nan Alia

Effect of proline on the production of singlet oxygen

Summary. Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (1O2) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of 1O2 with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20 mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.

Photo-CIDNP solid-state NMR on Photosystems I and II:what makes P680 special?

The origin of the extraordinary high redox potential of P680, the primary electron donor of Photosystem II, is still unknown. Photochemically induced dynamic nuclear polarisation (photo-CIDNP) 13C magic-angle spinning (MAS) NMR is a powerful method to study primary electron donors. In order to reveal the electronic structure of P680, we compare new photo-CIDNP MAS NMR data of Photosystem II to those of Photosystem I. The comparison reveals that the electronic structure of the P680 radical cation is a Chl a cofactor with strong matrix interaction, while the radical cation of P700, the primary electon donor of Photosystem I, appears to be a Chl a cofactor which is essentially undisturbed. Possible forms of cofactor–matrix interactions are discussed.

Magnetic field dependence of13C photo-CIDNP MAS NMR in plant photosystems I and II

Photochemically induced dynamic nuclear polarization is observed in the two photosynthetic reaction centers of plants, photosystem I (PSI) and photosystem II (PSII) by13C magic-angle spinning nuclear magnetic resonance (NMR) at three different magnetic fields 17.6, 9.4, and 4.7 T. There is a significant difference in field dependence detected in the light-induced signal pattern of the two photosystems. For PSII the optimal NMR enhancement factor of about 5000 is observed at 4.7 T. On the other hand, the maximal light-induced signals of PSI are observed at 9.4 T.

Bacteriochlorophyll/imidazole and chlorophyll/imidazole complexes are negatively charged in an apolar environment

Abstract 600 MHz 15 N-NMR and 2D homonuclear 1 H– 1 H and heteronuclear 1 H– 15 N-NMR data for bacteriochlorophyll a /imidazole and chlorophyll a /imidazole have been recorded. Unambiguous assignments of the 15 N signals of the two nitrogens of imidazole ligated to the Mg of the bacteriochlorophyll a or chlorophyll a were obtained. It follows that the imidazole in complex is deprotonated, which implies that the chlorophyll/imidazole carries a full negative charge in the apolar solvent environment. This information is potentially useful in characterizing the nature of the magnesium–histidine interaction and the charge state of chlorophylls coordinated by histidine in photosynthetic pigment protein complexes.

Probing the electronic structure of tyrosine radical YD in photosystem II by EPR spectroscopy using site specific isotope labelling in Spirodela oligorrhiza

Abstract Tyrosine (Y D ) in the D2 reaction centre polypeptide of photosystem II (PSII) is redox-active and, under illumination, forms a dark-stable radical Y D . The origin of its stability and the functional role of Y D are not well understood. For understanding the electronic structure and reactivity of Y D , it is crucial to unambiguosly establish its hyperfine structure. There is considerable variation in the hyperfine data of Y D and their interpretation in literature. In the present study, the hyperfine structure of tyrosine radical Y D in PSII was probed by EPR in conjunction with carefully designed site specific isotope labelling. A comprehensive series of different selectively 2 H-, 13 C- or 17 O-labeled tyrosine were synthesized and incorporated in Spirodela oligorrhiza with more than 95% enrichment. The 13 C- and 17 O-hyperfine interactions were obtained from spectral simulations. From the anisotropy of the hyperfine interactions the spin densities at all phenoxyl ring positions were precisely obtained. Comparison of the absolute differences in individual spin densities between Y D and neutral tyrosine radical in vitro with those of computationally calculated spin densities yield excellent agreement for a well ordered hydrogen bond between Y D and the surrounding protein matrix with a bond length of 1.5 A. Enantioselective labeling confirms that the β-methylene hydrogens of Y D in S. oligorrhiza are oriented in a highly constrained specific position making Y D strongly immobilized, thereby ensuring a firm hydrogen bond of the phenoxyl oxygen to the protein matrix.

Photochemically induced dynamic nuclear polarization in bacterial photosynthetic reaction centres observed by 13C solid-state NMR

Photosynthesis, the synthesis of organic compounds upon utilization of light energy, is one of the key reactions for life on earth. The first step of this complex process, a light induced electron transfer, occurs in photosynthetic reaction centre (RC) membrane proteins. The electron is emitted from a (bacterio)chlorophyll (BCh1) aggregate in its electronically excited state. In bacterial RCs, this aggregate is a strongly coupled BChl a dimer, the so-called “special pair” (P). When photoexcited to a higher electronic singlet state, P transfers an electron to the primary acceptor, a pheophytin molecule, within 3 ps. The electron is then transferred to QA in about 200 ps. Subsequently, in a much slower reaction taking about 100 µs, the electron is transferred to the ultimate electron acceptor QB. This light-induced electron transfer sequence is repeated after the special pair has been re-reduced by a cytochrome. Although the spatial structure and kinetics of several RCs are known to atomic resolution, there is no clear understanding of the process of electron emission from the electronically excited primary electron donor. In addition, a detailed picture of the molecular mechanism of the inhibition of the back reaction, which is probably due to the high exothermic reaction enthalpy pushing the system into the inverted Marcus region, is missing. The functionally crucial electronic structure can be probed by spectroscopic methods. Grosso modo, vibrational spectroscopy provides information about electron densities between nuclei, and NMR spectroscopy at the nuclei.

Selective 15 N-Isotope Labelling of Histidines in PSII Reaction Centers of Spirodela Oligorrhiza

The Dl-D2 polypeptide heterodimer in Photosystem II (PSII) binds an unusual variety of cofactors. These include chlorophylls (Chl), pheophytins, QA, non-heme iron, QB and probably also the inorganic cofactors associated with oxygen evolution: manganese, calcium and chloride (1). However, detailed information about the interaction between these co-factors and highly conserved amino acids of the D1 and D2 polypeptides is still missing. Histidine (His) is one of the important and highly conserved amino acids in the D1 and D2 polypeptides which is believed to take part in interaction with many of these redox components e.g. as axial ligands to the magnesium atoms of the putative primary donor Chls (His 198 for both D1 and D2 proteins) and to the ferrous-iron in the quinone (QA and QB)-iron acceptor complex (His-215 and His-272 on the D1 protein and His-215 and His-269 on D2 protein) (1,2). These interactions could be crucial for primary photochemistry in PSII. His is often found in active sites of enzymes where hydrogen bonding and switching between protonation states of the imidazole side chain can control reactivity of active sites (3,4). Because of the tautomeric nature of the imidazole ring of His, it is not clear which nitrogen of the imidazole ring is actually co-ordinating with redox components. Specific 15N labelling at either of the two nitrogen sites (τ:tele or π:pros) of the imidazole ring of His followed by incorporation of this specifically labelled His in the PSII reaction center can provide a tool for identification of interactions between the His nitrogen and redox components by isotope-sensitive spectroscopic techniques such as solid-state NMR. 15N CP/MAS NMR (cross-polarization/Magic angle spinning, nuclear magnetic resonance) spectroscopy of selectively enriched samples is the method of choice to obtain NMR access with atomic selectivity for large membrane protein (5). In the present study we developed methods to incorporate specific 15N-labelled His into PSII reaction centers in Spirodela oligorrhiza.