys. Acta 1058 (1992) 379-385).

Orientation Study on the Stable Tyrosyl Radical in Photosystem II by High Field EPR

The recently-built 285 GHz high-field EPR spectrometer in Saclay has been used to study the stable tyrosyl (Tyr D • ) radical in photosystem II (PSII). High fields allow the resolution of the g values which carry information concerning the chemical environment of the radical. Data are presented for intact and Mn-depleted PSII preparations, both in solution and oriented samples.