Dariusz M. Niedzwiedzki

Size and Structure Matter: Enhanced CO2 Photoreduction Efficiency by Size-Resolved Ultrafine Pt Nanoparticles on TiO2 Single Crystals

A facile development of highly efficient Pt-TiO(2) nanostructured films via versatile gas-phase deposition methods is described. The films have a unique one-dimensional (1D) structure of TiO(2) single crystals coated with ultrafine Pt nanoparticles (NPs, 0.5-2 nm) and exhibit extremely high CO(2) photoreduction efficiency with selective formation of methane (the maximum CH(4) yield of 1361 μmol/g-cat/h). The fast electron-transfer rate in TiO(2) single crystals and the efficient electron-hole separation by the Pt NPs were the main reasons attributable for the enhancement, where the size of the Pt NPs and the unique 1D structure of TiO(2) single crystals played an important role.

Phycobilisomes Supply Excitations to Both Photosystems in a Megacomplex in Cyanobacteria

Complexing Photosynthesis Photosynthesis operates through a series of protein complexes that harvest sunlight and turn it into chemical energy. The separate complexes—including photosystems I and II, phycobilisome antennae, and reaction centers—are understood for a number of photosynthetic organisms; however, the large-scale organization and interactions between them are less clear. Using protein cross-linking, Liu et al. (p. 1104) demonstrate how the individual components are organized when present as a megacomplex in the cyanobacterium Synechocystis PCC 6803. Time-resolved fluorescence spectroscopy indicated that the phycobilisomes transfer energy to both photosystems, which is consistent with their molecular arrangement. The organization of several protein complexes involved in photosynthesis elucidates their energy transfer mechanisms. In photosynthetic organisms, photons are captured by light-harvesting antenna complexes, and energy is transferred to reaction centers where photochemical reactions take place. We describe here the isolation and characterization of a fully functional megacomplex composed of a phycobilisome antenna complex and photosystems I and II from the cyanobacterium Synechocystis PCC 6803. A combination of in vivo protein cross-linking, mass spectrometry, and time-resolved spectroscopy indicates that the megacomplex is organized to facilitate energy transfer but not intercomplex electron transfer, which requires diffusible intermediates and the cytochrome b6f complex. The organization provides a basis for understanding how phycobilisomes transfer excitation energy to reaction centers and how the energy balance of two photosystems is achieved, allowing the organism to adapt to varying ecophysiological conditions.

Singlet and triplet excited state properties of natural chlorophylls and bacteriochlorophylls.

Ten naturally occurring chlorophylls (a, b, c2, d) and bacteriochlorophylls (a, b, c, d, e, g) were purified and studied using the optical spectroscopic techniques of both steady state and time-resolved absorption and fluorescence. The studies were carried out at room temperature in nucleophilic solvents in which the central Mg is hexacoordinated. The comprehensive studies of singlet excited state lifetimes show a clear dependency on the structural features of the macrocycle and terminal substituents. The wide-ranging studies of triplet state lifetime demonstrate the existence of an energy gap law for these molecules. The knowledge of the dynamics and the energies of the triplet state that were obtained in other studies allowed us to construct an energy gap law expression that can be used to estimate the triplet state energies of any (B)chlorophyll molecule from its triplet lifetime obtained in a liquid environment.

Molecular Mechanism of Photoactivation and Structural Location of the Cyanobacterial Orange Carotenoid Protein

The orange carotenoid protein (OCP) plays a photoprotective role in cyanobacterial photosynthesis similar to that of nonphotochemical quenching in higher plants. Under high-light conditions, the OCP binds to the phycobilisome (PBS) and reduces the extent of transfer of energy to the photosystems. The protective cycle starts from a light-induced activation of the OCP. Detailed information about the molecular mechanism of this process as well as the subsequent recruitment of the active OCP to the phycobilisome are not known. We report here our investigation on the OCP photoactivation from the cyanobacterium Synechocystis sp. PCC 6803 by using a combination of native electrospray mass spectrometry (MS) and protein cross-linking. We demonstrate that native MS can capture the OCP with its intact pigment and further reveal that the OCP undergoes a dimer-to-monomer transition upon light illumination. The reversion of the activated form of the OCP to the inactive, dark form was also observed by using native MS. Furthermore, in vitro reconstitution of the OCP and PBS allowed us to perform protein chemical cross-linking experiments. Liquid chromatography-MS/MS analysis identified cross-linking species between the OCP and the PBS core components. Our result indicates that the N-terminal domain of the OCP is closely involved in the association with a site formed by two allophycocyanin trimers in the basal cylinders of the phycobilisome core. This report improves our understanding of the activation mechanism of the OCP and the structural binding site of the OCP during the cyanobacterial nonphotochemical quenching process.

Ultrafast time-resolved carotenoid to-bacteriochlorophyll energy transfer in LH2 complexes from photosynthetic bacteria.

