Coordination of the N-Terminal Heme in the Non-Classical Peroxidase from Escherichia coli.

The non-classical bacterial peroxidase from Escherichia coli, YhjA, is proposed to deal with peroxidative stress in the periplasm when the bacterium is exposed to anoxic environments, defending it from hydrogen peroxide and allowing it to thrive under those conditions. This enzyme has a predicted transmembrane helix and is proposed to receive electrons from the quinol pool in an electron transfer pathway involving two hemes (NT and E) to accomplish the reduction of hydrogen peroxide in the periplasm at the third heme (P). Compared with classical bacterial peroxidases, these enzymes have an additional N-terminal domain binding the NT heme. In the absence of a structure of this protein, several residues (M82, M125 and H134) were mutated to identify the axial ligand of the NT heme. Spectroscopic data demonstrate differences only between the YhjA and YhjA M125A variant. In the YhjA M125A variant, the NT heme is high-spin with a lower reduction potential than in the wild-type. Thermostability was studied by circular dichroism, demonstrating that YhjA M125A is thermodynamically more unstable than YhjA, with a lower TM (43 °C vs. 50 °C). These data also corroborate the structural model of this enzyme. The axial ligand of the NT heme was validated to be M125, and mutation of this residue was proven to affect the spectroscopic, kinetic, and thermodynamic properties of YhjA.

Biochemical Characterization of the Copper Nitrite Reductase from Neisseria gonorrhoeae.

The copper-containing nitrite reductase from Neisseria gonorrhoeae has been shown to play a critical role in the infection mechanism of this microorganism by producing NO and abolishing epithelial exfoliation. This enzyme is a trimer with a type 1 copper center per subunit and a type 2 copper center in the subunits interface, with the latter being the catalytic site. The two centers were characterized for the first time by EPR and CD spectroscopy, showing that the type 1 copper center has a high rhombicity due to its lower symmetry and more tetragonal structure, while the type 2 copper center has the usual properties, but with a smaller hyperfine coupling constant (A// = 10.5 mT). The thermostability of the enzyme was analyzed by differential scanning calorimetry, which shows a single endothermic transition in the thermogram, with a maximum at 94 °C, while the CD spectra in the visible region indicate the presence of the type 1 copper center up to 80 °C. The reoxidation of the N. gonorrhoeae copper-containing nitrite reductase in the presence of nitrite were analyzed by visible spectroscopy and showed a pH dependence, being higher at pH 5.5-6.0. The high thermostability of this enzyme may be important to maintaining a high activity in the extracellular space and to making it less susceptible to denaturation and proteolysis, contributing to the proliferation of N. gonorrhoeae.

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production.

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) are responsible for approximately 150,000 envenoming cases per year in Brazil, of which 10% require antivenom treatment to reverse life-threatening venom effects. Therefore, thousands of T. serrulatus individuals are maintained under controlled captivity conditions for venom extraction, subsequently used in the production of the national supply of scorpion antivenom. Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing about 70,000 vials of scorpion antivenom for the Brazilian health system. Thus, the husbandry protocols and venom extraction methodologies are key points for the success of large-scale, standardized venom production. The objective of this article is to describe the captivity protocols of T. serrulatus husbandry, encompassing the husbandry routine and the venom extraction procedures, following good manufacturing practices, and ensuring animal welfare. These practices allow for the maintenance of up to 20,000 animals in captivity, with a routine of 3,000 to 5,000 scorpions milked monthly according to antivenom manufacturing demand, achieving an average of 90% of positive extraction.