34th Annual Mid-West Photosynthesis Conference at Turkey Run State Park, INSean Padden
The document reports on a study of mutations to the arginine residue at position 257 (R257) of the D1 protein in Photosystem II in Chlamydomonas reinhardtii. Three mutations were made - R257E, R257K, and R257Q. Thermoluminescence and fluorescence measurements showed that the redox potential of the QB/QB- pair was lowered by 20-40 mV in the mutants. This suggests the size and charge of the residue at position 257 modulates the redox potential of QB/QB-. Growth rates and oxygen evolution were negatively impacted in the mutants compared to wild type.
This document describes the crystal structure and characterization of a covalently cross-linked complex between cytochrome c peroxidase (CCP) and cytochrome c (cyt c) that was engineered by introducing cysteine mutations into the proteins. The 1.88 angstrom crystal structure of the cross-linked complex closely resembles the structure of the noncovalent complex and reveals ordered water molecules bridging the interface. Studies show the cross-linked complex maintains normal compound I formation and fast intramolecular electron transfer, indicating it closely mimics the physiological electron transfer complex.
This document summarizes research on engineering a cation-binding site into cytochrome c peroxidase (CcP) in order to study the effects on enzyme activity and structure. A key residue (Asn195) in the engineered cation-binding loop was mutated to proline to stabilize the loop conformation. Crystal structure analysis showed the loop is stabilized in the closed conformation when potassium is bound. While enzyme activity is reduced, it can be titrated based on potassium concentration. The goals were to better understand how cation binding and loop conformation impact electron transfer from cytochrome c and the stability of the tryptophan radical in the enzyme's active site.
1) Veratrole was electrooxidized on a platinum electrode in acetonitrile solutions containing either tetrabutylammonium perchlorate or lithium perchlorate.
2) A green deposit formed containing hexamethoxytriphenylene radical cation species associated with perchlorate counterions.
3) Spectroelectrochemical experiments showed that oxidation of perchlorate anions above 2.3V dissolved the deposit, but sweeping the potential back regenerated the solid, allowing the process to be cycled.
This document summarizes research on converting an engineered potassium-binding site in cytochrome c peroxidase (CCP) into a calcium-selective site through protein engineering and crystal structure analysis. The researchers previously engineered a potassium-binding site in CCP based on the structure of ascorbate peroxidase. They then designed mutants intended to bind calcium selectively instead. The crystal structure of the first mutant showed binding of a smaller cation like sodium rather than calcium due to disordering of a ligand. A second mutant was then designed and its crystal structure confirmed calcium binding with a fully coordinated ligand environment, demonstrating that an iterative engineering approach can switch cation selectivity in proteins.
Using natural abundance of isotopes to investigate chemical reaction mechanismsDaniel Morton
The document discusses using natural abundance of isotopes and kinetic isotope effect (KIE) measurements to investigate chemical reaction mechanisms. It provides examples of using these techniques to determine reaction mechanisms for Diels-Alder reactions, osmium-catalyzed dihydroxylation of olefins, and dirhodium-catalyzed cyclopropanation reactions. For the last example, Singleton's KIE data supported a conventional tetrabridged carbenoid mechanism over Corey's proposed tribridged intermediate mechanism.
Metal cu(ii) and zn(ii) bipyridyls as inhibitors of lactaterkkoiri
This document describes a study examining how metal complexes of copper (Cu-bpy) and zinc (Zn-bpy) interact with and modulate the activity of lactate dehydrogenase (LDH) in mouse tissues. The key findings are:
1) Both Cu-bpy and Zn-bpy were found to bind to LDH, with Zn-bpy showing a stronger binding constant.
2) Treatment of mouse tissues with non-toxic doses of the complexes resulted in a significant decline in LDH activity in various tissues, with Cu-bpy inhibiting LDH in all tissues studied and Zn-bpy inhibiting LDH in liver, kidney, and heart tissues.
3) Electrophoresis analysis suggested that both complexes equally inhibited
Vacancy and Copper-doping Effect on Superconductivity for Clathrate MaterialsYang Li
This document summarizes a study on the effects of vacancy and copper doping on superconductivity in clathrate materials. Key findings include:
1) Copper doping of Ba8Si46 decreases the superconducting critical temperature strongly as copper content increases. Theoretical simulations also show copper doping decreases electronic density of states at the Fermi level.
2) Comparison of Ba8Ge46 and Ba8Si46 using theoretical simulations found their electronic structures are very similar, suggesting superconductivity is intrinsic to fully occupied silicon and germanium clathrates.
3) The absence of superconductivity in most germanium clathrates is suggested to arise from vacancy defects which result in
34th Annual Mid-West Photosynthesis Conference at Turkey Run State Park, INSean Padden
The document reports on a study of mutations to the arginine residue at position 257 (R257) of the D1 protein in Photosystem II in Chlamydomonas reinhardtii. Three mutations were made - R257E, R257K, and R257Q. Thermoluminescence and fluorescence measurements showed that the redox potential of the QB/QB- pair was lowered by 20-40 mV in the mutants. This suggests the size and charge of the residue at position 257 modulates the redox potential of QB/QB-. Growth rates and oxygen evolution were negatively impacted in the mutants compared to wild type.
This document describes the crystal structure and characterization of a covalently cross-linked complex between cytochrome c peroxidase (CCP) and cytochrome c (cyt c) that was engineered by introducing cysteine mutations into the proteins. The 1.88 angstrom crystal structure of the cross-linked complex closely resembles the structure of the noncovalent complex and reveals ordered water molecules bridging the interface. Studies show the cross-linked complex maintains normal compound I formation and fast intramolecular electron transfer, indicating it closely mimics the physiological electron transfer complex.
This document summarizes research on engineering a cation-binding site into cytochrome c peroxidase (CcP) in order to study the effects on enzyme activity and structure. A key residue (Asn195) in the engineered cation-binding loop was mutated to proline to stabilize the loop conformation. Crystal structure analysis showed the loop is stabilized in the closed conformation when potassium is bound. While enzyme activity is reduced, it can be titrated based on potassium concentration. The goals were to better understand how cation binding and loop conformation impact electron transfer from cytochrome c and the stability of the tryptophan radical in the enzyme's active site.
1) Veratrole was electrooxidized on a platinum electrode in acetonitrile solutions containing either tetrabutylammonium perchlorate or lithium perchlorate.
2) A green deposit formed containing hexamethoxytriphenylene radical cation species associated with perchlorate counterions.
3) Spectroelectrochemical experiments showed that oxidation of perchlorate anions above 2.3V dissolved the deposit, but sweeping the potential back regenerated the solid, allowing the process to be cycled.
This document summarizes research on converting an engineered potassium-binding site in cytochrome c peroxidase (CCP) into a calcium-selective site through protein engineering and crystal structure analysis. The researchers previously engineered a potassium-binding site in CCP based on the structure of ascorbate peroxidase. They then designed mutants intended to bind calcium selectively instead. The crystal structure of the first mutant showed binding of a smaller cation like sodium rather than calcium due to disordering of a ligand. A second mutant was then designed and its crystal structure confirmed calcium binding with a fully coordinated ligand environment, demonstrating that an iterative engineering approach can switch cation selectivity in proteins.
Using natural abundance of isotopes to investigate chemical reaction mechanismsDaniel Morton
The document discusses using natural abundance of isotopes and kinetic isotope effect (KIE) measurements to investigate chemical reaction mechanisms. It provides examples of using these techniques to determine reaction mechanisms for Diels-Alder reactions, osmium-catalyzed dihydroxylation of olefins, and dirhodium-catalyzed cyclopropanation reactions. For the last example, Singleton's KIE data supported a conventional tetrabridged carbenoid mechanism over Corey's proposed tribridged intermediate mechanism.
Metal cu(ii) and zn(ii) bipyridyls as inhibitors of lactaterkkoiri
This document describes a study examining how metal complexes of copper (Cu-bpy) and zinc (Zn-bpy) interact with and modulate the activity of lactate dehydrogenase (LDH) in mouse tissues. The key findings are:
1) Both Cu-bpy and Zn-bpy were found to bind to LDH, with Zn-bpy showing a stronger binding constant.
2) Treatment of mouse tissues with non-toxic doses of the complexes resulted in a significant decline in LDH activity in various tissues, with Cu-bpy inhibiting LDH in all tissues studied and Zn-bpy inhibiting LDH in liver, kidney, and heart tissues.
