Analysis of electron donors in photosystems in oxygenic photosynthesis by photo-CIDNP MAS NMR.

Both photosystem I and photosystem II are considerably similar in molecular architecture but they operate at very different electrochemical potentials. The origin of the different redox properties of these RCs is not yet clear. In recent years, insight was gained into the electronic structure of photosynthetic cofactors through the application of photochemically induced dynamic nuclear polarization (photo-CIDNP) with magic-angle spinning NMR (MAS NMR). Non-Boltzmann populated nuclear spin states of the radical pair lead to strongly enhanced signal intensities that allow one to observe the solid-state photo-CIDNP effect from both photosystem I and II from isolated reaction center of spinach (Spinacia oleracea) and duckweed (Spirodela oligorrhiza) and from the intact cells of the cyanobacterium Synechocystis by (13)C and (15)N MAS NMR. This review provides an overview on the photo-CIDNP MAS NMR studies performed on PSI and PSII that provide important ingredients toward reconstruction of the electronic structures of the donors in PSI and PSII.

Solid-ionic liquid interfaces: pore filling revisited.

The properties of ionic liquids on ordered and non-ordered mesoporous silicas (silica gel, MCM-41, SBA-15) were studied by nitrogen sorption, mercury intrusion and thermogravimetric analyses, as well as (129)Xe-NMR spectroscopy. The ionic liquids investigated are based on the 1-hexyl-3-methylimidazolium cation, which was combined with anions of low (bis(trifluoromethanesulfonyl)imide; [NTf2](-)), medium (trifluoromethylsulfonate; [CF3SO3](-)) to high (acetate; [OAc](-)) basicity. The surface coverage depends on both the type of ionic liquid and support used. This results not only in layer or droplet formation, but also in different physico-chemical properties of the ionic liquid when compared to the bulk, depending mainly on the strength of interaction at the interface. Furthermore, the mercury intrusion analysis of mesopores is shown not to be suitable for supported ionic liquids.

Photo-CIDNP 13C magic angle spinning NMR on bacterial reaction centres: exploring the electronic structure of the special pair and its surroundings.

Photochemically induced dynamic nuclear polarisation (photo-CIDNP) in intact bacterial reaction centres has been observed by 13C-solid state NMR under continuous illumination with white light. Strong intensity enhancement of 13C NMR signals of the aromatic rings allows probing the electronic ground state of the two BChl cofactors of the special pair at the molecular scale with atomic selectivity. Differences between the two BChl cofactors are discussed. Several aliphatic 13C atoms of cofactors, as well as 13C atoms of the imidazole ring of histidine residue(s), show nuclear-spin polarisation to the same extent as the aromatic nuclei of the cofactors. Mechanisms and applications of polarisation transfer are discussed.

Effect of proline on the production of singlet oxygen.

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 20mM. 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.

Ultrahigh field MAS NMR dipolar correlation spectroscopy of the histidine residues in light-harvesting complex II from photosynthetic bacteria reveals partial internal charge transfer in the B850/His complex.

Low-temperature 15N and 13C CP/MAS (cross-polarization/magic angle spinning) NMR has been used to analyze BChl-histidine interactions and the electronic structure of histidine residues in the light-harvesting complex II (LH2) of Rhodopseudomonas acidophila. The histidines were selectively labeled at both or one of the two nitrogen sites of the imidazole ring. The resonances of histidine nitrogens that are interacting with B850 BChl a have been assigned. Specific 15N labeling confirmed that it is the tau-nitrogen of histidines which is ligated to Mg2+ of B850 BChl molecules (beta-His30, alpha-His31). The pi-nitrogens of these Mg2+-bound histidines were found to be protonated and may be involved in hydrogen bond interactions. Comparison of the 2-D MAS NMR homonuclear (13C-13C) dipolar correlation spectrum of [13C6,15N3]-histidines in the LH2 complex with model systems in the solid state reveals two different classes of electronic structures from the histidines in the LH2. In terms of the 13C isotropic shifts, one corresponds to the neutral form of histidine and the other resembles a positively charged histidine species. 15N-13C double-CP/MAS NMR data provide evidence that the electronic structure of the histidines in the neutral BChl a/His complexes resembles the positive charge character form. While the Mg...15N isotropic shift confirms a partial positive charge transfer, its anisotropy is essentially of the lone pair type. This provides evidence that the hybridization structure corresponding to the neutral form of the imidazole is capable of "buffering" a significant amount of positive charge.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680(.+) primary donor chlorophyll.

