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Proc Natl Acad Sci U S A. Jan 20, 2004; 101(3): 743–744.
Published online Jan 7, 2004. doi:  10.1073/pnas.0307635100
PMCID: PMC321751

CO2, not equation M1, facilitates oxidations by Cu,Zn superoxide dismutase plus H2O2


The Cu,Zn superoxide dismutase catalyzes equation M2 -dependent oxidations by H2O2. This activity has been shown to depend on the creation of a bound oxidant at the Cu(II) by interactions with H2O2. The bound oxidant was then thought to oxidize equation M3 to equation M4, which diffuses into the bulk solution and there oxidizes diverse substrates. We now find that CO2 rather than equation M5 facilitates the peroxidations catalyzed by Cu,Zn superoxide dismutase. This fact was shown by a lag in the rate of peroxidation of NADPH when equation M6 was added last and by a burst in the rate when aqueous CO2 was added last. Both the lag and the burst were eliminated by carbonic anhydrase.

Keywords: carbon dioxide, bicarbonate, carbonic anhydrase, Cu,Zn SOD, hydrogen peroxide

equation M7 dramatically increases the rate of oxidation of diverse substrates by Cu,Zn superoxide dismutase (SOD1) plus H2O2, while only modestly increasing the intrinsically slow inactivation of this enzyme by H2O2 (14). The following reactions have been advanced as part of a scheme to explain the behavior of this system (1).

equation M8

equation M9

equation M10

equation M11

Thus, the cuprous enzyme produced in reaction i is oxidized in reaction ii by a second H2O2 to yield a strong bound oxidant, which can be written as Cu(I)O, Cu(II)OH, or Cu(III). This bound oxidant can either oxidize an adjacent histidine residue and in so doing inactivate the enzyme, or it can oxidize equation M12 to the carbonate radical equation M13 that is free to diffuse into the bulk solution and cause further oxidations. Before leaving the active site, the carbonate radical can oxidize residues in the ligand field of the copper and thus cause inactivation.

equation M14 was assumed to be the species in the carbonate buffer that facilitated the oxidations observed. This was the case because, at neutral to mildly alkaline pH, it is the major species. It also seemed reasonable that an active site evolved to deal with the monovalent anion equation M15 would preferentially react with other monovalent anions. Nevertheless, the experiments described below establish that at pH 7.4, it is CO2 rather than equation M16 that profoundly facilitates the oxidation of NADPH by SOD1 plus H2O2.

Materials and Methods

SOD1 was from Chemie Grünenthal (Aachen, Germany); H2O2, from Mallinckrodt; and NaHCO3, from Fisher; whereas NADPH and carbonic anhydrase were from Sigma. NADPH at 0.1 mM was reacted with 0.1 or 0.2 mg/ml SOD1 and 10 mM H2O2 in 100 mM potassium phosphate at pH 7.4. When equation M17 was used to facilitate the oxidation of NADPH (followed at 340 nm), it was added to 20 mM from a 1.0 M stock of NaHCO3 in water. When CO2 was used, it was taken from ice-cold water saturated with CO2 gas at 1.0 atmosphere (atm) (1 atm = 101.3 kPa) that would contain 76 mM CO2. Carbonic anhydrase when used was present at 0.1 mg/ml. Additional experimental details are presented in the legends to Figs. Figs.11 and and22.

Fig. 1.
equation M44 requires dehydration before facilitating. Shown is the oxidation of NADPH by SOD1 plus H2O2. The final reaction mixtures contained 0.1 mM NADPH, 0.1 mg/ml SOD1, 20 mM equation M45, and 10 mM H2O2, plus 100 mM sodium phosphate at pH 7.4 and at 23°C. Line ...
Fig. 2.
CO2 added as such supports the peroxidation activity of SOD1. Reaction conditions shown are essentially as in Fig. 1, except that water at 0°C saturated with CO2 gas was the last component added in place of equation M47. Line 1, CO2 added at the arrow to ...


Line 1 in Fig. 1 demonstrates an immediately linear rate of NADPH oxidation that was seen when the HCO3-dependent oxidation by SOD1 plus H2O2 reaction was started by adding either SOD1 or H2O2. However, when NaHCO3 was the last component added, there was a distinct lag before the linear rate was achieved, shown by line 2. Because NaHCO3 was added from an alkaline stock solution into a reaction mixture buffered at pH 7.4, it seemed possible that this lag was due to the perceptibly slow dehydration of H2CO3 to CO2. That this was the case was shown by the effect of carbonic anhydrase (line 3), which eliminated this lag. These results indicated that CO2 rather than equation M18 was the essential reactant.

