Effective and Selective Ru(II)-Arene Complexes Containing 4,4'-Substituted 2,2' Bipyridine Ligands Targeting Human Urinary Bladder Cancer Cells.

Cisplatin-based chemotherapy is a common regimen for bladder cancer, a life-threatening cancer with more than 500,000 new cases worldwide annually. Like many other metallodrugs, cisplatin causes severe side effects for its general toxicity. Organoruthenium is known for its structural stability, good anticancer activity, and possible low general toxicity. Here, we have prepared and characterized a series of water-soluble ruthenium-arene complexes with N,N'-chelating ligands: [Ru(II)-η6-arene-(4,4'-(X)2-2,2'-bipyridine)Cl]Cl (arene = p-cymene, X = C4H9 (1), COOH (2), COOCH3 (3), COOC2H5 (4); arene = benzene, X = C4H9 (5), COOCH3 (6), COOC2H5 (7)). These complexes are carefully characterized using single-crystal X-ray diffraction, UV-vis, IR, 1H NMR, and MALDI-TOF MS spectroscopy. Their DFT-calculated structural and thermodynamic properties are consistent with the experimental observations. Biophysicochemical studies of complex interaction with CTDNA and BSA supported by molecular docking simulations reveal suitable properties of 1-7 as anticancer agents. Cytotoxicities of 1-7 are evaluated on healthy human MCF-10a-breast epithelial and African green monkey Vero cells, and carcinoma human HepG-2-hepatic, T24-bladder, and EAhy-926-endothelial cells. All complexes exhibit much higher cytotoxicity for T24 than cisplatin. Particularly, 1 and 2 are also highly selective toward T24. Fluorescence imaging and flow cytometry demonstrate that 1 and 2 penetrate T24 cell membrane and induce early apoptosis at their respective IC50 concentrations, which ultimately lead to cell death. Statistical analysis suggests that the order of importance for T24 cell antiproliferation is protein binding, Log p, Ru-Cl bond length, while DNA binding is the least important. This study is the first to report the anti-bladder cancer efficacy of Ru-arene-2,2'-bipyridine complexes, and may provide insights for rational design of organoruthenium drugs in the enduring search for new chemotherapeutic agents.

The Nitrogen Atom of Vitamin B6 Is Essential for the Catalysis of Radical Aminomutases.

Radical aminomutases are pyridoxal 5'-phosphate (PLP, a B6 vitamer)-dependent enzymes that require the generation of a 5'-deoxyadenosyl radical to initiate the catalytic cycle, to perform a 1,2 amino group shift reaction. The role of the nitrogen atom of PLP in radical aminomutases has not been investigated extensively yet. We report an alternative synthetic procedure to provide easy access to 1-deazaPLP (dAPLP), an isosteric analog of PLP which acts as a probe for studying the role of the nitrogen atom. Our results revealed that lysine 5,6-aminomutase (5,6-LAM), a radical aminomutase, reconstituted with dAPLP cannot turn over a substrate, demonstrating that the nitrogen atom is essential for radical aminomutases. In contrast, biochemical and spectroscopic studies on the S238A variant reconstituted with PLP revealed a minuscule loss of activity. This apparent anomaly can be explained by a water-mediated rescue of activity in S238A, as if mimicking the active site of lysine 2,3-aminomutase. This study leads to a better comprehension of how enzymes harness the optimum capability of PLP to realize catalysis.

Impact of Nonideal Nanoparticles on X-ray Photoelectron Spectroscopic Quantitation: An Investigation Using Simulation and Modeling of Gold Nanoparticles.

Quantitative X-ray photoelectron spectroscopic (XPS) analysis combined with spectral modeling of photoelectrons can be valuable while investigating the surface chemistry of nanoparticles (NPs) with different morphologies. Herein, with the use of NIST Simulation of Electron Spectra for Surface Analysis (SESSA), a comparative analysis of experimental and simulated photoelectron peak intensities in gold nanoparticles (AuNPs) of different morphologies is presented. Three sets of supported AuNPs with different morphologies were selected from a series of as synthesized Au-TiO2 catalyst samples. Using transmission electron microscopy (TEM) analyzed morphological information on the AuNPs as input model parameters in SESSA, XPS spectra were generated from the respective input NP morphologies. A degree of greater mismatch between SESSA simulated and experimental XPS spectra was observed while using the TEM obtained average diameter of the nanoparticles. The degree of mismatch lowered when the true nonspherical shape of the nanoparticles as obtained from TEM images was taken into account for the simulation. This demonstrates the impact of surface morphology on the XPS peak intensities which needs to be incorporated to obtain precise quantified information from the supported nanoparticles. This work demonstrates the applicability of SESSA in combination with experimental XPS and TEM measurements for precise quantification of XPS spectra from complex, nonideal shaped nanoparticles. This study can be extended to include a broad range of nanoparticles with ideal or nonideal geometries, thus providing a simple method to utilize quantitative XPS analysis to a wide range of nanomaterials.