We report (13)C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo-CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance (13)C provide information on the electronic structure of the primary electron donor P(680) (chlorophyll a molecules absorbing around 680 nm) and on the p(z) spin density pattern in its oxidized form, P(680)(.+). Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P(680)(.+). PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P(680) by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Induction of photochemical auto-reduction of cytochrome-c oxidase by an organic peroxide.

Cytochrome-c oxidase aa3 (CcO) from Paracoccus denitrificans interacts with tertiary butyl hydroperoxide (t-Bu-O-O-H, TBHP) by forming an adduct as indicated by an absorption shift at 408/432 nm and the induction of photochemical autoreduction. The adduct was stable at room temperature for several days even under aerobic conditions. Upon irradiation (413 nm) of the adduct, a photoproduct, similar to the oxygenated mixed valence species (607 nm form), was formed, as indicated by the 418/442 and 607 nm signals in the absorption-difference spectrum. It is concluded that the adduct formation changes the photochemical properties of heme a3. A molecular model for the binding mechanism of TBHP to CcO and for the photochemistry of heme a3-TBHP adduct is proposed.

Exploring the calcium-binding site in photosystem II membranes by solid-state (113)Cd NMR.

Calcium (Ca(2+)) is an essential cofactor for photosynthetic oxygen evolution. Although the involvement of Ca(2+) at the oxidizing side of photosystem II of plants has been known for a long time, its ligand interactions and mode of action have remained unclear. In the study presented here, (113)Cd magic-angle spinning solid-state NMR spectroscopy is used to probe the Ca(2+)-binding site in the water-oxidizing complex of (113)Cd(2+)-substituted PS2. A single NMR signal 142 ppm downfield from Cd(ClO(4))(2).2H(2)O was recorded from Cd(2+) present at the Ca(2+)-binding site. The anisotropy of the signal is small, as indicated by the absence of spinning side bands. The signal intensity is at its maximum at a temperature of -60 degrees C. The line width of the proton signal in a WISE (wide-line separation) two-dimensional (1)H-(113)Cd NMR experiment demonstrates that the signal arises from Cd(2+) in a solid and magnetically undisturbed environment. The chemical shift, the small anisotropy, and the narrow line of the (113)Cd NMR signal provide convincing evidence for a 6-fold coordination, which is achieved partially by oxygen and partially by nitrogen or chlorine atoms in otherwise a symmetric octahedral environment. The absence of a (113)Cd signal below -70 degrees C suggests that the Ca(2+)-binding site is close enough to the tetramanganese cluster to be affected by its electron spin state. To our knowledge, this is the first report for the application of solid-state NMR in the study of the membrane-bound PS2 protein complex.

A set-up to study photochemically induced dynamic nuclear polarization in photosynthetic reaction centres by solid-state NMR.

Recently, solid-state NMR spectroscopy became a viable method to investigate photosynthetic reaction centres (RCs) on the atomic level. To study the electronic structure of the radical cation state of the RC, occurring after the electron emission, solid-state NMR using an illumination set-up can be exploited. This paper describes the illumination set-up we designed for a standard Bruker wide-bore MAS NMR probe. In addition we demonstrate its application to get information from the active site in photosynthetic reaction centres of Rhodobacter sphaeroides R-26 by photochemically induced dynamic nuclear polarization (photo-CIDNP). Solid-state NMR spectra of natural abundance 13C in detergent solubilized quinone depleted photosynthetic reaction centres under continuous illumination showed exceptionally strong nuclear spin polarization in NMR lines. Both enhanced-absorptive and emissive polarization were seen in the carbon spectrum which could be assigned to a bacteriochlorophyll a (BChl a) cofactor, presumably the special pair BChl a. The sign and intensities of the 13C NMR signals provide information about the electron spin density distribution of the transiently formed radical P.+ on the atomic level.

Raman spectroscopic analysis of isomers of biliverdin dimethyl ester.