3) Electrophoresis analysis suggested that both complexes equally inhibited
Vacancy and Copper-doping Effect on Superconductivity for Clathrate MaterialsYang Li
This document summarizes a study on the effects of vacancy and copper doping on superconductivity in clathrate materials. Key findings include:
1) Copper doping of Ba8Si46 decreases the superconducting critical temperature strongly as copper content increases. Theoretical simulations also show copper doping decreases electronic density of states at the Fermi level.
2) Comparison of Ba8Ge46 and Ba8Si46 using theoretical simulations found their electronic structures are very similar, suggesting superconductivity is intrinsic to fully occupied silicon and germanium clathrates.
3) The absence of superconductivity in most germanium clathrates is suggested to arise from vacancy defects which result in
Superconductivity in Gallium-substituted Ba8Si46 ClathratesYang Li
We report a joint experimental and theoretical investigation of superconductivity in Ga-substituted type-I silicon clathrates. We prepared samples of the general formula Ba8Si46−xGax, with different values of x. We show that Ba8Si40Ga6 is a bulk superconductor, with an onset at TC=3.3 K. For x=10 and higher, no superconductivity was observed down to T=1.8 K. This represents a strong suppression of superconductivity with increasing Ga content, compared to Ba8Si46 with TC=8 K. Suppression of superconductivity can be attributed primarily to a decrease in the density of states at the Fermi level, caused by a reduced integrity of the sp3-hybridized networks as well as the lowering of carrier concentration. These results are corroborated by first-principles calculations, which show that Ga substitution results in a large decrease of the electronic density of states at the Fermi level, which explains the decreased superconducting critical temperature within the BCS framework. To further characterize the superconducting state, we carried out magnetic measurements showing Ba8Si40Ga6 to be a type-II superconductor. The critical magnetic fields were measured to be Hc1=35 Oe and Hc2=8.5 kOe.We deduce the London penetration depth 3700 Å and the coherence length 200 Å. Our estimate of the electron-phonon coupling reveals that Ba8Si40Ga6 is a moderate phonon-mediated BCS superconductor.
tuning the pH Response of i-Motif DNA Oligonucleotides_Lannes_et_al-2015-Chem...saheli halder
This document discusses tuning the pH response of i-motif DNA oligonucleotides. The authors introduced 5-methylcytosines (5-MeC) and 5-bromocytosines (5-BrC) into the human telomeric i-motif sequence to shift its pH response range. They found that 5-MeC shifted the pH response towards more basic values, while 5-BrC shifted it towards more acidic values. Additionally, lengthening the sequence shifted the pH response in a more basic direction. The modifications did not thermally destabilize the i-motifs. 5-BrC substitution led to a ten-fold increase in folding kinetics compared to the other sequences.
This document provides information on chemistry courses for general and special degrees at the University of Peradeniya in Sri Lanka. It lists the compulsory and elective chemistry courses required for each degree, including course codes, credits, prerequisites, and recommended textbooks. Descriptions are provided for several 200-level chemistry courses, including inorganic chemistry, organic chemistry, physical chemistry, and their associated laboratories. The courses cover topics such as coordination chemistry, organic reaction mechanisms, quantum mechanics, molecular spectroscopy, and physical chemistry experiments and measurements.
This study examines dissociation pathways in the dication of the amino acid cysteine after site-selective core ionization of the O 1s, N 1s, C 1s, and S 2p orbitals. A photoelectron-ion-ion coincidence experiment was performed. Several dissociation channels were identified, some showing strong site-selective dependence attributed to a combination of nuclear motion in the core-ionized state and Auger processes populating different electronic states in the dication. Fragmentation patterns were analyzed at each core ionization level to determine dissociation pathways and kinetic energy release partitioning between fragments through regression slope analysis.
1) The document describes a study investigating the roles of the K+/H+ antiporter KEA3, the Cl- channel/transporter CLCe, and the voltage-dependent Cl- channel VCCN1 in regulating photosynthesis in plants.
2) The results show that VCCN1 accelerates the activation of photoprotection, whereas KEA3 slows it down on transition from high to low light. CLCe regulates photosynthesis through a pH-independent mechanism involving Cl- homeostasis.
3) A proposed model indicates that in low light conditions, VCCN1 imports Cl- to build a pH gradient activating photoprotection, while KEA3 later dissipates this
This document summarizes two systems for modulating the rate of back electron transfer between guanine radicals and 2-aminopurine in duplex DNA. In the first system, increasing the distance between 2-AP and guanine slowed back electron transfer, allowing more DNA-protein crosslinks to form. In the second system, changing the local guanine sequence to alter oxidation potential facilitated or inhibited charge transport away from 2-AP, affecting oxidative reaction yields. Overall, the rate of back electron transfer compared to other reaction rates determines the fate of guanine radicals and the yield of permanent oxidative products.
This document summarizes the design and testing of a bisubstrate inhibitor for the insulin receptor tyrosine kinase (IRK). Researchers designed a compound that links ATPγS to a peptide substrate analog via a two-carbon spacer, setting the distance between the nucleophilic atom and phosphoryl group to mimic a dissociative transition state as suggested by previous studies of IRK's mechanism. Testing found this compound to be a potent and selective competitive inhibitor of IRK, with a Ki of 370 nM, making it the most potent inhibitor reported for this important signaling enzyme. A crystal structure confirmed the inhibitor bound as designed and validated the mechanism-inspired approach.
This document provides an overview of several sections on the topic of electrochemistry from a textbook or online course. It covers voltaic cells and how they generate electrical energy from redox reactions, different types of batteries like dry cells, lead-acid, and lithium batteries, fuel cells, corrosion and how to prevent it, and electrolysis and its applications in processes like metal smelting and electroplating. Diagrams and terminology related to these topics are also defined throughout the document sections.
1) Cryo-EM was used to determine the structure of the human nucleosome containing the histone variant H2A.B (H2A.B-NCP) at atomic resolution of 3.9 Angstroms.
2) The H2A.B-NCP structure is distinct from canonical nucleosomes, compacting around 103 base pairs of DNA in 1.2 superhelical turns compared to 145 base pairs in 1.7 turns for canonical nucleosomes.
3) A poly(ADP-ribose) polymerase 1 DNA binding domain was found to stabilize the dynamic H2A.B-NCP, allowing determination of its structure at higher resolution of 2.8 Ang
- Elongation Factor IV (EF4) is a translation factor found in nearly all eukaryotes and all sequenced prokaryotes. It was originally named LepA due to its location on the Lep operon.
- EF4 functions as a back-translocase during elongation, reversing the transition catalyzed by Elongation Factor G. It preferentially binds to post-translocation ribosomal complexes.
- EF4 and EF-G share structural homology and EF4 retains 55-68% amino acid identity among bacterial orthologs. EF4 binds competitively with EF-G to the ribosome.
Chapter 2 polar covalent bonds acids and basesWong Hsiung
This document summarizes key concepts about polar covalent bonds and acid-base chemistry from Chapter 2 of an organic chemistry textbook. It discusses how electronegativity differences between atoms lead to polar covalent bonds and molecular polarity. Bond polarity is quantified using dipole moments. The Brønsted-Lowry definition of acids and bases in terms of proton transfer is introduced. Acid strength is described using acid dissociation constants (Ka) and their pKa values. Factors that influence acid and base strength are also covered.
This document summarizes the rational design and generation of a catalytic antibody that selectively hydrolyzes a specific substrate. Researchers designed an antibody to bind a transition state analogue for the hydrolysis of a carbonate substrate. They generated monoclonal antibodies against a nitrophenyl phosphonate transition state analogue. One antibody was found to catalyze the hydrolysis of the carbonate substrate, displaying Michaelis-Menten kinetics. The antibody-catalyzed reaction had substrate specificity and was competitively inhibited by the corresponding phosphate transition state analogue, demonstrating the ability to rationally design catalytic antibodies.
This study investigated the structural changes that occur in the protein synaptotagmin-1 (syt1) when it binds lead instead of its native metal ion calcium. Researchers expressed a syt1-GST fusion protein in E. coli and purified it using affinity chromatography. Differential scanning calorimetry experiments showed syt1-GST unfolded in multiple stages both in the presence and absence of calcium, suggesting calcium does not stabilize the protein structure. Further experiments replacing calcium with lead are needed to understand how metal binding affects syt1 activity in neurotransmitter release.