Evidence of Diradicals Involved in the Yeast Transketolase Catalyzed Keto-Transferring Reactions.

Transketolase (TK) catalyzes a reversible transfer of a two-carbon (C2 ) unit between phosphoketose donors and phosphoaldose acceptors, for which the group-transfer reaction that follows a one- or two-electron mechanism and the force that breaks the C2"-C3" bond of the ketose donors remain unresolved. Herein, we report ultrahigh-resolution crystal structures of a TK (TKps) from Pichia stipitis in previously undiscovered intermediate states and support a diradical mechanism for a reversible group-transfer reaction. In conjunction with MS, NMR spectroscopy, EPR and computational analyses, it is concluded that the enzyme-catalyzed non-Kekulé diradical cofactor brings about the C2"-C3" bond cleavage/formation for the C2 -unit transfer reaction, for which suppression of activation energy and activation and destabilization of enzymatic intermediates are facilitated.

Magnetic field effects on coenzyme B12- and B6-dependent lysine 5,6-aminomutase: switching of the J-resonance through a kinetically competent radical-pair intermediate.

The environmental magnetic field is beneficial to migratory bird navigation through the radical-pair mechanism. One of the continuing challenges in understanding how magnetic fields may perturb biological processes is that only a very few field-sensitive examples have been explored despite the prevalence of radical pairs in enzymatic reactions. We show that the reaction of adenosylcobalamin- and pyridoxal-5'-phosphate-dependent lysine 5,6-aminomutase proceeds via radical-pair intermediates and is magnetic field dependent. The 5'-deoxyadenosyl radical from adenosylcobalamin abstracts a C5(H) from the substrate to yield a {cob(ii)alamin - substrate} radical pair wherein the large spin-spin interaction (2J = 8000 gauss) locks the radical pair in a triplet state, as evidenced by electron paramagnetic resonance spectroscopy. Application of an external magnetic field in the range of 6500 to 8500 gauss triggers intersystem crossing to the singlet {cob(ii)alamin - substrate} radical-pair state. Spin-conserved H back-transfer from deoxyadenosine to the substrate radical yields a singlet {cob(ii)alamin-5'-deoxyadenosyl} radical pair. Spin-selective recombination to adenosylcobalamin decreased the enzyme catalytic efficiency kcat/Km by 16% at 7600 gauss. As a mechanistic probe, observation of magnetic field effects successfully demonstrates the presence of a kinetically significant radical pair in this enzyme. The study of a pronounced high-field level-crossing characteristic through an immobilized radical pair with a constant exchange interaction deepens our understanding of how a magnetic field may interact with an enzyme.

Mutations of Cytochrome b559 and PsbJ on and near the QC Site in Photosystem II Influence the Regulation of Short-Term Light Response and Photosynthetic Growth of the Cyanobacterium Synechocystis sp. PCC 6803.

The characteristic features of two types of short-term light adaptations of the photosynthetic apparatus of the cyanobacterium Synechocystis sp. PCC 6803, state transition and blue-green light-induced fluorescence quenching, were compared in wild-type and cytochrome b559 and PsbJ mutant cells with mutations on and near the QC site in photosystem II (PSII). All mutant cells grew photoautotrophically and assembled stable PSII. Thermoluminescence emission experiments showed a decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the A16FJ, S28Aß, and V32Fß mutant cells. When dark-adapted wild-type and mutant cells were illuminated by medium-intensity blue light, the increase in the PSII fluorescence yield (indicating a transition to state 1) was more prominent in mutant than wild-type cells. Strong blue-light conditions induced a quenching of fluorescence corresponding to nonphotochemical fluorescence quenching (NPQ). The extension of NPQ decreased significantly in the mutants, and the kinetics appeared to be affected. When similar measures were repeated on an orange carotenoid protein (OCP)-deficient background, little or no quenching was observed, which confirms that the decrease in fluorescence under strong blue light corresponded to the OCP-dependent NPQ. Immunoblot results showed that the attenuated effect of blue light-induced NPQ in mutant cells was not due to a lack of OCP. Photosynthetic growth and biomass production were greater for A16FJ, S28Aß, and V32Fß mutant cells than for wild-type cells under normal growth conditions. Our results suggest that mutations of cytochrome b559 and PsbJ on and near the QC site of PSII may modulate the short-term light response in cyanobacteria.

