Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-dl-alanine (DOPA) has been studied. The ability of DOPA to react with has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by was observed using spin stabilization technique of ortho-semiquinones by Zn²⁺ ions. Simultaneously, the oxidation of DOPA by was found to produce hydrogen peroxide (H₂O₂). The analysis of H₂O₂ formation upon oxidation of DOPA by using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and being equal to (3.4±0.6)×10⁵?M^?1?s^?1.

The formation of H₂O₂ during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H₂O₂ production was found to be SOD-sensitive. The inhibition of H₂O₂ production by SOD was about 25% indicating that H₂O₂ is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with      

Extracellular H2O2 and not superoxide determines the compartment-specific activation of transferrin receptor by iron regulatory protein 1

Iron regulatory protein 1 (IRP1) functions as translational regulator that plays a central role in coordinating the cellular iron metabolism by binding to the mRNA of target genes such as the transferrin receptor (TfR)—the major iron uptake protein. Reactive oxygen species such as H₂O₂ and that are both co-released by inflammatory cells modulate IRP1 in opposing directions. While H₂O₂—similar to iron depletion—strongly induces IRP1 via a signalling cascade, inactivates the mRNA binding activity by a direct chemical attack. These findings have raised the question of whether compartmentalization may be an important mechanism for isolating these biological reactants when released from inflammatory cells during the oxygen burst cascade. To address this question, we studied cytosolic IRP1 and its downstream target TfR in conjunction with a tightly controlled biochemical modulation of extracellular and H₂O₂ levels mimicking the oxygen burst cascade of inflammatory cells. We here demonstrate that IRP1 activity and expression of TfR are solely dependent on H₂O₂ when co-released with from xanthine oxidase. Our findings confirm that extracellular H₂O₂ determines the functionality of the IRP1 cluster and its downstream targets while the reactivity of is limited to its compartment of origin.     

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase—contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of and H₂O₂ during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.