The document reports on a study of mutations to the arginine residue at position 257 (R257) of the D1 protein in Photosystem II in Chlamydomonas reinhardtii. Three mutations were made - R257E, R257K, and R257Q. Thermoluminescence measurements and fluorescence analysis showed that the redox potential of the QB/QB- pair was lowered by 20-40 mV in the mutants. This suggests the size and charge of the residue at position 257 modulates the redox potential of QB/QB-. The mutants also showed decreased growth rates and lowered maximum variable chlorophyll fluorescence compared to wild type.
1) Mutation studies of the D1H252 residue in photosystem II support the role of this residue in the proton transfer mechanism of the QB site two-electron gate. Specifically, protonation of D1H252 is needed to stabilize the QB- state.
2) A D1H252D mutation maintains some two-electron gate function, while D1H252Q/N mutations inhibit the second electron transfer, likely due to the need for protonation. A D1H252K mutation greatly inhibits electron flow.
3) Ser-264 is unlikely to directly ligand the quinone, but instead hydrogen bonds to D1H252 to relay protons during the proton transfer process.
This document discusses a study on the effects of molecular structure on the kinetics of electron transfer processes in quinones. Specifically, it examines the substitution effects of chlorine, methyl, and tert-butyl groups at the 2,5 and 2,6 positions of benzoquinones. Cyclic voltammetry and electron spin resonance spectroscopy were used to analyze differences in the inner reorganization energy for the first electron uptake between the structural isomers. The results provide insight into how substituents affect the stability and structure of the electrogenerated semiquinone intermediates, which influences the reactivity of quinones in biological systems.
This paper studies the dielectric properties of order-disorder type crystals like KH2PO4. It derives expressions for the dielectric constant and tangent loss using the soft mode dynamical model and double time temperature dependent Green's function techniques. It calculates the transverse dielectric constant, observed dielectric constant, and tangent loss for KH2PO4 in the paraelectric phase using model parameters. The calculated results agree well with experimental data from several other studies on the temperature dependence of the dielectric constant and tangent loss of KH2PO4. The paper finds that phonon anharmonicity contributes significantly to the observed loss at higher temperatures.
1) The document discusses light-dependent (photosynthetic) generators of proton potential, specifically focusing on the photosynthetic apparatus of purple bacteria.
2) Photosynthesis in purple bacteria involves a light-dependent cyclic redox chain where absorption of light by bacteriochlorophyll leads to electron transfer across the membrane, generating a proton gradient.
3) Key components of the redox chain include bacteriochlorophyll dimer and monomer, bacteriopheophytin, ubiquinone, cytochromes, and a nonheme iron-sulfur protein that facilitate electron transfer and proton pumping across the membrane.
1) The document discusses light-dependent (photosynthetic) generators of proton potential, specifically focusing on the photosynthetic apparatus of purple bacteria.
2) Photosynthesis in purple bacteria involves a light-dependent cyclic redox chain where absorption of light by bacteriochlorophyll leads to electron transfer across the membrane, generating a proton gradient.
3) Key components of the redox chain include bacteriochlorophyll dimer and monomer, bacteriopheophytin, ubiquinone, cytochromes, and a nonheme iron-sulfur protein that facilitate electron transfer and proton pumping across the membrane.
This document reports on a study that uses EPR spectroscopy to investigate proton-coupled electron transfer (PCET) reactions of the redox-active tyrosine Z (YZ) in photosystem II (PSII). The researchers isolated YZ radical reactions while retaining the manganese-calcium cluster at the oxygen-evolving complex (OEC) using low temperatures. They measured the pH and solvent isotope dependence of the YZ radical decay rate and found it exhibited a significant solvent isotope effect but was pH independent from pH 5 to 7.5. This is consistent with a rate-limiting coupled proton-electron transfer reaction for YZ and distinguishes it from the YD radical decay kinetics. The extensive hydrogen
Superconductivity in Gallium-substituted Ba8Si46 ClathratesYang Li
We report a joint experimental and theoretical investigation of superconductivity in Ga-substituted type-I silicon clathrates. We prepared samples of the general formula Ba8Si46−xGax, with different values of x. We show that Ba8Si40Ga6 is a bulk superconductor, with an onset at TC=3.3 K. For x=10 and higher, no superconductivity was observed down to T=1.8 K. This represents a strong suppression of superconductivity with increasing Ga content, compared to Ba8Si46 with TC=8 K. Suppression of superconductivity can be attributed primarily to a decrease in the density of states at the Fermi level, caused by a reduced integrity of the sp3-hybridized networks as well as the lowering of carrier concentration. These results are corroborated by first-principles calculations, which show that Ga substitution results in a large decrease of the electronic density of states at the Fermi level, which explains the decreased superconducting critical temperature within the BCS framework. To further characterize the superconducting state, we carried out magnetic measurements showing Ba8Si40Ga6 to be a type-II superconductor. The critical magnetic fields were measured to be Hc1=35 Oe and Hc2=8.5 kOe.We deduce the London penetration depth 3700 Å and the coherence length 200 Å. Our estimate of the electron-phonon coupling reveals that Ba8Si40Ga6 is a moderate phonon-mediated BCS superconductor.
tuning the pH Response of i-Motif DNA Oligonucleotides_Lannes_et_al-2015-Chem...saheli halder
This document discusses tuning the pH response of i-motif DNA oligonucleotides. The authors introduced 5-methylcytosines (5-MeC) and 5-bromocytosines (5-BrC) into the human telomeric i-motif sequence to shift its pH response range. They found that 5-MeC shifted the pH response towards more basic values, while 5-BrC shifted it towards more acidic values. Additionally, lengthening the sequence shifted the pH response in a more basic direction. The modifications did not thermally destabilize the i-motifs. 5-BrC substitution led to a ten-fold increase in folding kinetics compared to the other sequences.
This document provides information on chemistry courses for general and special degrees at the University of Peradeniya in Sri Lanka. It lists the compulsory and elective chemistry courses required for each degree, including course codes, credits, prerequisites, and recommended textbooks. Descriptions are provided for several 200-level chemistry courses, including inorganic chemistry, organic chemistry, physical chemistry, and their associated laboratories. The courses cover topics such as coordination chemistry, organic reaction mechanisms, quantum mechanics, molecular spectroscopy, and physical chemistry experiments and measurements.
This study examines dissociation pathways in the dication of the amino acid cysteine after site-selective core ionization of the O 1s, N 1s, C 1s, and S 2p orbitals. A photoelectron-ion-ion coincidence experiment was performed. Several dissociation channels were identified, some showing strong site-selective dependence attributed to a combination of nuclear motion in the core-ionized state and Auger processes populating different electronic states in the dication. Fragmentation patterns were analyzed at each core ionization level to determine dissociation pathways and kinetic energy release partitioning between fragments through regression slope analysis.
1) The document describes a study investigating the roles of the K+/H+ antiporter KEA3, the Cl- channel/transporter CLCe, and the voltage-dependent Cl- channel VCCN1 in regulating photosynthesis in plants.
2) The results show that VCCN1 accelerates the activation of photoprotection, whereas KEA3 slows it down on transition from high to low light. CLCe regulates photosynthesis through a pH-independent mechanism involving Cl- homeostasis.
3) A proposed model indicates that in low light conditions, VCCN1 imports Cl- to build a pH gradient activating photoprotection, while KEA3 later dissipates this
This document summarizes two systems for modulating the rate of back electron transfer between guanine radicals and 2-aminopurine in duplex DNA. In the first system, increasing the distance between 2-AP and guanine slowed back electron transfer, allowing more DNA-protein crosslinks to form. In the second system, changing the local guanine sequence to alter oxidation potential facilitated or inhibited charge transport away from 2-AP, affecting oxidative reaction yields. Overall, the rate of back electron transfer compared to other reaction rates determines the fate of guanine radicals and the yield of permanent oxidative products.
This document summarizes the design and testing of a bisubstrate inhibitor for the insulin receptor tyrosine kinase (IRK). Researchers designed a compound that links ATPγS to a peptide substrate analog via a two-carbon spacer, setting the distance between the nucleophilic atom and phosphoryl group to mimic a dissociative transition state as suggested by previous studies of IRK's mechanism. Testing found this compound to be a potent and selective competitive inhibitor of IRK, with a Ki of 370 nM, making it the most potent inhibitor reported for this important signaling enzyme. A crystal structure confirmed the inhibitor bound as designed and validated the mechanism-inspired approach.