4'-CyanoPLP presents better prospect for the experimental detection of elusive cyclic intermediate radical in the reaction of lysine 5,6-aminomutase.

The results of our calculations suggest that the reaction of 4'-cyanoPLP with lysine 5,6-aminomutase offers better prospect for the experimental detection of elusive cyclic azacyclopropylcarbinyl radical (I), which is proposed to be a key intermediate in the reaction of pyridoxal-5'-phosphate dependent radical aminomutases. We have calculated the corresponding hyperfine coupling constants (HFCCs) for (14)N and (13)C of cyano group using several basis sets to help the characterization of 4'-cyanoI.

Spectroscopic and functional characterization of cyanobacterium Synechocystis PCC 6803 mutants on the cytoplasmic-side of cytochrome b559 in photosystem II.

We performed spectroscopic and functional characterization on cyanobacterium Synechocystis PCC6803 with mutations of charged residues of the cytoplasmic side of cytochrome (Cyt) b559 in photosystem II (PSII). All of the mutant cells grew photoautotrophically and assembled stable PSII. However, R7Eα, R17Eα and R17Lß mutant cells grew significantly slower and were more susceptible to photoinhibition than wild-type cells. The adverse effects of the arginine mutations on the activity and the stability of PSII were in the following order (R17Lß>R7Eα>R17Eα and R17Aα). All these arginine mutants exhibited normal period-four oscillation in oxygen yield. Thermoluminescence characteristics indicated a slight decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the R7Eα and R17Lß mutant cells. R7Eα and R17Lß PSII core complexes contained predominantly the low potential form of Cyt b559. EPR results indicated the displacement of one of the two axial ligands to the heme of Cyt b559 in R7Eα and R17Lß mutant reaction centers. Our results demonstrate that the electrostatic interactions between these arginine residues and the heme propionates of Cyt b559 are important to the structure and redox properties of Cyt b559. In addition, the blue light-induced nonphotochemical quenching was significantly attenuated and its recovery was accelerated in the R7Lα and R17Lß mutant cells. Furthermore, ultra performance liquid chromatography-mass spectrometry results showed that the PQ pool was more reduced in the R7Eα and R17Lß mutant cells than wild-type cells in the dark. Our data support a functional role of Cyt b559 in protection of PSII under photoinhibition conditions in vivo.

Large-scale domain motions and pyridoxal-5'-phosphate assisted radical catalysis in coenzyme B12-dependent aminomutases.

Lysine 5,6-aminomutase (5,6-LAM) and ornithine 4,5-aminomutase (4,5-OAM) are two of the rare enzymes that use assistance of two vitamins as cofactors. These enzymes employ radical generating capability of coenzyme B12 (5'-deoxyadenosylcobalamin, dAdoCbl) and ability of pyridoxal-5'-phosphate (PLP, vitamin B6) to stabilize high-energy intermediates for performing challenging 1,2-amino rearrangements between adjacent carbons. A large-scale domain movement is required for interconversion between the catalytically inactive open form and the catalytically active closed form. In spite of all the similarities, these enzymes differ in substrate specificities. 4,5-OAM is highly specific for D-ornithine as a substrate while 5,6-LAM can accept D-lysine and L-ß-lysine. This review focuses on recent computational, spectroscopic and structural studies of these enzymes and their implications on the related enzymes. Additionally, we also discuss the potential biosynthetic application of 5,6-LAM.

FeoC from Klebsiella pneumoniae contains a [4Fe-4S] cluster.

Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe(2+)) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen- or iron-sensitive coordination of the Fe-S cluster.

Mechanism-based inhibition reveals transitions between two conformational states in the action of lysine 5,6-aminomutase: a combination of electron paramagnetic resonance spectroscopy, electron nuclear double resonance spectroscopy, and density functional theory study.

