Diane E. Cabelli

Reactivity of HO2/O−2 Radicals in Aqueous Solution

Kinetic data for the superoxide radical (HO2⇄O−2 +H+, pK=4.8) in aqueous solution have been critically assessed. Rate constants for reactions of O−2 and HO2 with more than 300 organic and inorganic ions, molecules and other transient species have been tabulated.

Superoxide dismutases—a review of the metal-associated mechanistic variations

Superoxide dismutases are enzymes that function to catalytically convert superoxide radical to oxygen and hydrogen peroxide. These enzymes carry out catalysis at near diffusion controlled rate constants via a general mechanism that involves the sequential reduction and oxidation of the metal center, with the concomitant oxidation and reduction of superoxide radicals. That the catalytically active metal can be copper, iron, manganese or, recently, nickel is one of the fascinating features of this class of enzymes. In this review, we describe these enzymes in terms of the details of their catalytic properties, with an emphasis on the mechanistic differences between the enzymes. The focus here will be concentrated mainly on two of these enzymes, copper, zinc superoxide dismutase and manganese superoxide dismutase, and some relatively subtle variations in the mechanisms by which they function.

Superoxide dismutases and superoxide reductases.

Superoxide, O2•–, is formed in all living organisms that come in contact with air, and, depending upon its biological context, it may act as a signaling agent, a toxic species, or a harmless intermediate that decomposes spontaneously. Its levels are limited in vivo by two different types of enzymes, superoxide reductase (SOR) and superoxide dismutase (SOD). Although superoxide has long been an important factor in evolution, it was not so when life first emerged on Earth at least 3.5 billion years ago. At that time, the early biosphere was highly reducing and lacking in any significant concentrations of dioxygen (O2), very different from what it is today. Consequently, there was little or no O2•– and therefore no reason for SOR or SOD enzymes to evolve. Instead, the history of biological O2•– probably commences somewhere around 2.4 billion years ago, when the biosphere started to experience what has been termed the “Great Oxidation Event”, a transformation driven by the increase in O2 levels, formed by cyanobacteria as a product of oxygenic photosynthesis.1 The rise of O2 on Earth caused a reshaping of existing metabolic pathways, and it triggered the development of new ones.2 Its appearance led to the formation of the so-called “reactive oxygen species” (ROS), for example, superoxide, hydrogen peroxide, and hydroxyl radical, and to a need for antioxidant enzymes and other antioxidant systems to protect against the growing levels of oxidative damage to living systems. Dioxygen is a powerful four-electron oxidizing agent, and the product of this reduction is water. 1 When O2 is reduced in four sequential one-electron steps, the intermediates formed are the three major ROS, that is, O2•–, H2O2, and HO•. 2 3 4 5 Each of these intermediates is a potent oxidizing agent. The consequences of their presence to early life must have been an enormous evolutionary challenge. In the case of superoxide, we find the SOD and SOR enzymes to be widely distributed throughout current living organisms, both aerobic and anaerobic, suggesting that, from the start of the rise of O2 on Earth, the chemistry of superoxide has been an important factor during evolution. The SORs and three very different types of SOD enzymes are redox-active metalloenzymes that have evolved entirely independently from one another for the purpose of lowering superoxide concentrations. SORs catalyze the one-electron reduction of O2•– to give H2O2, a reaction requiring two protons per superoxide reacted as well as an external reductant to provide the electron (eq 6). SODs catalyze the disproportionation of superoxide to give O2 and H2O2, a reaction requiring one proton per superoxide reacted, but no external reductant (eq 7). 6 7 All of the SOR enzymes contain only iron, while the three types of SODs are the nickel-containing SODs (NiSOD), the iron- or manganese-containing SODs (FeSOD and MnSOD), and the copper- and zinc-containing SODs (CuZnSOD). Although the structures and other properties of these four types of metalloenzymes are quite different, they all share several characteristics, including the ability to react rapidly and selectively with the small anionic substrate O2•–. Consequently, there are some striking similarities between these otherwise dissimilar enzymes, many of which can be explained by considering the nature of the chemical reactivity of O2•– (see below). Numerous valuable reviews describing the SOD and SOR enzymes have appeared over the years, but few have covered and compared all four classes of these enzymes, as we attempt to do here. Thus, the purpose of this Review is to describe, compare, and contrast the properties of the SOR and the four SOD enzymes; to summarize what is known about their evolutionary pathways; and to analyze the properties of these enzymes in light of what is known of the inherent chemical reactivity of superoxide.

Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis.

The presence of the copper ion at the active site of human wild type copper-zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyze the disproportionation of superoxide into dioxygen and hydrogen peroxide. Wild type CuZnSOD and several of the mutants associated with familial amyotrophic lateral sclerosis (FALS) (Ala4 → Val, Gly93 → Ala, and Leu38 → Val) were expressed inSaccharomyces cerevisiae. Purified metal-free (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification studies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis. From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in their proper locations in vitro. Similar studies of the wild type and FALS mutant CuZnSOD holoenzymes in the “as isolated” metallation state showed abnormally low copper-to-zinc ratios, although all of the copper acquired was located at the native copper binding sites. Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS). The loss of metal ion binding specificity of FALS mutant CuZnSODsin vitro may be related to their role in ALS.

Manganous phosphate acts as a superoxide dismutase.

A substantial body of evidence indicates that high intracellular concentrations of inorganic manganous ions render some cells resistant to ionizing radiation and provide substantial antioxidant protection to aerobic cells lacking superoxide dismutase (SOD) enzymes. We found that manganous phosphate is unique among those manganous salts studied in its ability to remove superoxide rapidly and catalytically from aqueous solution via a disproportionation mechanism that is entirely different from those of the SOD enzymes.