In that case, it could be predicted that starting the reaction with CO2-saturated water would yield an initially fast reaction that would slow to a linear rate as the hydration and ionization to equation M19 reached an equilibrium. Moreover, carbonic anhydrase should eliminate the burst seen when CO2 was added last, because it had eliminated the lag when equation M20 was added last. Fig. 2 demonstrates that these predictions were correct. Thus line 1 was obtained when 150 μl of CO2 solution was added to a 3-ml reaction mixture otherwise identical to that used in Fig. 1. The very evident initial burst was made even more pronounced when the concentration of SOD1 was doubled (line 2). Finally, carbonic anhydrase eliminated the initial burst (line 3). It should be emphasized that the initial CO2 concentration in the reaction mixture would have been 3.8 mM, substantially lower than the concentration of equation M21 (20 mM) used in Fig. 1, yet the initial rate was greater than that seen with equation M22.


These results establish that, at pH 7.4, the peroxidase activity of SOD1 depends on CO2 rather than on equation M23. The methods used to demonstrate this were virtually identical to those Cooper et al. (5) used to show that CO2 is the substrate for phosphoenol pyruvate carboxy kinase and carboxy transphosphorylase. In the case of the peroxidase activity of SOD1, we can envision two routes for the oxidation of CO2 to equation M24 by the bound oxidant. Thus,

equation M25

equation M26

and then

equation M27

Elam et al. (6) have proposed still another route to equation M28; thus,

equation M29

equation M30

However, the rate of formation of equation M31 from CO2 plus equation M32 is slow (7), yet we saw the same initial rate of oxidation of NADPH when the equation M33 and H2O2 were preincubated before the addition of SOD1 as we did when equation M34 and SOD1 were preincubated before the addition of H2O2, rendering the mechanism defined by reactions viii and ix less likely.

CO2 is a linear molecule that exists partially as a zwitterion, i.e., –O—CAn external file that holds a picture, illustration, etc.
Object name is tbond.jpgO+ (8). This structure is also supported by its considerable solubility in water (9), which results in 76 mM CO2 in water equilibrated with 1-atmosphere CO2 at 0°C compared with only 2.18 mM for O2 under the same conditions. Perhaps the entry of CO2 into the active site of SOD1 is facilitated by this zwitterionic form.

Carbonic anhydrase contains Zn(II) at its active site, and SOD1 contains Zn(II) joined to Cu(II) by the imidazolate moiety of a histidine residue. Here, it is possible that the first point of interaction of CO2 with SOD1 is at the Zn(II) rather than at the Cu center. The proximity of the Zn and Cu would allow CO2 interacting with the Zn to be oxidized by the Cu(II)—OH. While considering carbonic anhydrases, it is reasonable to ask whether this ubiquitous family of enzymes might play a defensive role by catalyzing the hydration of CO2, which can be oxidized to equation M35, in contrast to equation M36, which cannot. In support of this supposition is the report by Götz et al. (10) that deletion of the putative carbonic anhydrase from Saccharomyces cerevisiae caused an imposed oxygen sensitivity. Although it is certainly CO2 that is oxidized to equation M37 by SOD1 plus H2O2 at pH 7.4, there may be entities in cells that can similarly oxidize equation M38. In fact, it cannot be excluded that, at high pH, SOD1 plus H2O2 may be able to oxidize equation M39 or equation M40.

The results and discussion presented above preclude some hypotheses presented earlier, including the notions that equation M41 facilitates oxidations by SOD1 plus H2O2 by creating a binding site for H2O2 (4) and that equation M42 specifically binds in the solvent access channel of SOD1 and, while so bound, becomes oxidized, at which time the resulting equation M43 protrudes sufficiently to oxidize substrates in the bulk solution (6).


This work was supported by research grants from the Amyotrophic Lateral Sclerosis Association and by Grant R01 DK 59868 from the National Institute of Diabetes and Digestive and Kidney Diseases.


Abbreviation: SOD1, Cu,Zn superoxide dismutase.


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