Hydrostatic High-Pressure-Induced Denaturation of LH2 Membrane Proteins.

The denaturation of globular proteins by high pressure is frequently associated with the release of internal voids and/or the exposure of the hydrophobic protein interior to a polar aqueous solvent. Similar evidence with respect to membrane proteins is not available. Here, we investigate the impact of hydrostatic pressures reaching 12 kbar on light-harvesting 2 integral membrane complexes of purple photosynthetic bacteria using two types of innate chromophores in separate strategic locations: bacteriochlorophyll-a in the hydrophobic interior and tryptophan at both protein-solvent interfacial gateways to internal voids. The complexes from mutant Rhodobacter sphaeroides with low resilience against pressure were considered in parallel with the naturally robust complexes of Thermochromatium tepidum. In the former case, a firm correlation was established between the abrupt blue shift of the bacteriochlorophyll-a exciton absorption, a known indicator of the breakage of tertiary structure pigment-protein hydrogen bonds, and the quenching of tryptophan fluorescence, a supposed result of further protein solvation. No such effects were observed in the reference complex. While these data may be naively taken as supporting evidence of the governing role of hydration, the analysis of atomistic model structures of the complexes confirmed the critical part of the structure in the pressure-induced denaturation of the membrane proteins studied.

Establishment of the Qy Absorption Spectrum of Chlorophyll a Extending to Near-Infrared.

A weak absorption tail related to the Qy singlet electronic transition of solvated chlorophyll a is discovered using sensitive anti-Stokes fluorescence excitation spectroscopy. The quasi-exponentially decreasing tail was, at ambient temperature, readily observable as far as -2400 cm-1 from the absorption peak and at relative intensity of 10-7. The tail also weakened rapidly upon cooling the sample, implying its basic thermally activated nature. The shape of the spectrum as well as its temperature dependence were qualitatively well reproduced by quantum chemical calculations involving the pigment intramolecular vibrational modes, their overtones, and pairwise combination modes, but no intermolecular/solvent modes. A similar tail was observed earlier in the case of bacteriochlorophyll a, suggesting generality of this phenomenon. Long vibronic red tails are, thus, expected to exist in all pigments of light-harvesting relevance at physiological temperatures.

Absorption-emission symmetry breaking and the different origins of vibrational structures of the 1Qy and 1Qx electronic transitions of pheophytin a.

The vibrational structure of the optical absorption and fluorescence spectra of the two lowest-energy singlet electronic states (Qy and Qx) of pheophytin a were carefully studied by combining low-resolution and high-resolution spectroscopy with quantum chemical analysis and spectral modeling. Large asymmetry was revealed between the vibrational structures of the Qy absorption and fluorescence spectra, integrally characterized by the total Huang-Rhys factor and reorganization energy in absorption of Svib A = 0.43 ± 0.06, λA = 395 cm-1 and in emission of Svib E = 0.35 ± 0.06, λE = 317 cm-1. Time-dependent density-functional theory using the CAM-B3LYP, ωB97XD, and MN15 functionals could predict and interpret this asymmetry, with the exception of one vibrational mode per model, which was badly misrepresented in predicted absorption spectra; for CAM-B3LYP and ωB97XD, this mode was a Kekulé-type mode depicting aromaticity. Other computational methods were also considered but performed very poorly. The Qx absorption spectrum is broad and could not be interpreted in terms of a single set of Huang-Rhys factors depicting Franck-Condon allowed absorption, with Herzberg-Teller contributions to the intensity being critical. For it, CAM-B3LYP calculations predict that Svib A (for modes >100 cm-1) = 0.87 and λA = 780 cm-1, with effective x and y polarized Herzberg-Teller reorganization energies of 460 cm-1 and 210 cm-1, respectively, delivering 15% y-polarized intensity. However, no method was found to quantitatively determine the observed y-polarized contribution, with contributions of up to 50% being feasible.

Higher Order Vibronic Sidebands of Chlorophyll a and Bacteriochlorophyll a for Enhanced Excitation Energy Transfer and Light Harvesting.

Optical absorption and fluorescence spectra of molecules in condensed phases often show extensive sidebands. Originating from electron-vibrational and electron-phonon couplings, these spectral tails bear important information on the dynamics of electronic states and processes the molecules are involved in. The vibronic sidebands observed in conjugate Qy absorption and fluorescence spectra of chlorophyll a and bacteriochlorophyll a are relatively weak, characterized by the total Huang-Rhys factor which is less than one. Therefore, it is widely considered that only fundamental intramolecular modes are responsible for their formation. Here, we provide evidence for extra-long and structured fluorescence tails of chlorophyll a and bacteriochlorophyll a as far as 4000 cm-1 from respective spectral origins, far beyond the frequency range of fundamental modes. According to quantum chemical simulations, these sidebands extending to ∼960 nm in chlorophyll a and ∼1140 nm in bacteriochlorophyll a into the infrared part of the optical spectrum are mainly contributed to by vibrational overtones of the fundamental modes. Because energy transfer and relaxation processes generally depend on vibronic overlap integrals, these findings potentially contribute to better understanding of many vital photo-induced phenomena, including photosynthetic light harvesting.

