Electron spin echo electron-nuclear double resonance (ESE-ENDOR) experiments performed on a broad radical electron paramagnetic resonance (EPR) signal observed in photosystem II particles depleted of Ca2+ indicate that this signal arises from the redox-active tyrosine Yz. The tyrosine EPR signal width is increased relative to that observed in a manganese-depleted preparation due to a magnetic interaction between the photosystem II manganese cluster and the tyrosine radical. The manganese cluster is located asymmetrically with respect to the symmetry-related tyrosines Yz and YD. The distance between the Yz tyrosine and the manganese cluster is estimated to be approximately 4.5 A. Due to this close proximity of the Mn cluster and the redox-active tyrosine Yz, we propose that this tyrosine abstracts protons from substrate water bound to the Mn cluster.The photosystem II (PS II) component of the plant photosynthetic apparatus oxidizes water at an oxygen-evolving complex (OEC) that consists of a tetranuclear cluster of manganese ions along with essential cofactors calcium and chloride (1). The overall architecture of PS II shows homologies to the reaction centers of the purple non-oxygen-evolving bacteria, including the C2 symmetry found in these reaction centers (2-4). In PS II this symmetry appears to persist to the sites of two important tyrosine residues, Yz (tyrosine-161 of the Dl polypeptide, with residues designated for Synechocystis sp. 6803) and YD (tyrosine-160 of the D2 polypeptide) (5-7). The Yz tyrosine serves as an electron transfer intermediate between the chargeseparating chlorophyll moiety P680 and the manganese cluster of the OEC. The symmetry-related tyrosine YD, which is typically present as a dark-stable neutral radical (YD-), is bypassed in the fast electron transfer between P680 and the OEC. Whether the Mn cluster is located on the C2 symmetry axis has been the subject of much debate. Instead, the PS II symmetry may be broken at the OEC level, locating the Mn cluster closer to the active electron transfer intermediate Yz.Calcium depletion of PS II particles by NaCl/EGTA washing (8-10) or low-pH citrate treatment (11-13) eliminates oxygen-evolving activity. In such Ca2+-depleted PS II particles, illumination at a temperature of 273 K leads to the formation of a broad (130-180 G full width at half maximum) g = 2 EPR signal. Other treatments that block oxygen evolution such as acetate or fluoride incubation lead to similar signals upon such illumination, though the signal widths vary appreciably, depending on the details of the treatment and the resulting extrinsic polypeptide composition (14-18). The broad g = 2 signal is thought to arise from a radical center, with the large linewidth caused by a magnetic interaction with the Mn cluster (18,19). UV (19) and IR (20) absorption changes in PS II that are observed concomitantly with the formation of the radical have been interpreted to favor an oxidized histidine as the origin of the broad radical signal. Alternatively, on the basis of EPR s...

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.

A complex composed of presenilin (PS), nicastrin, PEN-2, and APH-1 is absolutely required for γ-secretase activity in vivo. Evidence has emerged to suggest a role for PS as the catalytic subunit of γ-secretase, but it has not been established that PS is catalytically active in the absence of associated subunits. We now report that bacterially synthesized, recombinant PS (rPS) reconstituted into liposomes exhibits γ-secretase activity. Moreover, an rPS mutant that lacks a catalytic aspartate residue neither exhibits reconstituted γ-secretase activity nor interacts with a transition-state γ-secretase inhibitor. Importantly, we demonstrate that rPS harboring mutations that cause early onset familial Alzheimer's disease (FAD) lead to elevations in the ratio of Aβ42 to Aβ40 peptides produced from a wild-type APP substrate and that rPS enhances the Aβ42∕ Aβ40 peptide ratio from FAD-linked mutant APP substrates, findings that are entirely consistent with the results obtained in in vivo settings. Thus, γ-secretase cleavage specificity is an inherent property of the polypeptide. Finally, we demonstrate that PEN2 is sufficient to promote the endoproteolysis of PS1 to generate the active form of γ-secretase. Thus, we conclusively establish that activated PS is catalytically competent and the bimolecular interaction of PS1 and PEN2 can convert the PS1 zymogen to an active protease.intermembrane-cleaving proteases | notch | presenilinase | reconstitution

The fabrication of de novo proteins able to self-assemble on the nano- to meso-length scales is critical in the development of protein-based biomaterials in nanotechnology and medicine. Here we report the design and characterization of a protein engineered coiled-coil that not only assembles into microfibers, but also can bind hydrophobic small molecules. Under ambient conditions, the protein forms fibers with nanoscale structure possessing large aspect ratios formed by bundles of α-helical homopentameric assemblies, which further assemble into mesoscale fibers in the presence of curcumin through aggregation. Surprisingly, these biosynthesized fibers are able to form in conditions of remarkably low concentrations. Unlike previously designed coiled-coil fibers, these engineered protein microfibers can bind the small molecule curcumin throughout the assembly, serving as a depot for encapsulation and delivery of other chemical agents within protein-based 3D microenvironments.