Structure and dynamics of photosystem II light-harvesting complex revealed by high-resolution FTICR mass spectrometric proteome analysis

Structure and dynamics of membrane-bound light-harvesting pigment-protein complexes (LHCs), which collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identified and characterized using high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LHCs from photosystem II (LHCII) were isolated from the thylakoid membrane of Arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel-filtration into LHCII subcomplexes. Using reversed-phase high-performance liquid chromatography and two-dimensional (2D) gel electrophoresis, the LHCII proteins, Lhcb1-6 and fibrillins, were efficiently separated and identified by FTICR-MS. Some of the LHCII subcomplexes were shown to migrate from photosystem II to photosystem I as a result of short-term adaptation to changes in light intensity. In the mobile LHCII subcomplexes, decreased levels of fibrillins and a modified composition of LHCII protein isoforms were identified compared to the tightly bound LHCII subcomplexes. In addition, FTICR-MS analysis revealed several oxidative modifications of LHCII proteins. A number of protein spots in 2D gels were found to contain a mixture of proteins, illustrating the feasibility of high-resolution mass spectrometry to identify proteins that remain unseparated in 2D gels even upon extended pH gradients.     

Biocatalytic organic synthesis of optically pure (S)-scoulerine and berbine and benzylisoquinoline alkaloids

A chemoenzymatic approach for the asymmetric total synthesis of the title compounds is described that employs an enantioselective oxidative C-C bond formation catalyzed by berberine bridge enzyme (BBE) in the asymmetric key step. This unique reaction yielded enantiomerically pure (R)-benzylisoquinoline derivatives and (S)-berbines such as the natural product (S)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates rac-1 required for the biotransformation were prepared in 4-8 linear steps using either a Bischler-Napieralski cyclization or a C1-Cα alkylation approach. The chemoenzymatic synthesis was applied to the preparation of fourteen enantiomerically pure alkaloids, including the natural products (S)-scoulerine and (R)-reticuline, and gave overall yields of up to 20% over 5-9 linear steps.     

A Bioorthogonal Chemical Reporter of Viral Infection

Pathogen‐selective labeling was achieved by using the novel gemcitabine metabolite analogue 2′‐deoxy‐2′,2′‐difluoro‐5‐ethynyluridine (dF‐EdU) and click chemistry. Cells infected with Herpes Simplex Virus‐1 (HSV‐1), but not uninfected cells, exhibit nuclear staining upon the addition of dF‐EdU and a fluorescent azide. The incorporation of the dF‐EdU into DNA depends on its phosphorylation by a herpes virus thymidine kinase (TK). Crystallographic analyses revealed how dF‐EdU is well accommodated in the active site of HSV‐1 TK, but steric clashes prevent dF‐EdU from binding human TK. These results provide the first example of pathogen‐enzyme‐dependent incorporation and labeling of bioorthogonal functional groups in human cells.     

Ternary Rare Earth Metal Palladium and Platinum Aluminides R4Pd9Al24 and R4Pt9Al24

The fifteen intermetallic compounds R₄Pd₉Al₂₄ (R = Gd–Tm) and R₄Pt₉Al₂₄ (R = Y, Gd–Lu) were prepared by reaction of the elemental components. Their crystal structure was determined from single-crystal X-ray data of Er₄Pt₉Al₂₄. It is pseudo-trigonal with triclinic symmetry: P 1, a = b = 747.5(2) pm, c = 1306.7(4) pm, α = 100.99(2)°, β = 95.47(2)°, γ = 60.00(3)°, Z = 1, R = 0.052 for 2593 structure factors and 110 variable parameters. The structure is closely related to that of Y₂Co₃Ga₉. Both may be described as stacking variants of each other. They consist of layers of the compositions PtAl₂ (CoGa₂), and Er₂Al₃ (Y₂Ga₃), designated A and B, respectively. These layers are stacked in the five- and four-layer sequences ABAAB (Er₄Pt₉Al₂₄) and ABAB (Y₂Co₃Ga₉). The layers PtAl₂ and CoGa₂ are similar to the hexagonal close packed layers in the TiSi₂-, CrSi₂-, and MoSi₂-type structures. The structure of Er₄Pt₉Al₂₄ contains a monoclinic subcell, where the layers Er₂Al₃ are disordered. A partial disorder of this kind, which could be ascribed to twinning or to the intergrowth with another stacking variant, was found during the structure refinement of the isotypic compound Y₄Pt₉Al₂₄: a = b = 749.0(2) pm. c = 1309.3(4) pm, α = 100.99(2)°, β = 95.48(2)°, γ = 60.00(3)°, R = 0.031 for 1435 structure factors and 128 variable parameters.     