An "open"-state crystal structure of lysine 5,6-aminomutase suggests that transition to a hypothetical "closed"-state is required to bring the cofactors adenosylcobalamin (AdoCbl) and pyridoxal-5'-phosphate (PLP) and the substrate into proximity for the radical-mediated 1,2-amino group migration. This process is achieved by transaldimination of the PLP-Lys144ß internal aldimine with the PLP-substrate external aldimine. A closed-state crystal structure is not available. UV-vis and electron paramagnetic resonance studies show that homologues of substrate D-lysine, 2,5-DAPn, 2,4-DAB, and 2,3-DAPr bind to PLP as an external aldimine and elicit the AdoCbl Co-C bond homolysis and the accumulations of cob(II)alamin and analogue-based radicals, demonstrating the existence of a closed state. (2)H- and (31)P-electron nuclear double resonance studies, supported by computations, show that the position for hydrogen atom abstraction from 2,5-DAPn and 2,4-DAB by the 5'-deoxyadenosyl radical occurs at the carbon adjacent to the imine, resulting in overstabilized radicals by spin delocalization through the imine into the pyridine ring of PLP. These radicals block the active site, inhibit the enzyme, and poise the enzyme into two distinct conformations: for even-numbered analogues, the cob(II)alamin remains proximal to and spin-coupled with the analogue-based radical in the closed state while odd-numbered analogues could trigger the transition to the open state of the enzyme. We provide here direct spectroscopic evidence that strongly support the existence of a closed state and its analogue-dependent transition to the open state, which is one step that was proposed to complete the catalytic turnover of the substrate lysine.

Self-Assembled Synthesis of Porous Iron-Doped Graphitic Carbon Nitride Nanostructures for Efficient Photocatalytic Hydrogen Evolution and Nitrogen Fixation.

In this study, Fe-doped graphitic carbon nitride (Fe-MCNC) with varying Fe contents was synthesized via a supramolecular approach, followed by thermal exfoliation, and was then used for accelerated photocatalytic hydrogen evolution and nitrogen fixation. Various techniques were used to study the physicochemical properties of the MCN (g-C3N4 from melamine) and Fe-MCNC (MCN for g-C3N4 and C for cyanuric acid) catalysts. The field emission scanning electron microscopy (FE-SEM) images clearly demonstrate that the morphology of Fe-MCNC changes from planar sheets to porous, partially twisted (partially developed nanotube and nanorod) nanostructures. The elemental mapping study confirms the uniform distribution of Fe on the MCNC surface. The X-ray photoelectron spectroscopy (XPS) and UV-visible diffuse reflectance spectroscopy (UV-DRS) results suggest that the Fe species might exist in the Fe3+ state and form Fe-N bonds with N atoms, thereby extending the visible light absorption areas and decreasing the band gap of MCN. Furthermore, doping with precise amounts of Fe might induce exfoliation and increase the specific surface area, but excessive Fe could destroy the MCN structure. The optimized Fe-MCNC nanostructure had a specific surface area of 23.6 m2 g-1, which was 8.1 times greater than that of MCN (2.89 m2 g-1). To study its photocatalytic properties, the nanostructure was tested for photocatalytic hydrogen evolution and nitrogen fixation; 2Fe-MCNC shows the highest photocatalytic activity, which is approximately 13.3 times and 2.4 times better, respectively, than MCN-1H. Due to its high efficiency and stability, the Fe-MCNC nanostructure is a promising and ideal photocatalyst for a wide range of applications.

Synthesis of 4-thia-[6-(13)C]lysine from [2- (13)C]glycine: access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine.

4-Thialysine (S-(2-aminoethyl)-L: -cysteine) is an analog of lysine. It has been used as an alternative substrate for lysine in enzymatic reactions. Site-directed isotopomers are often needed for elucidation of mechanism of reactions. 4-Thialysine can be synthesized by reacting cysteine with 2-bromoethylamine, an important reagent in chemical-modification rescue (CMR) of proteins. Here, we present the synthesis of 4-thia-[6-(13)C]lysine, one of the isotopomers of 4-thialysine, from commercially available starting material [2-(13)C]glycine via formation of five intermediates including 2-amino[2-(13)C]ethanol and 2-bromo[1-(13)C]ethylamine. The compounds were characterized using various spectroscopic techniques. Moreover, we discuss that our strategy would provide access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine. Biological activity of 4-thia-[6-(13)C]lysine was tested in the enzymatic reaction of lysine 5,6-aminomutase.

Electronic structures of ruthenium and osmium complexes of 9,10-phenanthrenequinone.

