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J. Biol. Chem., Vol. 275, Issue 49, 38482-38485, December 8, 2000

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Copper- and Zinc-containing Superoxide Dismutase Can Act as a Superoxide Reductase and a Superoxide Oxidase^*

Stefan I. Liochev and Irwin Fridovich

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, August 29, 2000

	ABSTRACT

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The copper- and zinc-containing superoxide dismutase can catalyze the oxidation of ferrocyanide by O₂ as well as the reduction of ferricyanide by O₂. Thus, it can act as a superoxide dismutase (SOD), a superoxide reductase (SOR), and a superoxide oxidase (SOO). The human manganese-containing SOD does not exert SOR or SOO activities with ferrocyanide or ferricyanide as the redox partners. It is possible that some biological reductants can take the place of ferrocyanide and can also interact with human manganese-containing superoxide dismutase, thus making the SOR activity a reality for both SODs. The consequences of this possibility vis à vis H₂O₂ production, the overproduction of SODs, and the role of copper- and zinc-containing superoxide dismutase mutations in causing familial amyotrophic lateral sclerosis are discussed, as well as the likelihood that the biologically effective SOD mimics, as described to date, actually function as SORs.

	INTRODUCTION

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The family of superoxide dismutases (SODs)¹ encompasses enzymes containing copper + zinc (1), manganese (2), iron (3), or nickel (4) at their active sites. They provide a defense against oxidative stress by catalyzing the dismutation of O₂ into H₂O₂ plus O₂ and do so at close to the limit imposed by diffusional encounters (5, 6). Their mechanism of action is based upon the reduction and reoxidation of the catalytic metal center by O₂ as illustrated in reactions 1 and 2 for the case of Cu,Zn-SOD.

and

If an electron donor other than O₂ was to reduce the active site Cu(II) to Cu(I), the SOD would then act as a reductant:O₂ oxidoreductase, i.e. as a superoxide reductase (SOR). On the other hand, if an electron acceptor was to replace O₂ in reoxidizing the Cu(I) to Cu(II), the enzyme would then act as a superoxide oxidase (SOO).

That these ruminations can have biological relevance is shown by the findings that desulfoferrodoxin substitutes for SOD in SOD-null Escherichia coli by acting as a SOR (7, 8) and by the similar action of neelaredoxin (9, 10). It had been proposed earlier that a manganic porphyrin studied as a mimic of SOD was actually acting as a SOR (11). It appeared possible that the Cu,Zn-SOD might itself act as a SOR and/or SOO under special conditions. If so, it would provide an explanation for the deleterious effects that have been associated with the overproduction or overadministration of this enzyme (12-14).

We chose to investigate the ferrocyanide (Fe(II))/ferricyanide (Fe(III)) couple as the electron donor/acceptor couple for studies of the SOR/SOO activity of Cu,Zn-SOD for several reasons. Thus, the conversion of Fe(II) to Fe(III) can be followed at 420 nm (15): Fe(II) is not autoxidizable to a noticeable degree; Fe(II) has already been shown to reduce the active site Cu(II) of Cu,Zn-SOD (16); and the rate of spontaneous reduction of Fe(III) by O₂ is slow with a rate constant at 25 °C of ~2.7 × 10² M¹ s¹ (17). In what follows, we demonstrated that Cu,Zn-SOD can act as a Fe(II):O₂ oxidoreductase (i.e. as a SOR) and an O₂:Fe(III) oxidoreductase (i.e. as a SOO), and we discussed the possible consequences of these activities.

	MATERIALS AND METHODS

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K₄Fe(CN)₆ was from Fisher Scientific, K₃Fe(CN)₆ was from J. T. Baker, Inc., MOPS and DETAPAC were from Sigma, acetaldehyde was from Aldrich, catalase and Cu,Zn-SOD were from Grunenthal Gmbh, and Mn-SOD was from Human Biotechnology General Corp., Inc. Bovine cream xanthine oxidase (XO) was prepared by Ralph Wiley (18). Acetaldehyde was freshly distilled each day. It was used as the substrate for XO in place of xanthine because urate rapidly reduced Fe(CN)₆³ (19) and also because acetaldehyde could be used at 20 mM, allowing relatively large fluxes of O₂ to be maintained without a significant depletion of the substrate. Reactions were performed at 23 °C in 50 mM MOPS, 0.1 mM DETAPAC, 20 mM acetaldehyde, 1.0 mM Fe(II) or 0.5 mM Fe(III), 0.2 mg/ml Cu,Zn-SOD, and enough XO to cause the production of 12 nmol of O₂/min/ml when Fe(II) oxidation was to be followed and 15 nmol of O₂/min/ml when Fe(III) reduction was to be followed. The rate of O₂ production was measured by replacing Fe(II) or Fe(III) with 0.020 mM ferricytochrome c and decreasing [XO] by a factor of 5. The molar rate of cytochrome c reduction, which could be 100% inhibited by SOD, was equal to the rate of O₂ production. Fe(II) oxidation or Fe(III) reduction was followed at 420 nm using E M¹ cm¹ = 1020 (15). Other components when present are specified in the figure legends.