INTERACTION OF HYDROXYLAMINE WITH THE WATER-OXIDIZING ENZYME INVESTIGATED VIA PROTON RELEASE*

Abstract— Photosynthetic water oxidation is a four-step redox process which is driven by a one-quantum-one-electron reaction center. Stepwise electron Abstract—ion from the water-oxidizing enzyme is accompanied by stepwise proton release with the following stoichiometric pattern at given half-rise times: 1 H⁺ (250 μs, S₀→ S₁):0 H⁺(S₁→ S₂): 1 H⁺ (200 μs, S₂→ S₃): 2 H⁺ (1.2 ms, S₃→ S₄→ S₀). (Förster and Junge, 1985, preceding article in this issue). Hydroxylamine at low concentrations (?100 μ M) appears to compete with water at the active site of the water- oxidizing enzyme. Its interference shifts the dark state of the water-oxidizing enzyme by two steps backwards (Bouges, 1971). We found that the hydroxylamine-induced shift was also reflected in the stoichiometric pattern and in the kinetics of proton-release. In the presence of hydroxylamine, two protons per reaction center were released with a half-rise time of ? 2 ms upon the first exciting flash given to dark adapted thylakoids. This was slower than observed for each of the protons released during unperturbed water oxidation. One proton was released upon the second flash. The half-rise time of the main component observed upon the second flash in hydroxylamine-treated samples agreed with the one observed upon the fourth flash in the absence of hydroxylamine, which had been attributed to the S₀→ S₁ transition. The two protons which were observed upon the first flash in hydroxylamine-treated thylakoids may be due to hydroxylamine oxidation or to the association of water to the catalytic manganese center after detachment of oxidized hydroxylamine from its binding site.     

Metal-free catalysts for the hydrolysis of RNA derived from guanidines, 2-aminopyridines, and 2-aminobenzimidazoles

2-aminopyridine and 2-aminobenzimidazole were chosen as structural analogues to substitute guanidinium groups in receptor molecules designed as phosphoryl transfer catalysts. Shifting the pKa of the guanidinium analogues toward 7 was expected to raise catalytic activities in aqueous buffer. Although the pKa's of both heterocycles are similar (6.2 and 7.0), only 2-aminobenzimidazole led to active RNA cleavers. All cleavage assays were run with fluorescently labeled substrates and a DNA sequencer. RNase contaminations would degrade RNA enantioselectively. In contrast, achiral catalysts such as 9b and 10b necessarily induce identical cleavage patterns in RNA and its mirror image. This principle allowed us to safely rule out contamination effects in this study. The most active catalysts, tris(2-aminobenzimidazoles) 9b and 10b, were shown by fluorescence correlation spectroscopy (FCS) to aggregate with oligonucleotides. However, at very low concentrations the compounds are still active in the nonaggregated state. Conjugates of 10b with antisense oligonucleotides or RNA binding peptides, therefore, will be promising candidates as site specific artificial ribonucleases.     

Consistent diffusion coefficients of ferrocene in some non-aqueous solvents: electrochemical simultaneous determination together with electrode sizes and comparison to pulse-gradient spin-echo NMR results

Cyclic voltammetry data recorded at disk macro- (millimeter dimension) and microelectrodes (10 and 100 μm) at various scan rates are used to simultaneously determine the diffusion coefficient D of ferrocene (fc) and the electroactive surfaces A and/or radii r of the electrodes. A case study with three electrodes of different sizes in CH₃CN- and propylene carbonate (PC)-based electrolytes shows the possibly large effect of incorrect D values. Diffusion coefficients of fc are determined for PC, CH₃CN, CH₂Cl₂, DMF, and DMSO electrolytes and (except for PC) compared to those from pulse-gradient spin-echo nuclear magnetic resonance experiments in the presence of supporting electrolyte in the respective deuterated solvents. The dependence of D _fc on solvent viscosity is shown to follow the Stokes–Einstein relation.