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Composed of the two bacteriochlorophyll cofactors, [P.sub.L] and [P.sub.M], the special pair functions as the primary electron donor in bacterial reaction centers of purple bacteria of Rhodobacter sphaeroides. Under light absorption, an electron is transferred to a bacteriopheophytin and a radical pair is produced. The occurrence of the radical pair is linked to the production of enhanced nuclear polarization called photochemically induced dynamic nuclear polarization (photo-CIDNP). This effect can be used to study the electronic structure of the special pair at atomic resolution by detection of the strongly enhanced nuclear polarization with laser-flash photo-CIDNP magic-angle spinning NMR on the carotenoid-less mutant R26. In the electronic ground state, PL is strongly disturbed, carrying a slightly negative charge. In the radical cation state, the ratio of total electron spin densities between [P.sub.L] and [P.sub.M] is 2:1, although it is 2.5:1 for the pyrrole carbons, 2.2:1 for all porphyrinic carbons, and 4:1 for the pyrrole nitrogen. It is shown that the symmetry break between the electronic structures in the electronic ground state and in the radical cation state is an intrinsic property of the special pair supermolecule, which is particularly attributable to a modification of the structure of [P.sub.L]. The significant difference in electron density distribution between the ground and radical cation states is explained by an electric polarization effect of the nearby histidine. electron transfer | nuclear polarization | photosynthesis | solid-state NMR | electronic structure doi/10.1073/pnas.0908608106

Both thermally stable states of phytochrome, Pr and Pfr, have been studied by [sup.13]C and [sup.15]N cross-polarization (CP) magic-angle spinning (MAS) NMR using cyanobacterial (Cphl) and plant (phyA) phytochrome sensory modules containing uniformly [sup.13]C and [sup.15]N-labeled bilin chromophores. Two-dimensional homo- and heteronudear experiments allowed most of the [sup.13]C chemical shifts to be assigned in both states. Chemical shift differences reflect changes of the electronic structure of the cofactor at the atomic level as well as its interactions with the chromophore-binding pocket. The chromophore in cyanobacterial and plant phytochromes shows very similar features in the respective Pr and Pfr states. The data are interpreted in terms of a strengthened hydrogen bond at the ring D carbonyl. The red shift in the Pfr state is explained by the increasing length of the conjugation network beyond ring C including the entire ring D. Enhanced conjugation within the [pi]-osystem stabilizes the more tensed chromophore in the Pfr state. Concomitant changes at the ring C propionate carboxylate and the ring D carbonyl are explained by a loss of hydrogen bonding to Cph1-His-290 and transmittance of conformational changes to the ring C propionate via a water network. These and other conformational changes may lead to modified surface interactions, e.g., along the tongue region contacting the bilin chromophore. photornorphogenesis | photoreceptors | chromophore-protein interaction phycocyanobilin | solid-state NMR

DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or [NAD.sup.+] and assistance of a divalent metal cofactor [Mg.sup.2+]. Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover [sup.31]p solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged [Mg.sup.2+], and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4-5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations. chemical movie | nucleotidyl transfer | structural reaction kinetics | time-resolved cryo-magic-angle-spinning NMR | transition state

In natural photosynthesis, the two photosystems that operate in series to drive electron transport from water to carbon dioxide are quite similar in structure and function, but operate at widely different potentials. In both systems photochemistry begins by photo-oxidation of a chlorophyll a, but that in photosystem II (PS2) has a 0.7 eV higher midpoint potential than that in photosystem I (PS1), so their electronic structures must be very different. Using reaction centers from [sup.15]N-labeled spinach, these electronic structures are compared by their photochemically induced dynamic nuclear polarization (photo-CIDNP) in magic-angle spinning (MAS) NMR measurements. The results show that the electron spin distribution in PS1, apart from its known delocalization over 2 chlorophyll molecules, reveals no marked disturbance, whereas the pattern of electron spin density distribution in PS2 is inverted in the oxidized radical state. A model for the donor of PS2 is presented explaining the inversion of electron spin density based on a tilt of the axial histidine toward pyrrole ring IV causing [pi]-[pi] overlap of both aromatic systems. photosynthesis | photosystem I | solid-state NMR | electron transfer | redox potential

A study demonstrates that photo-CIDNP MAS NMR is a method to study selectively the electronic structure of photosynthetic cofactors at the molecular and atomic resolution as well as in an intact cell at natural abundance. The method when employed in combination with [13.super.C]-isotope is expected to further increase the signal by a factor of 100 to a total enhancement factor of a million.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is observed in photosystem I of spinach by (super 13)C magic angle spinning solid-state NMR under continuous illumination with white light. The findings show since all light-induced (super 13)C NMR signals appear to be emissive, a predominance of the three-spin mixing mechanism over the differential decay mechanism is proposed.

Two sets of resonances were observed for carbons and nitrogens by 2-D NMR measurements and the two major fractions denoted by types A and B are identified. It 8is proposed that the CH2Cl2 precipitate represents a structural intermediate between the antiparallel dimer and the parallel stack as found in the chlorosome.

