Replacement Of The Axial Copper Ligand Methionine With Lysine In Amicyanin Converts It To A Zinc-Binding Protein That No Longer Binds Copper

The mutation of the axial ligand of the type I copper protein amicyanin from Met to Lys results in a protein that is spectroscopically invisible and redox inactive. M98K amicyanin acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals that its overall structure is very similar to native amicyanin but that the type I binding site is occupied by zinc. Anomalous difference Fourier maps calculated using the data collected around the absorption edges of copper and zinc confirm the presence of Zn 2+ at the type I site. The Lys98 NZ donates a hydrogen bond to a well-ordered water molecule at the type I site which enhances the ability of Lys98 to provide a ligand for Zn 2+. Attempts to reconstitute M98K apoamicyanin with copper resulted in precipitation of the protein. The fact that the M98K mutation generated such a selective zinc-binding protein was surprising as ligation of zinc by Lys is rare and this ligand set is unique for zinc. © 2011 Elsevier Inc. All rights reserved

Characterization Of Electron Tunneling And Hole Hopping Reactions Between Different Forms Of Maug And Methylamine Dehydrogenase Within A Natural Protein Complex

Respiration, photosynthesis, and metabolism require the transfer of electrons through and between proteins over relatively long distances. It is critical that this electron transfer (ET) occur with specificity to avoid cellular damage, and at a rate that is sufficient to support the biological activity. A multistep hole hopping mechanism could, in principle, enhance the efficiency of long-range ET through proteins as it does in organic semiconductors. To explore this possibility, two different ET reactions that occur over the same distance within the protein complex of the diheme enzyme MauG and different forms of methylamine dehydrogenase (MADH) were subjected to kinetic and thermodynamic analysis. An ET mechanism of single-step direct electron tunneling from diferrous MauG to the quinone form of MADH is consistent with the data. In contrast, the biosynthetic ET from preMADH, which contains incompletely synthesized tryptophan tryptophylquinone, to the bis-Fe(IV) form of MauG is best described by a two-step hole hopping mechanism. Experimentally determined ET distances matched the distances determined from the crystal structure that would be expected for single-step tunneling and multistep hopping. Experimentally determined relative values of electronic coupling (HAB) for the two reactions correlated well with the relative H AB values predicted from computational analysis of the structure. The rate of the hopping-mediated ET reaction is also 10-fold greater than that of the single-step tunneling reaction despite a smaller overall driving force for the hopping-mediated ET reaction. These data provide insight into how the intervening protein matrix and redox potentials of the electron donor and acceptor determine whether the ET reaction proceeds via single-step tunneling or multistep hopping. © 2012 American Chemical Society

A Bibliometric Analysis of Myopia Research in East Asia in the 21st Century: The Socio-Economic Status and Quantitative Analysis

The purpose of this study is to assess the quantity and quality of myopia related articles from 2001 to 2021 using bibliometric methods. The number of published articles and citations, the correlation analysis between gross domestic product (GDP) and annual publication number and citations was investigated. The proportion of myopia articles from East Asia accounted for 55.28% in 2021. The researchers from China published the most articles on myopia during 2001 to 2021, followed by Japan and South Korea. The annual number of articles and citations from China and South Korea showed an exponential increase with strong positive correlation with GDP. All 3 countries in East Asia are mainly researching refractive surgery, prevalence, and glaucoma, and research on children’s myopia is particularly active in China, and in Japan. The researchers from East Asia published more than half of articles on myopia since 2019, in order of China, Japan, and South Korea. The annual number of articles and citations from China and South Korea showed an exponential increase with strong positive correlation with GDP whereas those from Japan did not. All 3 countries are mainly researching refractive surgery, and glaucoma, and research on children’s myopia is particularly active in China, and in Japan

Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self‐cleavage exhibits enhanced enzymatic activity

Tobacco etch virus (TEV) protease is a 27‐kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S. Despite its substrate specificity, its use is limited by its autoinactivation through self‐cleavage and poor solubility during purification. It was previously reported that T17S/N68D/I77V mutations improve the solubility and yield of TEV protease and S219 mutations provide protection against self‐cleavage. In this study, we isolated TEV proteases with S219N and S219V mutations in the background of T17S, N68D, and I77V without the inclusion body, and measured their enzyme kinetics. The kcat of two isolated S219N and S219V mutants in the background of T17S, N68D, and I77V mutations was highly increased compared to that of the control, and S219N was twofold faster than S219V without Km change. This result indicates that combination of these mutations can further enhance TEV activity

A Simple Method To Engineer A Protein-Derived Redox Cofactor For Catalysis

The 6 ×-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co2 +. Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co 2 +-loaded 6 × His-tag could substitute for the hemes in the H2O2-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co2 +-loaded 6 × His-tag could mediate long range electron transfer, it was shown that addition of H2O2 to the Co2 +-loaded 6 × His-tagged Cu1 + amicyanin oxidizes the copper site which is 20 Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology. © 2014 Elsevier B.V

A T67A Mutation In The Proximal Pocket Of The High-Spin Heme Of Maug Stabilizes Formation Of A Mixed-Valent Fe\u3csup\u3eIi\u3c/sup\u3e/Fe\u3csup\u3eIii\u3c/sup\u3e State And Enhances Charge Resonance Stabilization Of The Bis-Fe\u3csup\u3eIv\u3c/sup\u3e State