The reaction of 9,10-phenanthrenequinone (PQ) with [M(II)(H)(CO)(X)(PPh(3))(3)] in boiling toluene leads to the homolytic cleavage of the M(II)-H bond, affording the paramagnetic trans-[M(PQ)(PPh(3))(2)(CO)X] (M = Ru, X = Cl, 1; M = Os, X = Br, 3) and cis-[M(PQ)(PPh(3))(2)(CO)X] (M = Ru, X = Cl, 2; M = Os, X = Br, 4) complexes. Single-crystal X-ray structure determinations of 1, 2·toluene, and 4·CH(2)Cl(2), EPR spectra, and density functional theory (DFT) calculations have substantiated that 1-4 are 9,10-phenanthrenesemiquinone radical (PQ(•-)) complexes of ruthenium(II) and osmium(II) and are defined as trans-[Ru(II)(PQ(•-))(PPh(3))(2)(CO)Cl] (1), cis-[Ru(II)(PQ(•-))(PPh(3))(2)(CO)Cl] (2), trans-[Os(II)(PQ(•-))(PPh(3))(2)(CO) Br] (3), and cis-[Os(II)(PQ(•-))(PPh(3))(2)(CO)Br] (4). Two comparatively longer C-O [average lengths: 1, 1.291(3) Å; 2·toluene, 1.281(5) Å; 4·CH(2)Cl(2), 1.300(8) Å] and shorter C-C lengths [1, 1.418(5) Å; 2·toluene, 1.439(6) Å; 4·CH(2)Cl(2), 1.434(9) Å] of the OO chelates are consistent with the presence of a reduced PQ(•-) ligand in 1-4. A minor contribution of the alternate resonance form, trans- or cis-[M(I)(PQ)(PPh(3))(2)(CO)X], of 1-4 has been predicted by the anisotropic X- and Q-band electron paramagnetic resonance spectra of the frozen glasses of the complexes at 25 K and unrestricted DFT calculations on 1, trans-[Ru(PQ)(PMe(3))(2)(CO)Cl] (5), cis-[Ru(PQ)(PMe(3))(2)(CO)Cl] (6), and cis-[Os(PQ)(PMe(3))(2)(CO)Br] (7). However, no thermodynamic equilibria between [M(II)(PQ(•-))(PPh(3))(2)(CO)X] and [M(I)(PQ)(PPh(3))(2)(CO)X] tautomers have been detected. 1-4 undergo one-electron oxidation at -0.06, -0.05, 0.03, and -0.03 V versus a ferrocenium/ferrocene, Fc(+)/Fc, couple because of the formation of PQ complexes as trans-[Ru(II)(PQ)(PPh(3))(2)(CO)Cl](+) (1(+)), cis-[Ru(II)(PQ)(PPh(3))(2)(CO)Cl](+) (2(+)), trans-[Os(II)(PQ)(PPh(3))(2)(CO)Br](+) (3(+)), and cis-[Os(II)(PQ)(PPh(3))(2)(CO)Br](+) (4(+)). The trans isomers 1 and 3 also undergo one-electron reduction at -1.11 and -0.96 V, forming PQ(2-) complexes trans-[Ru(II)(PQ(2-))(PPh(3))(2)(CO)Cl](-) (1(-)) and trans-[Os(II)(PQ(2-))(PPh(3))(2)(CO)Br](-) (3(-)). Oxidation of 1 by I(2) affords diamagnetic 1(+)I(3)(-) in low yields. Bond parameters of 1(+)I(3)(-) [C-O, 1.256(3) and 1.258(3) Å; C-C, 1.482(3) Å] are consistent with ligand oxidation, yielding a coordinated PQ ligand. Origins of UV-vis/near-IR absorption features of 1-4 and the electrogenerated species have been investigated by spectroelectrochemical measurements and time-dependent DFT calculations on 5, 6, 5(+), and 5(-).

Exploring the mechanism of action of lysine 5,6-aminomutase using EPR and ENDOR spectroscopies.

