Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O2 and H2O2. SOD also reacts with H2O2, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO2) and H2O2, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper. © 2012 Strange et al

Simultaneous identification of GSTP1 Ile105→Val105 and Ala114→Val114 substitutions using an amplification refractory mutation systempolymerase chain reactionassay: studies in patients with asthma

BACKGROUND: The glutathione S-transferase (GST) enzyme GSTP1 utilizes byproducts of oxidative stress. We previously showed that alleles of GSTP1 that encode the Ile105→Val105 substitution are associated with the asthma phenotypes of atopy and bronchial hyperresponsiveness (BHR). However, a further polymorphic site (Ala114→Val114) has been identified that results in the following alleles: GSTP1(*)A (wild-type Ile105→Ala114), GSTP1(*)B (Val105→Ala114), GSTP1(*)C (Val105→Val114) and GSTP1(*)D (Ile105→Val114). METHODS: Because full identification of GSTP1 alleles may identify stronger links with asthma phenotypes, we describe an amplification refractory mutation system (ARMS) assay that allows identification of all genotypes. We explored whether the GSTP1 substitutions influence susceptibility to asthma, atopy and BHR. RESULTS: Among 191 atopic nonasthmatic, atopic asthmatic and nonatopic nonasthmatic individuals, none had the BD, CD, or DD genotypes. GSTP1 BC was significantly associated with reduced risk for atopy (P = 0.031). Compared with AA, trend test analysis identified a significant decrease in the frequency of GSTP1 BC with increasing severity of BHR (P = 0.031). Similarly, the frequency of GSTP1 AA increased with increasing BHR. CONCLUSION: These data suggest that GSTP1(*)B and possibly GSTP1(*)C are protective against asthma and related phenotypes

Gene Electro Transfer of Plasmid Encoding Vascular Endothelial Growth Factor for Enhanced Expression and Perfusion in the Ischemic Swine Heart

Myocardial ischemia can damage heart muscle and reduce the heart\u27s pumping efficiency. This study used an ischemic swine heart model to investigate the potential for gene electro transfer of a plasmid encoding vascular endothelial growth factor for improving perfusion and, thus, for reducing cardiomyopathy following acute coronary syndrome. Plasmid expression was significantly greater in gene electro transfer treated tissue compared to injection of plasmid encoding vascular endothelial growth factor alone. Higher gene expression was also seen in ischemic versus non-ischemic groups with parameters 20 Volts (

QM/MM Simulations of Protein Crystal Reactivity Guided by MSOX Crystallography: A Copper Nitrite Reductase Case Study.

The recently developed multiple structures from one crystal (MSOX) serial crystallography method can be used to provide multiple snapshots of the progress of enzymatic reactions taking place within a protein crystal. Such MSOX snapshots can be used as a reference for combined quantum mechanical/molecular mechanical (QM/MM) simulations of enzyme reactivity within the crystal. QM/MM calculations are used to identify details of reference states that cannot be directly observed by X-ray diffraction experiments, such as protonation and oxidation states. These reference states are then used as known fixed endpoints for the modeling of reaction paths. We investigate the mechanism of nitrite reduction in an Achromobacter cycloclastes copper nitrite reductase crystal using MSOX-guided QM/MM calculations, identifying the change in nitrite binding orientation with a change in copper oxidation state, and determining the reaction path to the final NO-bound MSOX structure. The results are compared with QM/MM simulations performed in a solvated environment

Exceptional Preservation of Mid-Cretaceous Marine Arthropods and the Evolution of Novel Forms via Heterochrony

Evolutionary origins of novel forms are often obscure because early and transitional fossils tend to be rare, poorly preserved, or lack proper phylogenetic contexts. We describe a new, exceptionally preserved enigmatic crab from the mid-Cretaceous of Colombia and the United States, whose completeness illuminates the early disparity of the group and the origins of novel forms. Its large and unprotected compound eyes, small fusiform body, and leg-like mouthparts suggest larval trait retention into adulthood via heterochronic development (pedomorphosis), while its large oar-like legs represent the earliest known adaptations in crabs for active swimming. Our phylogenetic analyses, including representatives of all major lineages of fossil and extant crabs, challenge conventional views of their evolution by revealing multiple convergent losses of a typical “crab-like” body plan since the Early Cretaceous. These parallel morphological transformations may be associated with repeated invasions of novel environments, including the pelagic/necto-benthic zone in this pedomorphic chimera crab

Choosing the optimal spectroscopic toolkit to understand protein function

Spectroscopy was one of the earliest methods used to study the properties and reactions of proteins, and remains one of the most powerful and widely used approaches to this day. A sometimes bewildering range of spectroscopies is now available, applicable to different sample states, timescales and indeed biological questions. This editorial describes some of the most relevant spectroscopic methods together with a selection of illustrative examples.</jats:p

Fingerprinting redox and ligand states in haemprotein crystal structures using resonance Raman spectroscopy

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when appliedin situat macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochromec′ fromAlcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation-induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.</jats:p

Nature of the copper-nitrosyl intermediates of copper nitrite reductases during catalysis

Observation of side-on copper-nitrosyl intermediate and its confirmation by DFT during catalysis of copper nitrite reductases.</p