This document provides an overview of several sections on the topic of electrochemistry from a textbook or online course. It covers voltaic cells and how they generate electrical energy from redox reactions, different types of batteries like dry cells, lead-acid, and lithium batteries, fuel cells, corrosion and how to prevent it, and electrolysis and its applications in processes like metal smelting and electroplating. Diagrams and terminology related to these topics are also defined throughout the document sections.
1) Cryo-EM was used to determine the structure of the human nucleosome containing the histone variant H2A.B (H2A.B-NCP) at atomic resolution of 3.9 Angstroms.
2) The H2A.B-NCP structure is distinct from canonical nucleosomes, compacting around 103 base pairs of DNA in 1.2 superhelical turns compared to 145 base pairs in 1.7 turns for canonical nucleosomes.
3) A poly(ADP-ribose) polymerase 1 DNA binding domain was found to stabilize the dynamic H2A.B-NCP, allowing determination of its structure at higher resolution of 2.8 Ang
- Elongation Factor IV (EF4) is a translation factor found in nearly all eukaryotes and all sequenced prokaryotes. It was originally named LepA due to its location on the Lep operon.
- EF4 functions as a back-translocase during elongation, reversing the transition catalyzed by Elongation Factor G. It preferentially binds to post-translocation ribosomal complexes.
- EF4 and EF-G share structural homology and EF4 retains 55-68% amino acid identity among bacterial orthologs. EF4 binds competitively with EF-G to the ribosome.
Chapter 2 polar covalent bonds acids and basesWong Hsiung
This document summarizes key concepts about polar covalent bonds and acid-base chemistry from Chapter 2 of an organic chemistry textbook. It discusses how electronegativity differences between atoms lead to polar covalent bonds and molecular polarity. Bond polarity is quantified using dipole moments. The Brønsted-Lowry definition of acids and bases in terms of proton transfer is introduced. Acid strength is described using acid dissociation constants (Ka) and their pKa values. Factors that influence acid and base strength are also covered.
This document summarizes the rational design and generation of a catalytic antibody that selectively hydrolyzes a specific substrate. Researchers designed an antibody to bind a transition state analogue for the hydrolysis of a carbonate substrate. They generated monoclonal antibodies against a nitrophenyl phosphonate transition state analogue. One antibody was found to catalyze the hydrolysis of the carbonate substrate, displaying Michaelis-Menten kinetics. The antibody-catalyzed reaction had substrate specificity and was competitively inhibited by the corresponding phosphate transition state analogue, demonstrating the ability to rationally design catalytic antibodies.
This study investigated the structural changes that occur in the protein synaptotagmin-1 (syt1) when it binds lead instead of its native metal ion calcium. Researchers expressed a syt1-GST fusion protein in E. coli and purified it using affinity chromatography. Differential scanning calorimetry experiments showed syt1-GST unfolded in multiple stages both in the presence and absence of calcium, suggesting calcium does not stabilize the protein structure. Further experiments replacing calcium with lead are needed to understand how metal binding affects syt1 activity in neurotransmitter release.
The document reports on a study of mutations to the arginine residue at position 257 (R257) of the D1 protein in Photosystem II in Chlamydomonas reinhardtii. Three mutations were made - R257E, R257K, and R257Q. Thermoluminescence measurements and fluorescence analysis showed that the redox potential of the QB/QB- pair was lowered by 20-40 mV in the mutants. This suggests the size and charge of the residue at position 257 modulates the redox potential of QB/QB-. The mutants also showed decreased growth rates and lowered maximum variable chlorophyll fluorescence compared to wild type.
1) Mutation studies of the D1H252 residue in photosystem II support the role of this residue in the proton transfer mechanism of the QB site two-electron gate. Specifically, protonation of D1H252 is needed to stabilize the QB- state.
2) A D1H252D mutation maintains some two-electron gate function, while D1H252Q/N mutations inhibit the second electron transfer, likely due to the need for protonation. A D1H252K mutation greatly inhibits electron flow.
3) Ser-264 is unlikely to directly ligand the quinone, but instead hydrogen bonds to D1H252 to relay protons during the proton transfer process.
This document discusses a study on the effects of molecular structure on the kinetics of electron transfer processes in quinones. Specifically, it examines the substitution effects of chlorine, methyl, and tert-butyl groups at the 2,5 and 2,6 positions of benzoquinones. Cyclic voltammetry and electron spin resonance spectroscopy were used to analyze differences in the inner reorganization energy for the first electron uptake between the structural isomers. The results provide insight into how substituents affect the stability and structure of the electrogenerated semiquinone intermediates, which influences the reactivity of quinones in biological systems.
This paper studies the dielectric properties of order-disorder type crystals like KH2PO4. It derives expressions for the dielectric constant and tangent loss using the soft mode dynamical model and double time temperature dependent Green's function techniques. It calculates the transverse dielectric constant, observed dielectric constant, and tangent loss for KH2PO4 in the paraelectric phase using model parameters. The calculated results agree well with experimental data from several other studies on the temperature dependence of the dielectric constant and tangent loss of KH2PO4. The paper finds that phonon anharmonicity contributes significantly to the observed loss at higher temperatures.
1) The document discusses light-dependent (photosynthetic) generators of proton potential, specifically focusing on the photosynthetic apparatus of purple bacteria.
2) Photosynthesis in purple bacteria involves a light-dependent cyclic redox chain where absorption of light by bacteriochlorophyll leads to electron transfer across the membrane, generating a proton gradient.
3) Key components of the redox chain include bacteriochlorophyll dimer and monomer, bacteriopheophytin, ubiquinone, cytochromes, and a nonheme iron-sulfur protein that facilitate electron transfer and proton pumping across the membrane.
1) The document discusses light-dependent (photosynthetic) generators of proton potential, specifically focusing on the photosynthetic apparatus of purple bacteria.
2) Photosynthesis in purple bacteria involves a light-dependent cyclic redox chain where absorption of light by bacteriochlorophyll leads to electron transfer across the membrane, generating a proton gradient.
3) Key components of the redox chain include bacteriochlorophyll dimer and monomer, bacteriopheophytin, ubiquinone, cytochromes, and a nonheme iron-sulfur protein that facilitate electron transfer and proton pumping across the membrane.
This document reports on a study that uses EPR spectroscopy to investigate proton-coupled electron transfer (PCET) reactions of the redox-active tyrosine Z (YZ) in photosystem II (PSII). The researchers isolated YZ radical reactions while retaining the manganese-calcium cluster at the oxygen-evolving complex (OEC) using low temperatures. They measured the pH and solvent isotope dependence of the YZ radical decay rate and found it exhibited a significant solvent isotope effect but was pH independent from pH 5 to 7.5. This is consistent with a rate-limiting coupled proton-electron transfer reaction for YZ and distinguishes it from the YD radical decay kinetics. The extensive hydrogen
The document discusses substitution reactions of square-planar metal complexes. It states that these complexes typically undergo substitution via an associative mechanism involving a five-coordinate transition state. The rate of reaction depends on factors like the incoming ligand's nucleophilicity, polarizability, and ability to be oxidized. Ligands already present, particularly the trans ligand, also impact the rate. Spectator ligands exert a "trans effect" where they direct incoming groups to positions trans to themselves, based on their position in the trans effect sequence of Cl- > NO2- > Br- > NH3. Together, trans effects and bond strengths determine the stereochemistry of substitution products.
1. The document provides an overview of common elementary steps in bioorganic chemistry, including curved arrow notation, proton transfers, SN2 reactions, and addition and elimination steps.
2. It discusses the characteristics of nucleophiles and electrophiles and how they participate in reactions. Bond formation, bond breaking, rearrangements, and tautomerizations are described.
3. The driving forces of chemical reactions are explained as charge stability and bond energy. Ketone-enol tautomerization is used as an example where bond energies are the major driving force due to relative stabilities.
This document presents a new correlation for predicting the thermal conductivity of liquids based on their molar polarization. The correlation relates two parameters (A and b) from an existing thermal conductivity equation to molar polarization. Molar polarization takes into account molecular structure, polarity, and temperature effects. Experimental thermal conductivity data for various substances was used to develop a correlation between the parameters and molar polarization. The new correlation was found to predict thermal conductivity with average errors less than 5% for most substances tested, outperforming some existing methods.