Steady-state and ultrafast time-resolved optical spectroscopic investigations have been carried out at 293 and 10 K on LH2 pigment-protein complexes isolated from three different strains of photosynthetic bacteria: Rhodobacter (Rb.) sphaeroides G1C, Rb. sphaeroides 2.4.1 (anaerobically and aerobically grown), and Rps. acidophila 10050. The LH2 complexes obtained from these strains contain the carotenoids, neurosporene, spheroidene, spheroidenone, and rhodopin glucoside, respectively. These molecules have a systematically increasing number of pi-electron conjugated carbon-carbon double bonds. Steady-state absorption and fluorescence excitation experiments have revealed that the total efficiency of energy transfer from the carotenoids to bacteriochlorophyll is independent of temperature and nearly constant at approximately 90% for the LH2 complexes containing neurosporene, spheroidene, spheroidenone, but drops to approximately 53% for the complex containing rhodopin glucoside. Ultrafast transient absorption spectra in the near-infrared (NIR) region of the purified carotenoids in solution have revealed the energies of the S1 (2(1)Ag-)-->S2 (1(1)Bu+) excited-state transitions which, when subtracted from the energies of the S0 (1(1)Ag-)-->S2 (1(1)Bu+) transitions determined by steady-state absorption measurements, give precise values for the positions of the S1 (2(1)Ag-) states of the carotenoids. Global fitting of the ultrafast spectral and temporal data sets have revealed the dynamics of the pathways of de-excitation of the carotenoid excited states. The pathways include energy transfer to bacteriochlorophyll, population of the so-called S* state of the carotenoids, and formation of carotenoid radical cations (Car*+). The investigation has found that excitation energy transfer to bacteriochlorophyll is partitioned through the S1 (1(1)Ag-), S2 (1(1)Bu+), and S* states of the different carotenoids to varying degrees. This is understood through a consideration of the energies of the states and the spectral profiles of the molecules. A significant finding is that, due to the low S1 (2(1)Ag-) energy of rhodopin glucoside, energy transfer from this state to the bacteriochlorophylls is significantly less probable compared to the other complexes. This work resolves a long-standing question regarding the cause of the precipitous drop in energy transfer efficiency when the extent of pi-electron conjugation of the carotenoid is extended from ten to eleven conjugated carbon-carbon double bonds in LH2 complexes from purple photosynthetic bacteria.

Biohybrid Photosynthetic Antenna Complexes for Enhanced Light-Harvesting

Biohybrid antenna systems have been constructed that contain synthetic chromophores attached to 31mer analogues of the bacterial photosynthetic core light-harvesting (LH1) β-polypeptide. The peptides are engineered with a Cys site for bioconjugation with maleimide-terminated chromophores, which include synthetic bacteriochlorins (BC1, BC2) with strong near-infrared absorption and commercial dyes Oregon green (OGR) and rhodamine red (RR) with strong absorption in the blue-green to yellow-orange regions. The peptides place the Cys 14 (or 6) residues before a native His site that binds bacteriochlorophyll a (BChl-a) and, like the native LH proteins, have high helical content as probed by single-reflection IR spectroscopy. The His residue associates with BChl-a as in the native LH1 β-polypeptide to form dimeric ββ-subunit complexes [31mer(-14Cys)X/BChl](2), where X is one of the synthetic chromophores. The native-like BChl-a dimer has Q(y) absorption at 820 nm and serves as the acceptor for energy from light absorbed by the appended synthetic chromophore. The energy-transfer characteristics of biohybrid complexes have been characterized by steady-state and time-resolved fluorescence and absorption measurements. The quantum yields of energy transfer from a synthetic chromophore located 14 residues from the BChl-coordinating His site are as follows: OGR (0.30) < RR (0.60) < BC2 (0.90). Oligomeric assemblies of the subunit complexes [31mer(-14Cys)X/BChl](n) are accompanied by a bathochromic shift of the Q(y) absorption of the BChl-a oligomer as far as the 850-nm position found in cyclic native photosynthetic LH2 complexes. Room-temperature stabilized oligomeric biohybrids have energy-transfer quantum yields comparable to those of the dimeric subunit complexes as follows: OGR (0.20) < RR (0.80) < BC1 (0.90). Thus, the new biohybrid antennas retain the energy-transfer and self-assembly characteristics of the native antenna complexes, offer enhanced coverage of the solar spectrum, and illustrate a versatile paradigm for the construction of artificial LH systems.