Controlling Photosynthetic Excitons by Selective Pigment Photooxidation.

As a basis of photosynthesis, photoinduced oxidation of (bacterio)chlorophyll molecules in the special reaction center complexes has been a subject of extensive research. In contrast, the generally harmful photooxidation of antenna chromoproteins has received much less attention. Here, we have established the permanent structural changes in the LH2 antenna bacteriochlorophyll-protein complex from a sulfur photosynthetic purple bacterium Ectothiorhodospira haloalkaliphila taking place at physiological conditions upon intense optical irradiation. To this end, a crystal structure of the LH2 complex from E. haloalkaliphila was first resolved by X-ray diffraction to 3.7 Å, verifying a great similarity with the earlier structure from Phaesporillum molischianum. Analysis of the various steady-state and picosecond time-resolved optical spectroscopy data and related model simulations then confirmed that the major spectral effects observed-bleaching and blue-shifting of the B850 exciton band and correlated emergence of a higher-energy C700 exciton band-are associated with photooxidation of increasing numbers of B850 bacteriochlorophylls into 3-acetyl-chlorophylls, with no noticeable damage to the pigment-binding protein scaffold. A prospective noninvasive method for an in situ optical control of excitons by selective photooxidation of pigment chromophores was thus revealed and demonstrated in a structurally well-defined native system.

Vibronic Origin of the Qy Absorption Tail of Bacteriochlorophyll a Verified by Fluorescence Excitation Spectroscopy and Quantum Chemical Simulations.

The long-wavelength tail of the lowest-energy Qy singlet absorption band of bacteriochlorophyll a in triethylamine peaking at 768.6 nm was examined by means of fluorescence excitation spectroscopy at ambient temperature of 22 ± 1 °C. The tail, usually considered a Gaussian, does in fact weaken quasi-exponentially, being clearly evident as far as 940 nm, nearly 2400 cm-1 (∼12 kBT) away from the absorption peak. Quantum chemical simulations identified vibronic transitions from the thermally populated normal modes and their overtones in the ground electronic state as the origin of this tail. Because energy transfer and relaxation processes generally depend on vibronic overlap integrals, these findings may have important implications on the interpretation of numerous photoinduced phenomena that involve bacteriochlorophyll and similar molecules, including photosynthesis.

Superchiral Pd3 L6 Coordination Complex and Its Reversible Structural Conversion into Pd3 L3 Cl6 Metallocycles.

Large, non-symmetrical, inherently chiral bispyridyl ligand L derived from natural ursodeoxycholic bile acid was used for square-planar coordination of tetravalent Pd(II) , yielding the cationic single enantiomer of superchiral coordination complex 1 Pd3 L6 containing 60 well-defined chiral centers in its flower-like structure. Complex 1 can readily be transformed by addition of chloride into a smaller enantiomerically pure cyclic trimer 2 Pd3 L3 Cl6 containing 30 chiral centers. This transformation is reversible and can be restored by the addition of silver cations. Furthermore, a mixture of two constitutional isomers of trimer, 2 and 2', and dimer, 3 and 3', can be obtained directly from L by its coordination to trans- or cis-N-pyridyl-coordinating Pd(II) . These intriguing, water-resistant, stable supramolecular assemblies have been thoroughly described by (1) H DOSY NMR, mass spectrometry, circular dichroism, molecular modelling, and drift tube ion-mobility mass spectrometry.

Subtle spectral effects accompanying the assembly of bacteriochlorophylls into cyclic light harvesting complexes revealed by high-resolution fluorescence spectroscopy.

We have observed that an assembly of the bacteriochloropyll a molecules into B850 and B875 groups of cyclic bacterial light-harvesting complexes LH2 and LH1, respectively, results an almost total loss of the intra-molecular vibronic structure in the fluorescence spectrum, and simultaneously, an essential enhancement of its phonon sideband due to electron-phonon coupling. While the suppression of the vibronic coupling in delocalized (excitonic) molecular systems is predictable, as also confirmed by our model calculations, a boost of the electron-phonon coupling is rather unexpected. The latter phenomenon is explained by exciton self-trapping, promoted by mixing the molecular exciton states with charge transfer states between the adjacent chromophores in the tightly packed B850 and B875 arrangements. Similar, although less dramatic trends were noted for the light-harvesting complexes containing chlorophyll pigments.