Radical enzymes orchestrate challenging chemical transformations by devising strategies to tame the highly reactive radical intermediates. Electron paramagnetic resonance (EPR) spectroscopy is the most suitable technique to study various aspects of the radical enzymes. Lysine 5,6-aminomutase (5,6-LAM) is one such radical enzyme and employs coenzyme B12 and pyridoxal 5'-phosphate (PLP) to catalyze the 1,2-amino shift reaction through a radical mechanism. 5,6-LAM accepts either d-lysine or l-ß-lysine as the substrate. EPR and electron nuclear double resonance (ENDOR) spectroscopies have played major roles in deciphering the mechanism of action of 5,6-LAM, while density functional theoretical (DFT) computation and synthetic isotopologues have played supporting roles. This comprehensive toolkit has revealed that 5,6-LAM undergoes large-scale conformational movement to bring PLP and coenzyme B12 close together, which allows the reaction to progress. The conformational change also closes the active site, which protects the radical intermediates and enables their transformation to product without unwanted side reactions. The substrate-related radical (S•), which is spin-coupled with Co2+ generated from homolysis of the CoC bond in coenzyme B12, was unequivocally characterized when a substrate analog, 4-thia-l-lysine, and isotopologues of it were reacted with 5,6-LAM. Studies with substrate analogs revealed a unique "odd-even" correlation with opening of the closed state. Moreover, mutagenesis studies identified the contributions that conserved residues in 5,6-LAM make toward binding of the substrate. Further studies with a cofactor analog, PLP-N-oxide, have shed light on various aspects of the mechanism of action of 5,6-LAM.

Spectroscopic and functional characterizations of cyanobacterium Synechocystis PCC 6803 mutants on and near the heme axial ligand of cytochrome b559 in photosystem II.

The functional role of cytochrome (cyt) b(559) in photosystem II (PSII) was investigated in H22K alpha and Y18S alpha cyt b(559) mutants of the cyanobacterium Synechocystis sp. PCC6803. H22K alpha and Y18S alpha cyt b(559) mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b(559) in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b(559) in H22K alpha mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b(559) in Y18S alpha mutant PSII core complexes was shifted to 561 nm. Y18S alpha and H22K alpha mutant PSII core complexes contained predominately the low potential form of cyt b(559). The findings lend support to the concept that the redox properties of cyt b(559) are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b(559) mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b(559) in protection of PSII under photoinhibition conditions in vivo.

Radical stabilization is crucial in the mechanism of action of lysine 5,6-aminomutase: role of tyrosine-263α as revealed by electron paramagnetic resonance spectroscopy.

Adenosylcobalamin- and pyridoxal-5'-phosphate-dependent lysine 5,6-aminomutase utilizes free radical intermediates to mediate 1,2-amino group rearrangement, during which an elusive high-energy aziridincarbinyl radical is proposed to be central in the mechanism of action. Understanding how the enzyme participates in stabilizing any of the radical intermediates is fundamentally significant. Y263F mutation abolished the enzymatic activity. With isotope-edited EPR methods, the roles of the Tyr263α residue in the putative active site are revealed. The Tyr263α residue stabilizes a radical intermediate, which most likely is the aziridincarbinyl radical, either by acting as a spin-relay device or serving as an anchor for the pyridine ring of pyridoxal-5'-phosphate through aromatic π-stacking interactions during spin transfer. The Tyr263α residue also protects the radical intermediate from interception by molecular oxygen. This study supports the proposed reaction mechanism, including the aziridincarbinyl radical, which has eluded detection for more than two decades.

Spin-Orbit Coupling Effects in Au 4f Core-Level Electronic Structures in Supported Low-Dimensional Gold Nanoparticles.

Despite their many advantages, issues remain unresolved over the variability in catalytic activities in supported gold nanoparticle (AuNP)-based catalysts, which requires precise characterization to unravel the presence of any fine features. Herein, upon analyzing the Au 4f core-level spin-orbit components in many as-synthesized AuNP-based catalysts, we observed that like deviations in the Au 4f7/2 binding energy positions, both the Au 4f7/2-to-Au 4f5/2 peak intensity and linewidth ratios varied largely from the standard statistical bulk reference values. These deviations were observed in all the as-synthesized supported AuNPs irrespective of different synthesis conditions, variations in size, shape or morphology of the gold nanoparticles, and different support materials. On the other hand, the spin-orbit-splitting values remained almost unchanged and did not show any appreciable deviations from the atomic or bulk standard gold values. These deviations could originate due to alterations in the electronic band structures in the supported AuNPs and might be present in other NP-based catalyst systems as well, which could be the subject of future research interest.

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions.

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 microM DCMU. This mutant carries an R7L mutation on the alpha-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 A PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lalpha cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lalpha cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lalpha cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the alpha-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.