Determination of optimal conditions for synthesis of peroxynitrite by mixing acidified hydrogen peroxide with nitrite.

The measured parameters for the formation of peroxynitrous acid via the reaction of acidified hydrogen peroxide with nitrous acid and its self-decomposition corroborate with an earlier suggested mechanism in which H2NO2+ nitrosates H2O2. The activation energies for the formation and decay of peroxynitrous acid have been determined to be 15 and 19 kcal/mol, respectively. We found that perchlorate, nitrate, sulfate and phosphate ions have no effect on the formation and decay rates, whereas chloride ions enhance the rate of the formation of peroxynitrous acid at low peroxide concentrations, and have no effect at high peroxide concentrations. This suggests that at relatively low concentration of H2O2, Cl- competes with H2O2 for H2NO+ to yield NOCl, which may also nitrosate H2O2. Simulation of the experimentally observed parameters for the decay and formation rates suggests that it is not possible to obtain 100% yield of peroxynitrite under any condition. High yields of peroxynitrite were obtained at room temperature using an efficient double mixer where acidified peroxide was mixed with nitrite; after an appropriate delay, the reaction was quenched with strong alkali. An excess of more than 10% of H2O2 over nitrite, or vice versa, is sufficient to get ca. 85-90% of peroxynitrite, almost free from nitrite or H2O2, respectively. The results also suggest that conventional use of ice-cold solutions of the reactants and the alkali solutions is not required if an efficient mixer and appropriate quenching times are available.

Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines

Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability.

Nickel(II) macrocycles: highly efficient electrocatalysts for the selective reduction of CO2 to CO

A series of molecular materials that are structurally similar to the NiII macrocycle [Ni(cyclam)]2+ (cyclam = 1,4,8,11-tetraazacyclotetradecane) have been used as electrocatalysts for the reduction of CO2 at a mercury pool working electrode in aqueous solution. At pH 5, with an applied potential of −0.96 V vs. NHE (overpotential of −0.55 V), the complexes are highly efficient, having both high rate constants and Faradaic efficiencies (F.E.s) for the selective reduction of CO2 to CO. When the pH is below the pKa (pH < 2) of the Ni(H) species (pKas: 0.5–2), the F.E.s are still high but product selectivity changes to yield predominantly H2 from the reduction of water. At least two of the complexes investigated are better electrocatalysts than [Ni(cyclam)]2+, probably due to: (i) surface geometries that are suitable for adsorption onto the mercury electrode surface, and (ii) electronic effects of methyl groups or cyclohexane rings on the cyclam backbone. Mechanistic studies by pulse radiolysis show evidence of Ni(CO2) adducts for two of the catalysts, with KCO2 ∼ 10 M−1 for the reaction of NiI with CO2 in aqueous solution.

Biologically relevant mechanism for catalytic superoxide removal by simple manganese compounds.

Nonenzymatic manganese was first shown to provide protection against superoxide toxicity in vivo in 1981, but the chemical mechanism responsible for this protection subsequently became controversial due to conflicting reports concerning the ability of Mn to catalyze superoxide disproportionation in vitro. In a recent communication, we reported that low concentrations of a simple Mn phosphate salt under physiologically relevant conditions will indeed catalyze superoxide disproportionation in vitro. We report now that two of the four Mn complexes that are expected to be most abundant in vivo, Mn phosphate and Mn carbonate, can catalyze superoxide disproportionation at physiologically relevant concentrations and pH, whereas Mn pyrophosphate and citrate complexes cannot. Additionally, the chemical mechanisms of these reactions have been studied in detail, and the rates of reactions of the catalytic removal of superoxide by Mn phosphate and carbonate have been modeled. Physiologically relevant concentrations of these compounds were found to be sufficient to mimic an effective concentration of enzymatic superoxide dismutase found in vivo. This mechanism provides a likely explanation as to how Mn combats superoxide stress in cellular systems.

Mechanism of Hydride Donor Generation Using a Ru(II) Complex Containing an NAD+ Model Ligand : Pulse and Steady-State Radiolysis Studies

The mechanistic pathways of formation of the NADH-like [Ru(bpy) 2(pbnHH)] (2+) species from [Ru(bpy)2(pbn)](2+) were studied in an aqueous medium. Formation of the one-electron-reduced species as a result of reduction by a solvated electron (k=3.0 x 10(10) M(-1) s(-1)) or CO2(*-) (k=4.6 x 10(9) M(-1) s(-1)) or reductive quenching of an MLCT excited state by 1,4-diazabicyclo[2.2.2]octane (k=1.1 x 10(9) M(-1) s(-1)) is followed by protonation of the reduced species (p K a = 11). Dimerization (k7a=2.2 x 10(8) M(-1) s(-1)) of the singly reduced protonated species, [Ru(bpy) 2(pbnH(*))](2+), followed by disproportionation of the dimer as well as the cross reaction between the singly reduced protonated and nonprotonated species (k8= 1.2 x 10(8) M(-1) s(-1)) results in the formation of the final [Ru(bpy)2(pbnHH)](2+) product together with an equal amount of the starting complex, [Ru(bpy)2(pbn)](2+). At 0.2 degrees C, a dimeric intermediate, most likely a pi-stacking dimer, was observed that decomposes thermally to form an equimolar mixture of [Ru(bpy)2(pbnHH)](2+) and [Ru(bpy)2(pbn)](2+) (pH<9). The absence of a significant kinetic isotope effect in the disproportionation reaction of [Ru(bpy)2(pbnH(*))](2+) and its conjugate base (pH>9) indicates that disproportionation occurs by a stepwise pathway of electron transfer followed by proton transfer.