The document is Ian Blake Cooper's PhD dissertation from the Georgia Institute of Technology. It investigates the mechanism of photosynthetic water oxidation in photosystem II (PSII) using vibrational spectroscopy techniques. PSII is the membrane protein complex that uses light energy to oxidize water and produce molecular oxygen. Key findings of the dissertation include using time-resolved infrared spectroscopy to detect protein-based intermediates involved in the oxygen-evolving cycle, investigating proton-coupled electron transfer reactions associated with a redox-active tyrosine residue, examining the identity of the chloride binding site using bromide exchange, and probing proton transfer reactions at the oxygen-evolving complex using azide.
- The document examines proton uptake during the anaerobic reduction of cytochrome c oxidase. It uses computational methods to calculate the ionization states and pKas of residues in different redox states of the enzyme's active site.
- The calculations find that only a hydroxide coordinated to the CuB shifts its pKa above 7 upon reduction of the active site, allowing it to uptake a proton. Other proposed proton acceptors like Glu I-286 are found to have pKas above 7 and cannot uptake protons.
- This suggests that maintaining electroneutrality is not required during the anaerobic reduction of the active site, with approximately 2.5 protons taken up per 4 electrons
1) Low-lying excited states of the neutron-rich calcium isotopes 48-52Ca have been studied using gamma-ray spectroscopy following inverse kinematics proton scattering experiments.
2) The energies and strengths of the octupole states in these calcium isotopes are remarkably constant, indicating that these states are dominated by proton excitations of the calcium cores.
3) Specific excited states in 49,51,52Ca were analyzed, and spin and parity assignments were made or supported based on comparisons to previous reaction data and theoretical expectations.
PPT Describe elimination reactions, Evidences and mechanism of elimination reaction. Energy profile diagram. RELATIVE stabilities of intermediates. Comparison between E1 & E2 elimination reaction
The document discusses electron-acoustic solitary waves in a quantum plasma model that includes electrons at two different temperatures (hot and cold electrons) and stationary ions. It presents the following:
1) A quantum hydrodynamic model is used to derive fluid equations for a finite-temperature quantum plasma consisting of hot and cold electrons.
2) Linearizing the equations yields a dispersion relation for electron-acoustic waves that depends on factors like the quantum diffraction parameter and degeneracy parameter.
3) Nonlinear analysis is carried out to investigate solitary wave structures in the quantum plasma by deriving a Korteweg-de Vries equation.
This document presents the theory and simulation of EPR spectra of Cu2+ ions in Cd2(NH4)2(SO4)3 single crystals at different temperatures ranging from 15-180K. The author derives an expression for the derivative line shape function of the EPR spectra accounting for the orthorhombic Jahn-Teller effect of Cu2+. Computer simulation is performed by fitting the line shape function to the observed spectra. The simulation shows excellent agreement and allows extraction of spectroscopic parameters like g-values and hyperfine coupling constants. Analysis of the temperature-dependent spectra provides insight into the Jahn-Teller splitting and dynamics of Cu2+ in this system.
This document summarizes a study investigating how the oxidation state of the oxygen-evolving complex (OEC) in photosystem II affects proton-coupled electron transfer (PCET) reactions of the tyrosine Z radical (YZ•). The researchers measured the rate of YZ• recombination with QA- in the S0 and S2 states of the OEC using EPR spectroscopy. They found the rate was faster in S2 than S0 and that ammonia, which disrupts hydrogen bonding, slowed the rate more in S2. This suggests alterations in the hydrogen bonding network between YZ and the OEC influence the YZ midpoint potential and pKa during the S state cycle.
Stillwell_ Strongly coupled electronic, magnetic, and lattice degrees of free...Ryan Stillwell, Ph.D.
This document summarizes research on the ferromagnetic compound LaCo5 under high pressure. X-ray diffraction measurements show an anisotropic lattice collapse of the c axis near 10 GPa, consistent with theoretical predictions. High-pressure magnetotransport measurements reveal changes in the Hall effect signatures near 10 GPa, providing the first experimental evidence of changes in the electronic and magnetic properties associated with the predicted magnetoelastic collapse. The coupling of structural, electronic, and magnetic behaviors in LaCo5 under pressure substantiates the theoretical model of an electronic topological transition driving the magnetoelastic collapse.
This document discusses evidence for a possible shape transition from prolate to oblate in neutron-rich ruthenium isotopes based on spectroscopy experiments of 109,110,111,112Ru nuclei. The experiments extended existing level schemes to higher spin and excitation energy, allowing observation of band crossings. A band crossing was observed in 111Ru at a similar rotational frequency as the second band crossing in 112Ru, suggesting the alignment of proton g9/2 pairs and a triaxial to oblate shape transition in 111Ru. Comparisons are made to cranked shell model predictions to help interpret the results.
My_papers_Nastishin_PRL_2012_Elasticity of Lyotropic Chromonic Liquid Crystal...Myroslava Omelchenko
This document reports on a study that uses a magnetic Frederiks transition technique to measure the elastic constants (splay K1, twist K2, bend K3) of the lyotropic chromonic liquid crystal sunset yellow formed through reversible aggregation of organic molecules in water. The key findings are:
1) K1 and K3 are comparable in magnitude and about an order of magnitude higher than K2.
2) At higher concentrations and lower temperatures, K1 and the ratios K1/K3 and K1/K2 increase, which is attributed to elongation of the self-assembled lyotropic chromonic liquid crystal aggregates.
3) This concentration and temperature dependence of the elastic constants
Similar to 28th Eastern Regional Photosynthesis Conference (20)
LinkedIn Strategic Guidelines for June 2024Bruce Bennett
LinkedIn is a powerful tool for networking, researching, and marketing yourself to clients and employers. This session teaches strategic practices for building your LinkedIn internet presence and marketing yourself. The use of # and @ symbols is covered as well as going mobile with the LinkedIn app.
Delta International is an ISO Certified top recruiting agency in Pakistan, recognized for its highly experienced recruiters. With a diverse range of international jobs for Pakistani workers, Delta International maintains extensive connections with overseas employers, making it one of the top 10 recruitment agencies in Pakistan. It stands out in the list of recruitment agencies in Pakistan for its exceptional services.
https://www.ditrc.com/
Known for its expertise in the Gulf region, Delta International is among the top 10 international recruitment agencies, specializing in expert headhunting and candidate sourcing. This prominence places it in the list of top 10 overseas recruitment agencies in Pakistan. As one of the best overseas recruitment agencies in Pakistan, Delta International is a trusted name for manpower recruitment, particularly from Pakistan.
The agency is not just a leading name in Karachi but also recognized as one of the best recruitment agencies in Islamabad. Delta International consistently ranks as the top recruitment agency in Pakistan, earning its reputation among the top recruiting agencies in Pakistan. It is also regarded as one of the top overseas employment agencies in Pakistan.
For those seeking foreign jobs, Delta International is listed among the top overseas employment companies in Pakistan. Their extensive network and expertise make them a go-to for anyone looking at the list of overseas employment agencies in Pakistan. As a leading foreign jobs recruitment agency in Pakistan, they offer opportunities across various sectors.
Delta International is consistently listed among the top recruitment companies in Pakistan, known for providing the best recruitment services. It’s considered one of the best recruitment agencies in Pakistan and a prominent recruitment agency in Pakistan. The company excels in international recruitment, making it a key player among international recruitment agencies in Pakistan.
Their inclusion in the list of international recruitment agencies further attests to their excellence. As a top manpower agency in Pakistan, Delta International specializes in recruiting skilled professionals and labor for various industries, including construction, healthcare, IT, engineering, and hospitality.
Delta International is a leader among recruitment agencies in Pakistan, with a particular focus on overseas employment. They are one of the foremost overseas employment agencies in Pakistan, catering to technical jobs and other employment opportunities. Their role as overseas employment promoters highlights their commitment to connecting Pakistani talent with global opportunities.
In summary, Delta International is not only one of the best recruitment agencies in Pakistan but also a distinguished name among overseas employment agencies. Their extensive network and experienced recruiters make them a top choice for anyone seeking employment both locally and internationally.
Khushi Saini, An Intern from The Sparks Foundationkhushisaini0924
This is my first task as an Talent Acquisition(Human resources) Intern in The Sparks Foundation on Recruitment, article and posts.
