At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

/pdf/free-radicals-in-the-physiological-control-of-cell-function-g3w3iyr0ph.pdf

Free Radicals in the Physiological Control of Cell Function

Intact spinach chloroplasts scavenge hydrogen peroxide with a peroxidase that uses a photoreductant as the electron donor, but the activity of ruptured chloroplasts is very low [Nakano and Asada (1980) Plant & Cell Physiol. 21: 1295]. Ruptured spinach chloroplasts recovered their ability to photoreduce hydrogen peroxide with the concomitant evolution of oxygen after the addition of glutathione and dehydroascorbate (DHA). In ruptured chloroplasts, DHA was photoreduced to ascorbate and oxygen was evolved in the process in the presence of glutathione. DHA reductase (EC 1.8.5.1) and a peroxidase whose electron donor is specific to L-ascorbate are localized in chloroplast stroma. These observations confirm that the electron donor for the scavenging of hydrogen peroxide in chloroplasts is L-ascorbate and that the L-ascorbate is regenerated from DHA by the system: photosystem I-*ferredoxin-*NADP^>glutathione. A preliminary characterization of the chloroplast peroxidase is given.

Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts

The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2−•). Even though O2−• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments. We also discuss various physiological and pathological scenarios resulting from an increased steady state concentration of mitochondrial oxidants.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-7793.2003.00335.x

Mitochondrial formation of reactive oxygen species.

Beckman, Kenneth B., and Bruce N. Ames. The Free Radical Theory of Aging Matures. Physiol. Rev. 78: 547–581, 1998. — The free radical theory of aging, conceived in 1956, has turned 40 and is rapidl...

/pdf/the-free-radical-theory-of-aging-matures-3b861ahk8h.pdf

The Free Radical Theory of Aging Matures

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Superoxide Radical and Superoxide Dismutases

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide

/pdf/essential-role-of-thiol-groups-in-aldehyde-dehydrogenases-4rrtqwhuo3.pdf

Essential role of thiol groups in aldehyde dehydrogenases

1. Pyruvate carboxylase from baker's yeast acts with either MgATP2− or MnATP2− as substrate. The optimum pH for the enzyme reaction is 8·0 with MgATP2− and 7·0 with MnATP2−. 2. When the reaction velocity is plotted against MgATP2− (or MnATP2−) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg2+ (or Mn2+) the curves turn into hyperbolae, whereas in the presence of excess of free ATP4− the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP2− (or MnATP2−) concentration can be explained by the inhibitory effect of free ATP4−, the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP–magnesium chloride (or ATP–manganese chloride) mixture. Free ATP4− behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg2+ (or Mn2+) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP4−, instead of a direct enzyme activation. 5. At pH6·5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP2− complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6·25–6·75; 12mm-magnesium chloride and 0·75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP4− is doubtful since the Mg2+ and Mn2+ concentrations reported to exist inside the yeast cell are sufficient to decrease ATP4− concentrations to ineffective values.

/pdf/effects-of-magnesium-manganese-and-adenosine-triphosphate-bg1kinu1zg.pdf

Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker's yeast

/pdf/the-role-of-sulphydryl-groups-of-yeast-carboxylase-k9hc80cmgv.pdf

The role of sulphydryl groups of yeast carboxylase.

1. Pyruvate carboxylase from baker9s yeast is inhibited by ADP, AMP and adenosine at pH8·0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD + , NADP + and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD + , NADH, NADP + and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6·5, in the presence of 12mm-Mg 2+ , 0·75mm-Mn 2+ and 0·5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.

/pdf/effects-of-adenosine-phosphates-and-nicotinamide-nucleotides-1i08mkr4c3.pdf

A. O. M. Stoppani

Papers

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide

Essential role of thiol groups in aldehyde dehydrogenases

Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker's yeast

The role of sulphydryl groups of yeast carboxylase.

Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker's yeast.