Analysis of the ground-state electronic structures of the 'special pair' bacteriochlorophyll dimer reveals three bacteriochlorophyll species with comparable chemical shifts and one bacteriochlorophyll exhibiting a different shift. Data indicate that the structural changes of the special pair do not impact reorganization energy associated with the charge-transfer process.

Histidine residues in light harvesting complex from photosynthetic bacterial were studied using ultrahigh field MAS NMR dipolar correlation spectroscopy.

Research has been conducted on calcium (Ca (super)2+). The use of the cadmium ((super)113 Cd) magic-angle spinning solid-state NMR spectroscopy in probing Ca (super)2+ -binding site is discussed.

The low-temperature Fourier-transform resonance Raman spectrum of the intermediary forms Pr and Pfr of the phytochrome is given and a mechanism is proposed for the changes in the phytochrome during the photocycle. The signal-to-noise ratio and the resolution of the spectrum obtained by this method is better than that obtained by other methods. The interconversion of these forms is by the absorption of light and involves the a Z to E isomerization in the methine C-D bridge and torsion around two single bonds. In Pr the configuration of the exocyclic bonds is ZZZ and in Pfr it is ZZE.

Resonance Raman spectroscopy and near-infrared Fourier transform Raman spectroscopy of the blue copper protein halocyanin in Natronobacterium haraonis reveal that the amide I band frequency at 1676 cm-1 exhibits the presence of a prominent beta-sheet protein configuration that is characteristic of blue copper proteins. The spectrum of the oxidized protein after excitation in the 4.5-7.7 pH range reveals the vibrational band patterns in the Cu-cystein segment. A downshift due to pH reduction to 4.5 occurs through the disruption of a hydrogen bond that undergoes protonation at low pH. The spectra also reveal the absence of major redox-linked secondary structure modifications.

[Super 13]C magic-angle spinning (MAS) NMR is applied to observe the solid-state-photo-CIDNP effect in a nonphotosynthetic system, the blue-light photoreceptor phototropin LOV1-C57S. The solid-state photo-CIDNP effect was observed selectively in photosynthetic systems.

Magic-angle spinning (MAS) NMR spectroscopy was used to understand the photochemically induced Pfr to Pr back-reaction at low temperature. Uniformly labeled [super 13]C- and [super 15]N-open-chain tetrapyrrole chromophores was used to structurally characterize all four states of Pfr, Lumi-F, Meta-F, and Pr pf cyanobacterial phytochrome Cph1.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is seen in membrane fragments of heliobacterium Heliobacillus mobilis without further isolation by [super 13]C magic-angle spinning (MAS) solid-state NMR under continuous illumination with white light. The occurrence of the solid-state photo-CIDNP effect is shown to be a general feature of primary radical pairs in natural photosynthesis.

The effect of [super 13]C photochemically induced dynamic nuclear polarization (photo-CIDNP) in the solid state on the recovery rate of the signal is examined. In reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroids R26, the photochemical active channels have allowed for the accelerated signal scanning by a factor of 5 and hence photo-CIDNP MAS NMR has provided the possibility to drive the NMR method beyond the [T.sub.1] limit.

A complete set of [super 13]C chemical shifts of the aromatic systems of all active BChl and BPhe cofactors is presented, thus allowing for a comprehensive map of the molecular electronic ground state of the photochemically active cofactors with atomic selectivity. The electronic structure of two BChl cofactors forming the special pair in reaction centers (RCs) of Rhodobacter sphaeroides is distinguished in their electronic ground state and the primary acceptor BPhe has appeared to be relatively undisturbed.

Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy is used to investigate the self-organization of bacteriochlorophylls in chlorosomal light-harvesting antennae. The results reveal a balance between relatively strong local interactions and contributions to the free energy of the system associated with a longer length scale.

We report [sup.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 [sup.13]C provide information on the electronic structure of the primary electron donor [P.sub.680] (chlorophyll a molecules absorbing around 680 nm) and on the [p.sub.z] spin density pattern in its oxidized form, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. 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.sub.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.

MAS NMR, which is refined with molecular modeling, is used for obtaining the detailed structural model of microcrystalline Cd-bacteriochlorophyll d analogue structure. Results indicate that the structure determined for Cd-bacteriochlorophyll d analogue compares well with structures of well-ordered chlorophyllide crystals determined using X-ray diffraction.

Studies BChl-histidine interactions and the electronic structure of histidine residues in the light-harvesting complex II of Rhodopseudomonas acidophila using nuclear magnetic resonance measurements. Homonuclear dipolar correlation spectrum; Hybridization structure corresponding to the neutral form of the imidazole.

Explores the calcium(Ca)-binding site in photosystem II membranes using a solid-state 113 cadmium (Cd) nuclear magnetic resonance spectroscopy. Identification of Ca2+ ligands and ligand geometry of coordination; Ability of Cd2+ to compete with Ca2+ for the binding site in oxygen-evolving activity; Evidence of chemical shift of Cd2+ signal.