I invitr everyone to look into my work and provide me a quick feedback.
We recently hosted the much-anticipated Community Skill Builders Workshop during our June online meeting. This event was a culmination of six months of listening to your feedback and crafting solutions to better support your PMI journey. Here’s a look back at what happened and the exciting developments that emerged from our collaborative efforts.
A Gathering of Minds
We were thrilled to see a diverse group of attendees, including local certified PMI trainers and both new and experienced members eager to contribute their perspectives. The workshop was structured into three dynamic discussion sessions, each led by our dedicated membership advocates.
Key Takeaways and Future Directions
The insights and feedback gathered from these discussions were invaluable. Here are some of the key takeaways and the steps we are taking to address them:
• Enhanced Resource Accessibility: We are working on a new, user-friendly resource page that will make it easier for members to access training materials and real-world application guides.
• Structured Mentorship Program: Plans are underway to launch a mentorship program that will connect members with experienced professionals for guidance and support.
• Increased Networking Opportunities: Expect to see more frequent and varied networking events, both virtual and in-person, to help you build connections and foster a sense of community.
Moving Forward
We are committed to turning your feedback into actionable solutions that enhance your PMI journey. This workshop was just the beginning. By actively participating and sharing your experiences, you have helped shape the future of our Chapter’s offerings.
Thank you to everyone who attended and contributed to the success of the Community Skill Builders Workshop. Your engagement and enthusiasm are what make our Chapter strong and vibrant. Stay tuned for updates on the new initiatives and opportunities to get involved. Together, we are building a community that supports and empowers each other on our PMI journeys.
Stay connected, stay engaged, and let’s continue to grow together!
About PMI Silver Spring Chapter
We are a branch of the Project Management Institute. We offer a platform for project management professionals in Silver Spring, MD, and the DC/Baltimore metro area. Monthly meetings facilitate networking, knowledge sharing, and professional development. For more, visit pmissc.org.
5 key differences between Hard skill and Soft skillsRuchiRathor2
𝐓𝐡𝐞 𝐏𝐞𝐫𝐟𝐞𝐜𝐭 𝐁𝐥𝐞𝐧𝐝:
𝐖𝐡𝐲 𝐘𝐨𝐮 𝐍𝐞𝐞𝐝 𝐁𝐨𝐭𝐡 𝐇𝐚𝐫𝐝 & 𝐒𝐨𝐟𝐭 𝐒𝐤𝐢𝐥𝐥𝐬 𝐭𝐨 𝐓𝐡𝐫𝐢𝐯𝐞 💯
In today's dynamic and competitive market, a well-rounded skillset is no longer a luxury - it's a necessity.
While technical expertise (hard skills) is crucial for getting your foot in the door, it's the combination of hard and soft skills that propels you towards long-term success and career advancement. ✨
Think of it like this: Imagine a highly skilled carpenter with a masterful understanding of woodworking (hard skills). But if they struggle to communicate effectively with clients, collaborate with builders, or adapt to project changes (soft skills), their true potential remains untapped. 😐
The synergy between hard and soft skills is what creates true value in the workplace. Strong communication allows you to clearly articulate your technical expertise, while problem-solving skills help you navigate complex challenges alongside your team. 💫
By actively developing both sets of skills, you position yourself as a well-rounded professional who can not only perform tasks efficiently but also contribute meaningfully to a collaborative and dynamic work environment.
Go through the carousel and let me know your views 🤩
1. Site Specific Mutagenesis Reveals a Critical Role of Histidine 252 of the D1 subunit in the Two-Electron Gate of Photosystem II Sean Padden1, Jun Minagawa2, Atsuko Kanazawa3, Govindjee4, Antony Crofts5 1Program in Physiological and Molecular Plant Biology , School of Integrative Biology, University of Illinois at Urbana-Champaign, 286 Morrill Hall, 505 South Goodwin Ave., Urbana, IL 61801, USA; 2Division of Environmental Photobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan 3: Michigan State University, East Lansing, MI 48824-1312 USA; 4 Department of Plant Biology, University of Illinois at Urbana-Champaign 265 Morrill Hall, 505 S. Goodwin Ave., Urbana IL 61801, USA5: Department of Biochemistry, University of Illinois at Urbana-Champaign, 419 Roger Adams Lab, 600 S. Morrill Ave., Urbana IL 61801, USA Abstract: Site-directed mutagenesis of histidine 252 in the QB site of the D1 protein in Photosystem II of the green alga Chlamydomonas reinhardtii has revealed a plausible route for the first proton transfer to the plastosemiquinone at the QB site. When D1H252 was mutated, only one mutation retained photosynthetic capacity (H252D) while three others were rendered non-photosynthetic (H252K, N, Q). In all mutants, the first electron transfer from QA- to QB was altered to various degrees, the timing of the back-reaction from the acceptor side to the oxygen evolving complex was modified in the presence of diuron, and only the H252D mutant could produce oxygen under constant light. Thermoluminescence data from the H252 mutants showed changes in the thermodynamic profile of the QB-site and an effect on the QA-/S2equilibrium constant. The results suggest that H252 facilitates protonic stabilization of the semiquinone intermediate, and is required for H+ transfer associated with the reduction of QB- on the second electron transfer. When compared to the QB site of the bacterial reaction center, a semi-conserved route of proton transfer is revealed. Table 1: Kinetic parameters determined from first flash of the fluorescence decay curves of the WT and mutant strains of C. reinhardtii Introduction: The two – electron gate is the current paradigm for the electron transfer on the acceptor side of Photosystem II (PS II) (Bouges-Bocquet, 1973; Velthyus, 1974, 1981). In this model, the tightly bound quinone QA passes electrons in two sequential transfers to the weakly bound quinone at the QB site. Electron transfer from QA- to QB produces a semiquinone anion (QB-) which is stabilized at the QB site. On the basis of a model to account for the pH dependence of the QAQB reaction (Robinson et. al., 1984), Crofts et al., (1987) suggested that D1H252 in the D1 protein of PSII might be the ‘putative ligand for the proton which stabilizes the semiquinone’. This proposal was based on a model of the PSII acceptor side using the B. viridis structure (Michel et. al., 1986) as a template for the D1 protein from Anacystisnidulans(Golden et. al. 1985). In the bacterial reaction center (BRC) the route for the first protonation event has been termed the ‘serine hydrogen bond switch’ (Wraight, 2004). In this mechanism when in the dark LS223 is hydrogen bonded to LD213, and the quinone is in the distal position. When exposed to light LD213 protonates allowing LS223 to hydrogen bond to the quinone, forming a network from LD213 – LS223 –QBfor proton transfer as the quinone moves into the proximal position (Fig. 1A). In PSII the crystal structures (Loll, et. al. 2005, Guskov et. al. 2009) are in agreement with the model of Crofts et al. 1987 where D1H252 corresponds to LD213 and D1S264 corresponds to LD223. Thus a similar system exists in PSII (Fig. 1B) but with one caveat. In the BRC LE212 protonates on electron transfer stabilizing the anionic semiquinone (Paddock et. al. 1989), but the QB site of PSII does not have an equilivalent residue. Instead D1H252 must serve as both the stabilizing influence for the anionic semiquinone and the initial proton. In order to test if D1H252 was the ligand used to stabilize the semiquinone as proposed we mutated the residue in the unicellular green alga, ChlamydomonasreinhardtiiThe mutations constructed were D1H252D, D1H252K, D1H252N and D1H252Q. In each of these mutants the following parameters were measured: QA- fluorescence decay out to 10 ms (Fig. 2), the binary oscillation of the two-electron gate (Fig. 3), production of oxygen under continuous light (Table 1), the kinetics of back reaction from DCMU inhibited cells (Fig. 4), and the thermoluminescence properties of the acceptor side of PSII (Fig. 5). These measurements were used to calculate the binding constants of quinone at the QB site, the individual rate constants of the electron transfer, and the overall equilibrium constant