Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different... more Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics.
The mechanism for carbon-based superoxide dismutase (SOD) nano-mimetics is not known, hindering o... more The mechanism for carbon-based superoxide dismutase (SOD) nano-mimetics is not known, hindering optimization of this potentially clinically useful feature. Here we studied oxidized activated charcoal (OAC) prepared by fuming nitric acid oxidation of activated charcoal and characterized its properties. The OAC nanoparticles have size 5-nm and are highly water-soluble. The OACs are strong SOD mimetics with a k cat = 2.1 × 10 5 s-1 at pH 12.7 and ~ 10 8 M-1 s-1 rate constants at pH 8.5, having great potential in therapeutic application for disorders with pathological superoxide levels. Electron paramagnetic resonance (EPR) indicates that resting OACs are fully oxidized, exhibiting a stoichiometric level of intrinsic radical. The OACs can be reduced with superoxide, leading to a decreased level of intrinsic radical; however, the reduction is incomplete even at high superoxide levels. This outcome was predicted by a simple two-step SOD reaction mechanism using the species containing the intrinsic radical as the fully oxidized state. Pretreatment of the OACs with superoxide causes little change to its SOD activity, indicating a stoichiometric amount of intrinsic radical is not mandatory for full activity. This study indicates direct participation of the intrinsic radical in the catalytic turnover of a highly active SOD-like nanozyme. Keywords nanozyme, superoxide dismutase mimetics, oxidized activated charcoal, intrinsic radical, good manufacturing practice An important field of application of many nanomaterials is based on their enzyme-like activities. These enzyme mimetic nanomaterials are referred to as nanozymes and they exhibit a wide range of enzyme-like activities such as peroxidase, oxidase, catalase, superoxide dismutase (SOD), and many others. 1,2 Among these, nano-SOD mimetics are of particular interest owing to their
Journal of the American Chemical Society, Jan 6, 2019
The precise size-and shape-controlled synthesis of monodisperse Al nanocrystals remains an open c... more The precise size-and shape-controlled synthesis of monodisperse Al nanocrystals remains an open challenge, limiting their utility for numerous applications that would take advantage of their size and shape-dependent optical properties. Here we pursue a molecular-level understanding of the formation of Al nanocrystals by titanium(IV) isopropoxide-catalyzed decomposition of AlH3 in Lewis base solvents. As determined by electron paramagnetic resonance spectroscopy of intermediates, the reaction begins with the formation of Ti 3+-AlH3 complexes. Proton nuclear magnetic resonance spectroscopy indicates isopropoxy ligands are removed from Ti by Al, producing aluminum(III) isopropoxide and low-valent Ti 3+ catalysts. These Ti 3+ species catalyze elimination of H2 from AlH3 inducing the polymerization of AlH3 into colloidally unstable lowvalent aluminum hydride clusters. These clusters coalesce and grow while expelling H2 to form colloidally stable Al nanocrystals. The colloidal stability of the Al nanocrystals and their size are determined by the molecular structure and density of coordinating atoms in the reaction, which is controlled by choice of solvent composition.
bioRxiv (Cold Spring Harbor Laboratory), Mar 18, 2023
Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated longchain fatt... more Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated longchain fatty acid and the reaction is catalyzed by a diiron center, which is well-coordinated by conserved histidine residues and is thought to remain with enzyme. However, we find that SCD1 progressively loses its activity during catalysis and becomes fully inactive after nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center, and that the addition of free ferrous ions (Fe 2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe 2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe 2+ in cells could regulate SCD1 activity, and hence lipid metabolism. .
EAAT3 is expressed not only throughout the brain, but in many organs such as intestines, liver an... more EAAT3 is expressed not only throughout the brain, but in many organs such as intestines, liver and heart. Here it seems to provide the main pathway of aspartate. Several connections of EAAT3 to severe neuronal disorders like epilepsy and schizophrenia have been described, as well as to metabolic disturbances concerning in the maintenance of aspartate and cysteine levels. This makes EAAT 3 not only an important target for functional research, but also a potential drug target. Here, we present data on EAAC1, a mouse homologue of EAAT3, using a novel high throughput instrument for SSM (solid supported membrane)based electrophysiology, the SURFE 2 R 96SE. SSM-based electrophysiology is a label-free electrical measuring method with very high sensitivity which enables the resolution of low turnover transport and even binding-events. Using the purified membrane of EAAC1 expressing CHO cells, we were able to determine substrate affinities and their interaction and to compare the effect of six known inhibitors directly with each other. We evaluated the assay stability and success rate. Furthermore, we were able to resolve substrate binding and to confirm the described anion conductance of the transporter. Using the SURFE 2 R 96SE with CHO cells we were able to generate an efficient, robust and very flexible assay, which is an ideal tool for the biophysical and pharmacological characterization of EAAC1 and even suitable for drug screening approaches.