Identification of a single peridinin sensing Chl-a excitation in reconstituted PCP by crystallography and spectroscopy

The peridinin-chlorophyll a-protein (PCP) of dinoflagellates is unique among the large variety of natural photosynthetic light-harvesting systems. In contrast to other chlorophyll protein complexes, the soluble PCP is located in the thylakoid lumen, and the carotenoid pigments outnumber the chlorophylls. The structure of the PCP complex consists of two symmetric domains, each with a central chlorophyll a (Chl-a) surrounded by four peridinin molecules. The protein provides distinctive surroundings for the pigment molecules, and in PCP, the specific environment around each peridinin results in overlapping spectral line shapes, suggestive of different functions within the protein. One particular Per, Per-614, is hypothesized to show the strongest electronic interaction with the central Chl-a. We have performed an in vitro reconstitution of pigments into recombinant PCP apo-protein (RFPCP) and into a mutated protein with an altered environment near Per-614. Steady-state and transient optical spectroscopic experiments comparing the RFPCP complex with the reconstituted mutant protein identify specific amino acid-induced spectral shifts. The spectroscopic assignments are reinforced by a determination of the structures of both RFPCP and the mutant by x-ray crystallography to a resolution better than 1.5 Å. RFPCP and mutated RFPCP are unique in representing crystal structures of in vitro reconstituted light-harvesting pigment-protein complexes.

Spectroscopic Investigation of Peridinin Analogues Having Different π-Electron Conjugated Chain Lengths: Exploring the Nature of the Intramolecular Charge Transfer State

The lifetime of the lowest excited singlet (S(1)) state of peridinin and many other carbonyl-containing carotenoids and polyenes has been reported to depend on the polarity of the solvent. This effect has been attributed to the presence of an intramolecular charge transfer (ICT) state in the manifold of excited states for these molecules. The nature of this ICT state has yet to be elucidated. In the present work, steady-state and ultrafast time-resolved optical spectroscopy have been performed on peridinin and three synthetic analogues, C(33)-peridinin, C(35)-peridinin, and C(39)-peridinin, which have different numbers of conjugated carbon-carbon double bonds. Otherwise, the molecules are structurally similar in that they possess the same functional groups. The trends in the positions of the steady-state and transient spectral profiles for this systematic series of molecules allow an assignment of the spectral features to transitions involving the S(0), S(1), S(2), and ICT states. A kinetics analysis reveals the lifetimes of the excited states and the dynamics of their excited state deactivation pathways. The most striking observation in the data is that the lifetime of the ICT state converges to the same value of 10.0 +/- 2.0 ps in the polar solvent, methanol, for all the peridinin analogues, regardless of the extent of pi-electron conjugation. This suggests that the ICT state is highly localized on the lactone ring, which is a common structural feature in all the molecules. The data further suggest that the S(1) and ICT states behave independently and that the ICT state is populated from both S(1) and S(2), the rate and efficiency from S(1) being dependent on the length of the pi-electron chain of the carotenoid and the solvent polarity.

Ultrafast time-resolved spectroscopy of xanthophylls at low temperature.

Many of the spectroscopic features and photophysical properties of xanthophylls and their role in energy transfer to chlorophyll can be accounted for on the basis of a three-state model. The characteristically strong visible absorption of xanthophylls is associated with a transition from the ground state S0 (1(1)Ag-) to the S2 (1(1)Bu+) excited state. The lowest lying singlet state denoted S1 (2(1)Ag-), is a state into which absorption from the ground state is symmetry forbidden. Ultrafast optical spectroscopic studies and quantum computations have suggested the presence of additional excited singlet states in the vicinity of S1 (2(1)Ag-) and S2 (1(1)Bu+). One of these is denoted S* and has been suggested in previous work to be associated with a twisted molecular conformation of the molecule in the S1 (2(1)Ag-) state. In this work, we present the results of a spectroscopic investigation of three major xanthophylls from higher plants: violaxanthin, lutein, and zeaxanthin. These molecules have systematically increasing extents of pi-electron conjugation from nine to eleven conjugated carbon-carbon double bonds. All-trans isomers of the molecules were purified by high-performance liquid chromatography (HPLC) and studied by steady-state and ultrafast time-resolved optical spectroscopy at 77 K. Analysis of the data using global fitting techniques has revealed the inherent spectral properties and ultrafast dynamics of the excited singlet states of each of the molecules. Five different global fitting models were tested, and it was found that the data are best explained using a kinetic model whereby photoexcitation results in the promotion of the molecule into the S2 (1(1)Bu+) state that subsequently undergoes decay to a vibrationally hot S1 (1(1)Ag-) state and with the exception of violaxanthin also to the S* state. The vibrationally hot S1 (1(1)Ag-) state then cools to a vibrationally relaxed S1 (2(1)Ag-) state in less than a picosecond. It was also found that a portion of the S* population is converted into S1 (2(1)Ag-) during deactivation, but this process and the relative yield of S* was found to depend on temperature, consistent with it being associated with a twisted conformation of the xanthophyll. The results of the global fitting suggest that subpopulations of twisted conformers of xanthophylls already exist in the ground state prior to photoexcitation.

Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon–carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N = 10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2′-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC–LH1–PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2′-diketo-spirilloxanthin (15 conjugated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds; N = 15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N = 9; 94%), spheroidene (N = 10; 96%) and spheroidenone (N = 11; 95%), whereas intermediate values were measured for lycopene (N = 11; 64%), rhodopin (N = 11; 62%) and spirilloxanthin (N = 13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.