for the QAQB electron transfer (Crofts et. al. 1993, Petrouleas et. al. 2005 and see the Kinetics section). ‘The Acetate Effect’: The acetate effect on the growth of Chlamydomonas is of paramount importance to the study of the D1H252 mutations. As E. coli preferentially chooses glucose over fructose as a carbon source when mixed, Chlamydomonas will choose acetate over photosynthetic growth (Heifitz, 2000). Since all D1H252 mutations are lethal (even the D1H252D) once the acetate is consumed the mutants begin a downward spiral towards death. This makes analysis of the kinetics of the two electron gate problematic . The acetate effect may even have an effect on the WT strain, as the photosynthetic capacity of the organism will mature as the acetate is consumed. AO and BO are the amplitudes of the slow and fast phase respectively obtained directly from the fluorescence decay curves (Fig. 1 panel A – E). Ft-(AO+BO) is the residual of the curves and is equal to “C” in the binary decay equation: y = AO(e-x*r1) + BO(e-x*r2) + C. KO is the ratio (AO/BO), r1 and r2 are the rates of electron transfer for the slow and fast phase, while t1 and t2 are the half-times for the slow and fast phase respectively. (Crofts et. al. 1993, Petrouleas, 2005) Results: Kinetic Analysis of the D1H252 Mutants: Kinetic analysis of the two-electron gate in Photosystem II, based on the data in Figs. 2, 3, and 4 for the WT and the D1H252 mutants, is presented in Table 1 above. The collected data shows the D1H252 mutants affect the slow and fast rates of electron transfer; where the rate for electron transfer for the fast phase fit of the D1H252K, D1H252N and D1H252Q mutants at pH 7.0 was similar to that of WT, with similar half-times; whereas in the D1H252D mutant the half time of the fast phase was more than doubled. In the case of the slow phase, the D1H252D, D1H252N, and D1H252Q had similar half times, whereas the D1H252K increased the half time more than five times longer than the WT. Fig. 5: TL analysis of the WT and D1H252 mutant strains of C. reinhardtii WT and D1H252 mutant cells. Fig 5A and 5B: black: H252D; red: H252K; blue: H252Q; teal: pBA158 (WT): A: TAP pH 7.0 100 µM BQ wash, 10 µM 18crown6; B: as A plus 1 µM DCMU C: Fig 5C: mutant – WT from Fig 5A; Fig 5D: mutant – WT from Fig 5B where black: H252D; red: H252K; blue: H252Q. Results: Thermoluminesence: Thermoluminescence was measured on the D1H252 mutants and the wild type cells. A major observation was that the B-band of thermoluminescence (due to S2/QBrecombination, Rutherford et al. (1982)), from the mutants was shifted to lower temperatures (Fig. 5A, Table IV),while the position of the Q-band remained unchanged (Fig. 5B, Table IV). When the WT data was subtracted from the mutant data, scaled and normalized, the position of the Q-Band did not change, and the height of the peaks represent the population of electrons left on QA- (Fig 5C). When the WT curve was subtracted from the mutant curve in the presence of DCMU (Fig 5D) it was observed that the height of the Q-band relative to the WT was increased when a negative charge was placed in the QB-site (D1H252D) leaving a positive peak, and decreased when a positive charge was placed in the QB-site (D1H252K), forming a negative trough. The D1H252Q strain retains a Q-band which has a Tm much lower than the other two mutants, and also displays a B-band indicating DCMU resistance. Results: Chlorophyll a fluorescence decay. [When the D1H252 mutants were struck on media without a reduced carbon source, the WT strain showed colonies in ~ 14 days, the D1H252D mutation in ~ 21 days, while the D1H252K, N, and Q mutants failed to show colonies out to ~28 days (data not shown). ] After dark adaptation, the D1H252D mutant showed electron transfer from QA- to QB on both the odd and even flashes (Fig. 2. panel B), but the rate of the electron transfer after the first flash was slower than after the second flash, leading to a reversed phase pattern of the binary oscillation at the two-electron gate (Fig.3 panel A). The D1H252N and D1H252Q mutants showed electron transfer from QA- to QB with reduced yield on the first flash; and both were severely inhibited on the second flash (Fig. 2 panel D, E). The D1H252K mutant was severely inhibited even on the first flash. (Fig. 2, panel C). In addition to D1H252D only the D1H252Q mutation had any binary oscillation present. (Fig. 3, panel d) in the same phase as the WT (WT curve not shown). The data derived from the fluorescence decay is presented in Table 1 in the Kinetics section) Results : Kinetic Analysis of the D1 H252 mutants (cont.) The effect of the D1H252 mutations can be seen in the equilibrium constants KO, KE and Kapp (Table 2 below, see scheme above for the description of the equilibrium constants). In the case of KO, it appears as if each of the mutations, except the D1H252N mutation, has an effect on the binding of plastoquinone to the QB site. The D1H252K mutation had the most dramatic effect, decreasing the binding of plastoquinone by almost twenty times, thereby practically eliminating the fast phase of kinetics (Fig. 2, panel B), and binary oscillation (Fig 3, panel B). The D1H252Q mutant inhibited plastoquinone binding by only a factor of ~3, whereas the D1H252D mutant was approximately five times worse than the WT. The D1H252N mutant showed a similar affinity for quinone binding, yet was non-photosynthetic. The effect of the mutations on the binding of the plastoquinone also had an impact on the equilibrium constant between QA-QB and QAQB- (KE). As seen in Table 2, in the WT the QAQB- state is heavily favored with a KE value of ~ 29, while both the D1H252D and D1H252Q values for KE were much closer to equilibrium, 4.9 and 3.9 respectively. As the apparent equilibrium constant, Kapp is calculated from KO and KE, it follows that changes which affect the binding of plastoquinone in the QB-site along with an increase in the QA-QB population relative to the QAQB- population will lead to a decrease in Kapp. Table 4: Determination of maximal peak height at temperature of the thermoluminescence bands Table 2: Parameters of the two electron gate in wild type and mutant strain of C. reinhardtii Concluding Remarks: A Hypothesis The effect of the QB-site mutations on the timing of the back reaction QA/S2 is dependent on the charge, polarity and position of the mutation relative to the plastoquinone molecule. Under the assumption that DCMU binds similar to plastoquinone, the model interaction is presented in Fig 6. The WT model is presented in Fig. 6,panel a, and shows the ‘serine hydrogen bond switch’ in PSII where a hydrogen bond network travels from D1H252 to D1S264 and onto O1 carbonyl group of the plastoquinone (O1). The D1H252D (Fig. 6, panel B) mutation disrupts this network by forming a second hydrogen bond to O1, and eliminating the D1H252 to D1S264 hydrogen bond. The D1H252K (Fig. 6, panel C) mutation can form a hydrogen bond to O1, but the lysine’s pkA is not suited to lose a proton. Correspondingly both of these mutations introduce a charge in to QB-site and rapidly increase the rate of the QA-/S2 back reaction (Fig. 4), yet have an opposite effect on the height of the TL peak in the presence of DCMU (Fig. 5, panel D). The D1H252N was the only mutation to have two minimum energy states for the model (Fig. 6, panels d and e). The D1H252N mutant was also the least stable (hence no TL analysis). The two models show that the hydrogen bond between D1S264 and D1N252 is disrupted (Fig. 6, panel d) or weak (Fig. 6, panel e). In either case the D1H252N mutation will not release a proton to D1S264. The D1H252N mutation speeds up the QA-/S2 back reaction by about 2x compared to WT (Fig. 4). The extra –CH2– group in the side chain of the D1H252Q mutant forces that mutant away from the O1, precluding the possibility of a hydrogen bond (Fig. 6, panel f). Without any quinone interaction, the half time of the QA/S2 back reaction is most similar to WT (Fig. 4). It is the lack of protonation of the anionic semiquinone in the D1H252K, D1H252N, and D1H252Q mutants which leads to the strains being non-photosynthetic and dependent on acetate for survival. The effect of mutation of the ‘serine hydrogen bond shift’ (Wraight, 2004) is dependent on the position of the mutation. If the ‘front end’ of the switch is mutated (LD213 or D1H252 Fig. 1A and 1B) R. sphaeroides will resort to suppressor mutations in order to survive. No suppression mutants for the D1H252 mutations were seen in C. reinhardtii. Conversely, if the ‘back-end’ of the serine hydrogen bond switch (LS223 or D1S264 Fig 1A and 1B) is mutated there is an effect on the two-electron gate but both R. sphaeroides and C. reinhardtii survive without further modification. This relationship further emphasizes the importance of the ‘front-end’ of the ‘serine hydrogen bond switch’ in its relation to the first proton transfer to the anionic semiquinone at the QB-site. Fig 1: VMD model of the ‘serine hydrogen bond switch’ (Wraight, 2004) . Final assembly of each figure was performed by Photoshop CS5 Extended. 