Mammalian cytochrome b 5 (cyt b 5) and cytochrome b 5 reductase (b 5 R) are electron carrier prot... more Mammalian cytochrome b 5 (cyt b 5) and cytochrome b 5 reductase (b 5 R) are electron carrier proteins for membrane-embedded oxidoreductases. Both b 5 R and cyt b 5 have a cytosolic domain and a single transmembrane (TM) helix. The cytosolic domains of b 5 R and cyt b 5 contain cofactors required for electron transfer, but it is not clear if the TM helix has function beyond being an anchor to the membrane. Here we show that b 5 R and cyt b 5 form a stable binary complex, and so do cyt b 5 and stearoyl-CoA desaturase-1 (SCD1). We also show that b 5 R, cyt b 5 and SCD1 form a stable ternary complex. We demonstrate that the TM helices are required for the assembly of stable binary and ternary complexes where electron transfer rates are greatly enhanced. These results reveal a role of the TM helix in cyt b 5 and b 5 R, and suggest that an electron transport chain composed of a stable ternary complex may be a general feature in membrane-embedded oxidoreductases that require cyt b 5 and b 5 R.
Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and-2) each have a peroxidase activity and also... more Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and-2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and-2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4°c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k 1) was 2.3 ؋ 10 7 M ؊1 s ؊1 for PGHS-1 and 2.5 ؋ 10 7 M ؊1 s ؊1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k 2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10 2-10 3 s ؊1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k 2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.
The complexity of Alzheimer's disease (AD) complicates the search for effective treatments. While... more The complexity of Alzheimer's disease (AD) complicates the search for effective treatments. While the key roles of pathologically modified proteins has occupied a central role in hypotheses of the pathophysiology, less attention has been paid to the potential role for transition metals Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fat... more Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid and the reaction is catalyzed by a diiron center, which is well-coordinated by conserved histidine residues and is thought to remain with enzyme. However, we find that SCD1 progressively loses its activity during catalysis and becomes fully inactive after nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center, and that the addition of free ferrous ions (Fe2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe2+is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe...
Biochimica Et Biophysica Acta - Bioenergetics, Feb 1, 1983
Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint p... more Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome ct, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and-20 mV at room temperature (MCD) compared to 116 and-4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 inV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23°C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c I was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycbolate buffers, while a variation of-30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in E m of greater than 300 mV was found in potentiometric measurements of cytochrome c I at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c t but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by uhiquinone.
The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alp... more The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alpha-acetyl microperoxidase-8 (Ac-MP-8) have been studied by examining its optical, resonance Raman, electron paramagnetic resonance, and magnetic circular dichroism spectra. An optical titration indicates that Ac-MP-8 exists in three major ionization forms over the pH 1-12 range that are linked by pK alpha values of approximately 3 and 9. The acid form that is present at pH 1.5 exists as a mixture of five- and six-coordinate high-spin species and most likely has water or buffer ions as axial ligand(s). On titration to pH 7, the His18 residue is deprotonated and becomes the proximal ligand to the iron to give a six-coordinate neutral form that has water as the sixth ligand. This form exists in a thermal high-spin intermediate-spin state equilibrium. On raising the pH to 10, an alkaline form is generated which is predominantly a five-coordinate high-spin species. It is formed by ionization of the proximal His18 residue to its imidazolate form with concomitant dissociation of the water ligand at the sixth site. At concentrations of Ac-MP-8 greater than 10 microM, some six-coordinate low-spin species are formed that are attributed to a dimer in which a His18 residue from a second molecule of Ac-MP-8 coordinates to the sixth site of another to give a bis-His complex. Raising the pH to 11.5 does not produce an appreciable amount of the six-coordinate complex with hydroxide as the sixth ligand. These studies show that Ac-MP-8 is a good water-soluble model for the peroxidases that exhibits minimal aggregation at concentrations below 10 microM in the neutral and alkaline pH regions.
ABSTRACT Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of pro... more ABSTRACT Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[35S]methionine incorporation into PGH synthase that was induced by IL-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.
Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different... more Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics.
The mechanism for carbon-based superoxide dismutase (SOD) nano-mimetics is not known, hindering o... more The mechanism for carbon-based superoxide dismutase (SOD) nano-mimetics is not known, hindering optimization of this potentially clinically useful feature. Here we studied oxidized activated charcoal (OAC) prepared by fuming nitric acid oxidation of activated charcoal and characterized its properties. The OAC nanoparticles have size 5-nm and are highly water-soluble. The OACs are strong SOD mimetics with a k cat = 2.1 × 10 5 s-1 at pH 12.7 and ~ 10 8 M-1 s-1 rate constants at pH 8.5, having great potential in therapeutic application for disorders with pathological superoxide levels. Electron paramagnetic resonance (EPR) indicates that resting OACs are fully oxidized, exhibiting a stoichiometric level of intrinsic radical. The OACs can be reduced with superoxide, leading to a decreased level of intrinsic radical; however, the reduction is incomplete even at high superoxide levels. This outcome was predicted by a simple two-step SOD reaction mechanism using the species containing the intrinsic radical as the fully oxidized state. Pretreatment of the OACs with superoxide causes little change to its SOD activity, indicating a stoichiometric amount of intrinsic radical is not mandatory for full activity. This study indicates direct participation of the intrinsic radical in the catalytic turnover of a highly active SOD-like nanozyme. Keywords nanozyme, superoxide dismutase mimetics, oxidized activated charcoal, intrinsic radical, good manufacturing practice An important field of application of many nanomaterials is based on their enzyme-like activities. These enzyme mimetic nanomaterials are referred to as nanozymes and they exhibit a wide range of enzyme-like activities such as peroxidase, oxidase, catalase, superoxide dismutase (SOD), and many others. 1,2 Among these, nano-SOD mimetics are of particular interest owing to their
Journal of the American Chemical Society, Jan 6, 2019
The precise size-and shape-controlled synthesis of monodisperse Al nanocrystals remains an open c... more The precise size-and shape-controlled synthesis of monodisperse Al nanocrystals remains an open challenge, limiting their utility for numerous applications that would take advantage of their size and shape-dependent optical properties. Here we pursue a molecular-level understanding of the formation of Al nanocrystals by titanium(IV) isopropoxide-catalyzed decomposition of AlH3 in Lewis base solvents. As determined by electron paramagnetic resonance spectroscopy of intermediates, the reaction begins with the formation of Ti 3+-AlH3 complexes. Proton nuclear magnetic resonance spectroscopy indicates isopropoxy ligands are removed from Ti by Al, producing aluminum(III) isopropoxide and low-valent Ti 3+ catalysts. These Ti 3+ species catalyze elimination of H2 from AlH3 inducing the polymerization of AlH3 into colloidally unstable lowvalent aluminum hydride clusters. These clusters coalesce and grow while expelling H2 to form colloidally stable Al nanocrystals. The colloidal stability of the Al nanocrystals and their size are determined by the molecular structure and density of coordinating atoms in the reaction, which is controlled by choice of solvent composition.
bioRxiv (Cold Spring Harbor Laboratory), Mar 18, 2023
Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated longchain fatt... more Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated longchain fatty acid and the reaction is catalyzed by a diiron center, which is well-coordinated by conserved histidine residues and is thought to remain with enzyme. However, we find that SCD1 progressively loses its activity during catalysis and becomes fully inactive after nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center, and that the addition of free ferrous ions (Fe 2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe 2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe 2+ in cells could regulate SCD1 activity, and hence lipid metabolism. .
EAAT3 is expressed not only throughout the brain, but in many organs such as intestines, liver an... more EAAT3 is expressed not only throughout the brain, but in many organs such as intestines, liver and heart. Here it seems to provide the main pathway of aspartate. Several connections of EAAT3 to severe neuronal disorders like epilepsy and schizophrenia have been described, as well as to metabolic disturbances concerning in the maintenance of aspartate and cysteine levels. This makes EAAT 3 not only an important target for functional research, but also a potential drug target. Here, we present data on EAAC1, a mouse homologue of EAAT3, using a novel high throughput instrument for SSM (solid supported membrane)based electrophysiology, the SURFE 2 R 96SE. SSM-based electrophysiology is a label-free electrical measuring method with very high sensitivity which enables the resolution of low turnover transport and even binding-events. Using the purified membrane of EAAC1 expressing CHO cells, we were able to determine substrate affinities and their interaction and to compare the effect of six known inhibitors directly with each other. We evaluated the assay stability and success rate. Furthermore, we were able to resolve substrate binding and to confirm the described anion conductance of the transporter. Using the SURFE 2 R 96SE with CHO cells we were able to generate an efficient, robust and very flexible assay, which is an ideal tool for the biophysical and pharmacological characterization of EAAC1 and even suitable for drug screening approaches.