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. One heme is low-spin with ligands provided by His205 and Tyr294, and the other is high-spin with a ligand provided by His35. The side chain methyl groups of Thr67 and Leu70 are positioned at a distance of 3.4 Å on either side of His35, maintaining a hydrophobic environment in the proximal pocket of the high-spin heme and restricting the movement of this ligand. Mutation of Thr67 to Ala in the proximal pocket of the high-spin heme prevented reduction of the low-spin heme by dithionite, yielding a mixed-valent state. The mutation also enhanced the stabilization of the charge-resonance-transition of the high-valent bis-FeIV state that is generated by addition of H2O2. The rates of electron transfer from TTQ biosynthetic intermediates to the high-valent form of T67A MauG were similar to that of wild-type MauG. These results are compared to those previously reported for mutation of residues in the distal pocket of the high-spin heme that also affected the redox properties and charge resonance transition stabilization of the high-valent state of the hemes. However, given the position of residue 67, the structure of the variant protein and the physical nature of the T67A mutation, the basis for the effects of the T67A mutation must be different from those of the mutations of the residues in the distal heme pocket

Mixed Exposure of Persistent Organic Pollutants Alters Oxidative Stress Markers and Mitochondrial Function in the Tail of Zebrafish Depending on Sex

Persistent organic pollutants (POPs) are lipid-soluble toxins that are not easily degraded; therefore, they accumulate in the environment and the human body. Several studies have indicated a correlation between POPs and metabolic diseases; however, their effects on mitochondria as a central organelle in cellular metabolism and the usage of mitochondria as functional markers for metabolic disease are barely understood. In this study, a zebrafish model system was exposed to two subclasses of POPs, organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs), under two different conditions (solitary OCPs or OCPs with PCBs (Aroclor 1254)), and changes in the oxidative stress marker levels and mitochondrial enzyme activities in the electron transport chain of the tail were measured to observe the correlation between POPs and representative biomarkers for metabolic disease. The results indicated different responses upon exposure to OCPs and OCPs with Aroclor 1254, and accelerated toxicity was observed following exposure to mixed POPs (OCPs with Aroclor 1254). Males were more sensitive to changes in the levels of oxidative stress markers induced by POP exposure, whereas females were more susceptible to the toxic effects of POPs on the levels of mitochondrial activity markers. These results demonstrate that the study reflects real environmental conditions, with low-dose and multiple-toxin exposure for a long period, and that POPs alter major mitochondrial enzymes’ functions with an imbalance of redox homeostasis in a sex-dependent manner

The Sole Tryptophan Of Amicyanin Enhances Its Thermal Stability But Does Not Influence The Electronic Properties Of The Type 1 Copper Site

The cupredoxin amicyanin possesses a single tryptophan residue, Trp45. Its fluorescence is quenched when copper is bound even though it is separated by 10.1 Å. Mutation of Trp45 to Ala, Phe, Leu and Lys resulted in undetectable protein expression. A W45Y amicyanin variant was isolated. The W45Y mutation did not alter the spectroscopic properties or intrinsic redox potential of amicyanin, but increased the pKa value for the pH-dependent redox potential by 0.5 units. This is due to a hydrogen-bond involving the His95 copper ligand which is present in reduced W45Y amicyanin but not in native amicyanin. The W45Y mutation significantly decreased the thermal stability of amicyanin, as determined by changes in the visible absorbance of oxidized amicyanin and in the circular dichroism spectra for oxidized, reduced and apo forms of amicyanin. Comparison of the crystal structures suggests that the decreased stability of W45Y amicyanin may be attributed to the loss of a strong interior hydrogen bond between Trp45 and Tyr90 in native amicyanin which links two of the β-sheets that comprise the overall structure of amicyanin. Thus, Trp45 is critical for stabilizing the structure of amicyanin but it does not influence the electronic properties of the copper which quenches its fluorescence. © 2014 Elsevier Ltd. All rights reserved

Mori Ramulus Inhibits Pancreatic β-Cell Apoptosis and Prevents Insulin Resistance by Restoring Hepatic Mitochondrial Function

Type 2 diabetes mellitus is characterized by insulin resistance and pancreatic beta (β)-cell dysfunction. Accumulating evidence suggests that mitochondrial dysfunction may cause insulin resistance in peripheral tissues. As commercial hypoglycemic drugs have side effects, it is necessary to develop safe and effective natural compound-based hypoglycemic treatments. This study aimed to investigate the hypoglycemic effects of Mori Ramulus ethanol extract (ME) in a high-fat diet (HFD)-induced diabetes mouse model to decipher the underlying mechanisms focusing on apoptosis and mitochondrial function. ME significantly decreased tunicamycin-induced apoptotic cell death and increased insulin secretion following glucose stimulation in NIT-1 pancreatic β-cells. Tunicamycin-exposed NIT-1 pancreatic β-cells showed elevated reactive oxygen species levels and reduced mitochondrial membrane potential, which were reversed by ME treatment. ME inhibited the tunicamycin-induced apoptosis cascade in tunicamycin-exposed NIT-1 pancreatic β-cells. In HFD diabetic mice, the serum-free fatty acid and insulin levels decreased following a 15-week ME administration. Glucose and insulin tolerance tests showed that ME improved insulin sensitivity. Moreover, ME ameliorated pancreatic β-cell mass loss in diabetic mice. Finally, ME-treated HFD-fed mice showed improved hepatic mitochondrial function resulting in insulin sensitivity in target tissues. Thus, ME provides protection against pancreatic β-cell apoptosis and prevents insulin resistance by improving mitochondrial function