1A: PDB 1AIG, (Stowell et. al. 1997). Bacterial reaction center. The green surf and coil (transparent) corresponds to the L sub-unit, the ice-blue surf represents the M sub-unit, and the blue surf shows the H sub-unit. The ubiquinone molecule is drawn as ‘licorice’, with the highlighted residues drawn as ball and stick. Carbon is yellow, oxygen red, nitrogen blue. The white dashed lines are modeled hydrogen bonds between the residues with the distance posted close by. 1B: PDB 3BZ1 (Guskov et. al. 2009). Photosystem II. The green surf and coil (transparent) corresponds to the D1 protein and the ice-blue surf represents the D2 protein. The plastoquinone molecule is drawn as ‘licorice’ with the highlighted residues drawn as ball and stick. Carbon is yellow, oxygen red, nitrogen blue. The white dashed lines are modeled hydrogen bonds between the residues with the distance posted close by. The t1/2 QA is the half-time of the DCMU back reaction QA- S2 (Fig. 4), and the t1/2 QB is the half-time of the back reaction from QB S2 obtained from the rephrasing of the binary oscillation (Fig. 3). KO is equilibrium constant for the binding of the semiquinone to the QB-site, KE is the equilibrium constant for the sharing of the electron between QA and QB, Kapp is the apparent equilibrium constant between QA and QB. The rate constants kAB, kBA, kAV, and kVA are for the forward and reverse rate constants of the KE and KO equilibrium constants respectively. The binary oscillation for the D1H252K and D1H252N mutants was not detected (N/D). Thus the kinetic parameters could not be calculated and are not available (N/A) (Crofts et. al. 1993, Petrouleas et al., 2005). Results: Oxygen Evolution Steady-state oxygen evolution activity of the cells was measured in TAP medium with a Clark-type oxygen electrode in the presence of 0.5 mM 2,5-dimethyl-p-benzoquinone (DMBQ) and 2 mM K3Fe(CN)6 at 25°C under illumination at 3,000 μmol photons m−2 s−1 using dichlorophenol indophenol (DCPIP) as an electron acceptor. Each strain was assayed in at least two separate cultures, with an average of three measurements for a single culture. Oxygen evolution data show that the pBA158 strain produced the most oxygen at 262 µmol O2 mg[Chl]-1 / hr, while the D1H252D mutant produced ~50% of the WT value, ~145 µmol O2 mg[Chl]-1/ hr.The D1H252N and D1H252Q mutants failed to produce any oxygen. (Table 3). We conclude that the disruption of oxygen production was a result of the D1H252 mutations on the equilibrium constant between QA and S2 of the OEC. Figure 3: Binary oscillation in chlorophyll a fluorescence decay, measured at 195 µs after the flash, as a function of flash number in mutant strains of Chlamydomonas reinhardtii (ac-µ-ε pBA158 and pBA155). Flashes were given at 1 Hz. Panel A:D1H252D; panel B:D1H252K; panel C:D1H252N; and panel D:D1H252Q. Whole cells of the mutant strains were treated as in Figure 1, with the inclusion of 100 µM NH4Cl as done with the WT. For panel A, the delay between the actinic flash and the first flash is: 0.8 s (black squares), 0.9 s (open squares), 1 s (black circles), and 5s (open circles). For panelsB, C and D, the delay is 1s (black squares), 5 s (open squares), 10 s (black circles), 20 s (open circles), and 30 s (black triangles). Each curve is an average of 3 samples from 7 day old secondary cultures, with an average [Chl] of 7 - 10 µg/mL. All binary oscillation curves are offset for clarity. Fig 2 The [QA-] fluorescence decay of the C. reinhardtii pBA158 strain and the D1H252 mutants where the first flash is a black line with black squares and the second flash is a red line with red circles. Panel A: pBA158 (WT); panel BD1H252D; panel CD1H252K; panel DD1H252N; panel ED1H252Q. Table 3: Values for the production of oxygen, with DCPIP as an electron acceptor, in cells of C. reinhardtii Results: The QA/S2 Back Reaction When the WT and mutants strains were treated with 10 µM DCMU the timing of the back-reaction from the acceptor side to the oxygen evolving complex was modified (Fig. 4, Table 3) in each of the mutants. The D1H252D and D1H252K mutants had the fastest back reaction, ~5X sooner than WT. The D1H252N had a back reaction ~ one-half that of WT, where the D1H252Q mutant had the most similar time. Fig, 6: The PSII crystal structure 2AXT from (Loll et al., 2005) was used to model the QB site of PSII. Using the mutation analysis software within Swiss PBV Viewer 4.01 PC, the mutation was modeled in place of the WT histidine. The energy minimization was performed by Swiss PDB-viewer and the figures were re-modeled using POV-Ray 6.2 software. The images created were assembled and converted to .tiff format using Photoshop CS4 Extended. The golden helices indicate the helices d (with H215), and de (with H252 and S264) of the D1 protein of PSII with the random coil between the helices colored brown. Panel a:D1H252 (WT); panel b:D1H252D; panel c:D1H252K, panel d:D1H252N 1st minimization; panel e:D1H252N 2nd minimization; panel f:D1H252Q. The dashed green lines are modeled hydrogen bonds, the dashed purple line is a modeled steric hindrance (D1H252N panel d), and the dashed gray line is a weak hydrogen bond due to distance (D1H252N panel e). Only the D1H252N has two images as it was the only mutant to have two equal calculated energy minimizations. References: Bouges-Bocquet, B. (1973). "Electron transfer between the two photosystems in spinach chloroplasts." Biochimica et Biophysica Acta 314(2): 250-256. Crofts, A., I. Baroli, et al. (1993). "Kinetics of Electron Transfer Between QA and QB in Wild Type and Herbicide-Resistant Mutants of Chlamydomonas reinhardtii." Z. Naturforsch48c: 259-266. Crofts, A., H. Robinson, et al., Eds. (1987). Catalytic Sites for Reduction and Oxidation of Quinones. Cytochrome Systems: Molecular Biology and Bioenergetics, Plenum Publishing Corporation, New York. Michel H, Epp O, J. 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"Pathway of proton transfer in bacterial reaction centers: replacement of glutamic acid 212 in the L subunit by glutamine inhibits quinone (secondary acceptor) turnover.“ PNAS 86(17): 6602-6606. Petrouleas, V. and A. Crofts, Eds. (2005). The Iron-Quinone Acceptor Complex. Photosystem II: The Light-Driven Water:Plastoquinone Oxidoreductase. Dordrecht, The Netherlands, Springer. Robinson, H. H. and A. R. Crofts (1983). "Kinetics of the oxidation--reduction reactions of the photosystem II quinone acceptor complex, and the pathway for deactivation." FEBS Letters153(1): 221. Rutherford, A. W., A. R. Crofts, et al. (1982). "Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks." Biochimica et Biophysica Acta (682(3): 457. Stowell, M. H. B., T. M. McPhillips, et al. (1997). "Light-Induced Structural Changes in Photosynthetic Reaction Center: Implications for Mechanism of Electron-Proton Transfer." Science276(5313): 812-816. Velthuys, B. and J. Amesz (1974). "Charge accumulation at the reducing side of system 2 of photosynthesis." Biochimica et Biophysica Acta333(1): 85-94. Velthuys, B. R. (1981). "Electron-dependent competition between plastoquinone and inhibitors for binding to photosystem II." FEBS Letters126(2): 277-281. Wraight, C. A. (2004). "Proton and Electron Transfer in the Acceptor Quinone Complex of Photosynthetic Reaction Centers from Rhodobacter sphaeroides." Frontiers in Biosciences9: 309-337. Fig. 4. Variable Chl a fluorescence decay Whole cells of four mutants (H252D, H252K, H252N, H252Q) and the wild type (pBA158) C. reinhardtii were treated as in Fig 3. DCMU was added to a final concentration of 10 μM. A single actinic flash was delivered after 10 min dark adaptation with the DCMU present. A train of measuring pulses was used up to 10 seconds. Ft is fluorescence at time t; and F0 is the initial minimal fluorescence. [Chl], 7 -10 μg/mL. Half-times are reported in Table 3. curves in the presence of DCMU.