The constitutional isomers of biliverdin dimethyl ester, IX alpha and XIII alpha, were studied by resonance Raman spectroscopy. The far-reaching spectral similarities suggest that despite the different substitution patterns, the compositions of the normal modes are closely related. This conclusion does not hold only for the parent state (ZZZ, sss configuration) but also for the configurational isomers which were obtained upon double-bond photoisomerization. Based on a comparison of the resonance Raman spectra, a EZZ configuration is proposed for one of the two photoisomers of biliverdin dimethyl ester IX alpha, while a ZZE, ssa configuration has been assigned previously to the second isomer.

Fourier-transform resonance Raman spectroscopy of intermediates of the phytochrome photocycle.

The parent states of the 124-kDa phytochrome (phy A from Avena sativa) and intermediates of its photocycle were studied by low-temperature Fourier-transform resonance Raman spectroscopy. Spectra of the primary photoproducts I700 and lumi-F and of the thermal intermediate meta-F have been obtained for the first time. The spectra of the stable photochromic forms of photochrome, Pr and Pfr, presented in this work are significantly better in signal-to-noise ratio and resolution than previously published spectra, demonstrating the distinct advantages of our experimental approach. The high spectral quality allows for the identification of subtle details of the vibrational band pattern so that the resonance Raman spectra, which have been measured from samples in H2O and D2O, constitute a solid basis for the structural analysis of the various forms of phytochrome. Notwithstanding the current uncertainty in the vibrational assignment of many resonance Raman bands, the spectral changes of the tetrapyrrole chromophore can plausibly be interpreted in terms of conformational changes at two different methine bridges, i.e., torsions around two single bonds and the E/Z isomerization of a double bond. Within the framework of this interpretation, which is based on a vibrational analysis of biliverdin dimethyl ester (Smith, K. Matysik, J., Hlldebrandt, P., & Mark, F. (1993) J. Phys. Chem. 97, 11887-11900), a consistent model is proposed to describe the molecular events in the chromophore during the photocycle. The involvement of a proton transfer in the primary photoprocess of Pr can safely be ruled out. However, previous conclusions concerning the chromophore protonation in the individual states appear premature at the present state of the vibrational assignment. In particular, the attribution of a broad band at 1100 cm-1 to the N-H out-of-plane bending of the protonated pyrrolenin nitrogen (Hildebrandt, P., Hoffmann, A., Lindemann, P., Heibel, G., Braslavsky, S. E., Schaffner, K., & Schrader, B. (1992) Biochemistry 32, 7957-7962) has now been found to be erroneous.

Raman spectroscopic study of the blue copper protein halocyanin from Natronobacterium pharaonis.

Halocyanin, a blue copper protein from Natronobacterium pharaonis was studied by Raman spectroscopic techniques. Near-infrared Fourier transform Raman spectra, which for the first time have been employed to study copper proteins in both oxidation states, display the Raman bands of the protein and, in the oxidized state, also the preresonance-enhanced bands of the copper center. The frequency of the amide I band at 1676 cm-1 indicates a predominant beta-sheet protein structure, which is typical for small blue copper proteins. The Raman spectra provide no evidence for major redox-linked changes of the secondary structure. Resonance Raman spectra of the oxidized protein obtained upon excitation close to the 600-nm absorption band were measured in the pH range between 7.7 and 4.5. The vibrational band pattern in the Cu-cysteine stretching region is closely related to that of azurin, indicating far-reaching similarities of the coordination geometry of the copper center in both proteins. Significantly lower frequencies, however, are noted for the Cu-histidine stretches, which appear as a closely spaced doublet at ca. 260 cm-1. Lowering the pH to 4.5 leads to an increase of this band splitting with one component shifting down to 247 cm-1. This downshift is attributed to the rupture of a hydrogen bond between one of the histidine ligands and a nearby carboxyl group, which becomes protonated at such a low pH. On the other hand, no major changes in the Cu-cysteine stretching region are noted at pH 4.5, implying that the coordination geometry remains largely unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic voltammetric determination of adrenaline in the presence of copper(II) ions.

Low concentrations of adrenaline (10(-5)-10(-4)M) have been found to give rise to quite marked current peaks in the cyclic voltammograms recorded for ammoniacal solutions containing a copper(II) salt at around 10(-4)M. Fast sweep-rates (20 V/sec) are generated by a cathode-ray polarograph and applied to a DME. Peak heights are proportional to the adrenaline concentration, the procedure is rapid and simple, and the reproducibility is good.