The Role of H2O2-Scavenging Enzymes (Ascorbate Peroxidase and Catalase) in the Tolerance of Lemna minor to Antibiotics: Implications for Phytoremediation

We investigated the individual and combined contributions of two distinct heme proteins namely, ascorbate peroxidase (APX) and catalase (CAT) on the tolerance of Lemna minor plants to antibiotics. For our investigation, we used specific inhibitors of these two H2O2-scavenging enzymes (p-aminophenol, 3-amino,1,2,4-triazole, and salicylic acid). APX activity was central for the tolerance of this aquatic plant to amoxicillin (AMX), whereas CAT activity was important for avoiding oxidative damage when exposed to ciprofloxacin (CIP). Both monitored enzymes had important roles in the tolerance of Lemna minor to erythromycin (ERY). The use of molecular kinetic approaches to detect and increase APX and/or CAT scavenging activities could enhance tolerance, and, therefore, improve the use of L. minor plants to reclaim antibiotics from water bodies.

Exploring the folding energy landscapes of heme proteins using a hybrid AWSEM-heme model

Heme is an active center in many proteins. Here we explore computationally the role of heme in protein folding and protein structure. We model heme proteins using a hybrid model employing the AWSEM Hamiltonian, a coarse-grained forcefield for the protein chain along with AMBER, an all-atom forcefield for the heme. We carefully designed transferable force fields that model the interactions between the protein and the heme. The types of protein–ligand interactions in the hybrid model include thioester covalent bonds, coordinated covalent bonds, hydrogen bonds, and electrostatics. We explore the influence of different types of hemes (heme b and heme c) on folding and structure prediction. Including both types of heme improves the quality of protein structure predictions. The free energy landscape shows that both types of heme can act as nucleation sites for protein folding and stabilize the protein folded state. In binding the heme, coordinated covalent bonds and thioester covalent bonds for heme c drive the heme toward the native pocket. The electrostatics also facilitates the search for the binding site.

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins

Hemoglobin oxidation due to oxidative stress and disease conditions leads to generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the RBC membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2, 2-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-β-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.