Mammalian cytochrome b 5 (cyt b 5) and cytochrome b 5 reductase (b 5 R) are electron carrier prot... more Mammalian cytochrome b 5 (cyt b 5) and cytochrome b 5 reductase (b 5 R) are electron carrier proteins for membrane-embedded oxidoreductases. Both b 5 R and cyt b 5 have a cytosolic domain and a single transmembrane (TM) helix. The cytosolic domains of b 5 R and cyt b 5 contain cofactors required for electron transfer, but it is not clear if the TM helix has function beyond being an anchor to the membrane. Here we show that b 5 R and cyt b 5 form a stable binary complex, and so do cyt b 5 and stearoyl-CoA desaturase-1 (SCD1). We also show that b 5 R, cyt b 5 and SCD1 form a stable ternary complex. We demonstrate that the TM helices are required for the assembly of stable binary and ternary complexes where electron transfer rates are greatly enhanced. These results reveal a role of the TM helix in cyt b 5 and b 5 R, and suggest that an electron transport chain composed of a stable ternary complex may be a general feature in membrane-embedded oxidoreductases that require cyt b 5 and b 5 R.
Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and-2) each have a peroxidase activity and also... more Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and-2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and-2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4°c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k 1) was 2.3 ؋ 10 7 M ؊1 s ؊1 for PGHS-1 and 2.5 ؋ 10 7 M ؊1 s ؊1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k 2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10 2-10 3 s ؊1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k 2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.
The complexity of Alzheimer's disease (AD) complicates the search for effective treatments. While... more The complexity of Alzheimer's disease (AD) complicates the search for effective treatments. While the key roles of pathologically modified proteins has occupied a central role in hypotheses of the pathophysiology, less attention has been paid to the potential role for transition metals Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fat... more Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid and the reaction is catalyzed by a diiron center, which is well-coordinated by conserved histidine residues and is thought to remain with enzyme. However, we find that SCD1 progressively loses its activity during catalysis and becomes fully inactive after nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center, and that the addition of free ferrous ions (Fe2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe2+is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe...
Biochimica Et Biophysica Acta - Bioenergetics, Feb 1, 1983
Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint p... more Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome ct, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and-20 mV at room temperature (MCD) compared to 116 and-4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 inV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23°C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c I was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycbolate buffers, while a variation of-30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in E m of greater than 300 mV was found in potentiometric measurements of cytochrome c I at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c t but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by uhiquinone.
The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alp... more The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alpha-acetyl microperoxidase-8 (Ac-MP-8) have been studied by examining its optical, resonance Raman, electron paramagnetic resonance, and magnetic circular dichroism spectra. An optical titration indicates that Ac-MP-8 exists in three major ionization forms over the pH 1-12 range that are linked by pK alpha values of approximately 3 and 9. The acid form that is present at pH 1.5 exists as a mixture of five- and six-coordinate high-spin species and most likely has water or buffer ions as axial ligand(s). On titration to pH 7, the His18 residue is deprotonated and becomes the proximal ligand to the iron to give a six-coordinate neutral form that has water as the sixth ligand. This form exists in a thermal high-spin intermediate-spin state equilibrium. On raising the pH to 10, an alkaline form is generated which is predominantly a five-coordinate high-spin species. It is formed by ionization of the proximal His18 residue to its imidazolate form with concomitant dissociation of the water ligand at the sixth site. At concentrations of Ac-MP-8 greater than 10 microM, some six-coordinate low-spin species are formed that are attributed to a dimer in which a His18 residue from a second molecule of Ac-MP-8 coordinates to the sixth site of another to give a bis-His complex. Raising the pH to 11.5 does not produce an appreciable amount of the six-coordinate complex with hydroxide as the sixth ligand. These studies show that Ac-MP-8 is a good water-soluble model for the peroxidases that exhibits minimal aggregation at concentrations below 10 microM in the neutral and alkaline pH regions.
ABSTRACT Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of pro... more ABSTRACT Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[35S]methionine incorporation into PGH synthase that was induced by IL-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.
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