Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels☆

The Role of Superoxide Anion in the Autoxidation of Epinephrine and a Simple Assay for Superoxide Dismutase

Photoreduction of dioxygen in photosystem I (PSI) of chloroplasts generates superoxide radicals as the primary product. In intact chloroplasts, the superoxide and the hydrogen peroxide produced via the disproportionation of superoxide are so rapidly scavenged at the site of their generation that the active oxygens do not inactivate the PSI complex, the stromal enzymes, or the scavenging system itself. The overall reaction for scavenging of active oxygens is the photoreduction of dioxygen to water via superoxide and hydrogen peroxide in PSI by the electrons derived from water in PSII, and the water-water cycle is proposed for these sequences. An overview is given of the molecular mechanism of the water-water cycle and microcompartmentalization of the enzymes participating in it. Whenever the water-water cycle operates properly for scavenging of active oxygens in chloroplasts, it also effectively dissipates excess excitation energy under environmental stress. The dual functions of the water-water cycle for protection from photoinihibition are discussed.

THE WATER-WATER CYCLE IN CHLOROPLASTS: Scavenging of Active Oxygens and Dissipation of Excess Photons

The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen

Byproducts of normal mitochondrial metabolism and homeostasis include the buildup of potentially damaging levels of reactive oxygen species (ROS), Ca2+, etc., which must be normalized. Evidence suggests that brief mitochondrial permeability transition pore (mPTP) openings play an important physiological role maintaining healthy mitochondria homeostasis. Adaptive and maladaptive responses to redox stress may involve mitochondrial channels such as mPTP and inner membrane anion channel (IMAC). Their activation causes intra- and intermitochondrial redox-environment changes leading to ROS release. This regenerative cycle of mitochondrial ROS formation and release was named ROS-induced ROS release (RIRR). Brief, reversible mPTP opening-associated ROS release apparently constitutes an adaptive housekeeping function by the timely release from mitochondria of accumulated potentially toxic levels of ROS (and Ca2+). At higher ROS levels, longer mPTP openings may release a ROS burst leading to destruction of mitochondria, and if propagated from mitochondrion to mitochondrion, of the cell itself. The destructive function of RIRR may serve a physiological role by removal of unwanted cells or damaged mitochondria, or cause the pathological elimination of vital and essential mitochondria and cells. The adaptive release of sufficient ROS into the vicinity of mitochondria may also activate local pools of redox-sensitive enzymes involved in protective signaling pathways that limit ischemic damage to mitochondria and cells in that area. Maladaptive mPTP- or IMAC-related RIRR may also be playing a role in aging. Because the mechanism of mitochondrial RIRR highlights the central role of mitochondria-formed ROS, we discuss all of the known ROS-producing sites (shown in vitro) and their relevance to the mitochondrial ROS production in vivo.

Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release

The production of superoxide anion radicals in the reaction of reduced flavins and flavoproteins with molecular oxygen

The Purification and Properties of the Flavoprotein Melilotate Hydroxylase

The Mechanism of Action of the Flavoprotein Melilotate Hydroxylase

Melilotate has been synthesized from melilotate by iodiantion followed by reductive deiodination in the presence of deuterated hydrazine. The deuterated melilotate has been employed in investigations of the reaction mechanism of melilotate hydroxylase. Stopped-flow spectrophotometry has revealed no isotope effect in the formation or decay of the oxygenated intermediate which is observed when reduced melilotate hydroxylase reacts with molecular oxygen. Steady-state analysis has corroborated this result, and in addition shows that there is no isotope effect in the reductive cycle of the enzyme mechanism. This analysis does reveal a reproducible 8% decrease in Vmax for the enzyme when using deuterated melilotate. These observations are compatible with the thesis that the above intermediate is an oxygenated form of the reduced flavine prosthetic group and that the last step of the proposed mechanism is rapid and involves a primary isotope effect. The existence of the NIH shift mechanism has been studied using combined gas chromatography-mass spectrometry. No evidence could be obtained for intramolecular migration of deuterium during the hydroxylation reaction. However, the small amount of migration expected when phenols are hydroxylated precludes elimination of the NIH shift as a possibility.

Kinetic and mechanistic studies on the reaction of melilotate hydroxylase with deuterated melilotate.

Publisher Summary This chapter focuses on the role of flavins in hydroxylase reactions. The mechanism of hydroxylation has been investigated using two enzyme systems, p -hydroxybenzoate hydroxylase from Pseudomonas fluorescens and melilotate hydroxylase from Pseudomonas spp., as well as two model systems. A comparison of the rate of reduction of the enzyme flavin in the presence and absence of melilotate is shown. The analogues which have been investigated simulated many of the characteristics of p -hydroxybenzoate, such as spectral perturbations on binding, but none of the previously investigated analogues mimic the effector role. The data may be compared to the fluorescence titration of p -hydroxybenzoate hydroxylase with p -hydroxybenzoate monitoring the quenching of flavin fluorescence upon formation of the enzyme- p -hydroxybenzoate complex. The nature of the active oxygen produced during the hydroxylation phase of the catalytic cycle is of great interest.

Sidney Strickland

Papers

The production of superoxide anion radicals in the reaction of reduced flavins and flavoproteins with molecular oxygen

The Purification and Properties of the Flavoprotein Melilotate Hydroxylase

The Mechanism of Action of the Flavoprotein Melilotate Hydroxylase

Kinetic and mechanistic studies on the reaction of melilotate hydroxylase with deuterated melilotate.

The role of flavins in hydroxylase reactions