	RESULTS

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Cu,Zn-SOD Catalyzes the Oxidation of Fe(II) by O₂-- SOR activity with Fe(II) as the reductant would entail the catalysis of the O₂-dependent oxidation of Fe(II). Line 1 in Fig. 1A shows that Cu,Zn-SOD alone caused only a stoichiometric oxidation of Fe(II) to Fe(III), whereas the provision of a flux of O₂ allowed the enzyme to catalyze that oxidation. Line 2 in Fig. 1A shows that the flux of O₂ did not cause the oxidation of Fe(II) until Cu,Zn-SOD was added. Thus, Cu,Zn-SOD can act as a SOR with Fe(II) serving as the reductant of the active site Cu(II). Under the conditions used, the rate of Fe(II) oxidation was ~45% of the rate of O₂ production. Hence, 1.0 mM Fe(II) was unable to largely outcompete the much lower steady state concentration of O₂ as the reductant for the active site Cu(II), and the enzyme was acting simultaneously as a SOD and SOR.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   A, Cu,Zn-SOD catalyzes the O2-dependent oxidation of Fe(II). Reaction mixtures contained 20 mM acetaldehyde, 0.1 mM DETAPAC, 0.2 mg/ml Cu,Zn-SOD, 50 mM MOPS, pH 7.8, at 25 °C. Line 1, 1.0 mM Fe(II) was added at the first arrow, and XO was added at the second arrow. Line 2, all components were present except 0.2 mg/ml Cu,Zn-SOD (SOD), which was added at the arrow. B, effects of varying [XO], [Fe(II)], and [Cu,Zn-SOD]. Line 3, buffer, DETAPAC, acetaldehyde, and Fe(II) were present at the outset. 0.2 mg/ml Cu,Zn-SOD was added at the first arrow, one-fifth the usual amount of XO was added at the second arrow, and four-fifths the usual amount of XO was added at the third arrow. Line 4, all components except Cu,Zn-SOD were present at the outset. 0.02 mg/ml Cu,Zn-SOD was added at the first arrow, and 0.20 mg/ml Cu,Zn-SOD was added at the second arrow. An additional 4.0 mM Fe(II) was added at the third arrow. C, effect of Mn-SOD (see reaction mixture in A). Line 5, 0.04 mg/ml Cu,Zn-SOD was added at the arrow. Line 6, 0.04 mg/ml Cu,Zn-SOD was added with 0.15 mg/ml Mn-SOD present at the outset. Line 7, 0.04 mg/ml Cu,Zn-SOD was added with 0.67 mg/ml Mn-SOD present at the outset.

The Effects of Varying the Concentration of Cu,Zn-SOD and the Supply of O₂-- Raising the [Cu,Zn-SOD] at a constant [Fe(II)] should cause a directly proportional increase in the rate of the reduction of the active site Cu(II) by Fe(II) but a less than proportional increase in the reduction of the active site Cu(II) by O₂ because [O₂] falls as [SOD] increases. Thus, at a constant Fe(II) and constant flux of O₂, raising [Cu,Zn-SOD] should increase the ratio of SOR to SOD activities. Conversely, raising [O₂] with other factors that are held constant should favor SOD activity over SOR activity. Hence, increasing the rate of production of O₂ does not proportionately increase SOR activity because a larger fraction of the O₂ is eliminated by the SOD reactions. In keeping with those expectations, line 3 in Fig. 1B shows again that Cu,Zn-SOD caused a stoichiometric oxidation of Fe(II) and that the subsequent addition of graded amounts of XO caused a less than proportional increase in the rate of Fe(II) oxidation. Thus, increasing [XO] 5-fold, which would increase the O₂ flux 5-fold, caused only a 2.3-fold increase in the rate of Fe(II) oxidation. Line 4 shows again that a flux of O₂ per se was not able to oxidize Fe(II) and that the subsequent addition of graded amounts of Cu,Zn-SOD caused a less than proportional increase in the rate of Fe(II) oxidation as did raising the [Fe(II)]. These results are in accordance with expectations based on the effects of these manipulations on the [O₂] and on the ratio of Cu(II) to Cu(I) at the active site, and they are further considered under "Discussion."

Effect of Mn-SOD-- Mn-SOD does not catalyze the oxidation for Fe(II) by O₂. It could be used to test the effect of [O₂] on the SOD:SOR ratio. Thus, 0.15 mg/ml Mn-SOD slowed the oxidation of Fe(II) by the SOR activity of Cu,Zn-SOD (compare lines 5 and 6 in Fig. 1C), whereas 0.66 mg/ml caused further inhibition, which was less than proportional (line 7) because the SOR:SOD ratio increases as [O₂] decreases. Such high concentrations of Mn-SOD were needed also because it was competing for O₂ with the Cu,Zn-SOD.

Catalase added to 100 units/ml was without effect on the O₂-dependent oxidation of Fe(II) by Cu,Zn-SOD. Moreover, 0.1 mM H₂O₂ did not replace the flux of O₂ in facilitating the oxidation of Fe(II) by Cu,Zn-SOD (data not shown). It follows that there was no detectable oxidation of Fe(II) by the "peroxidase" activity of Cu,Zn-SOD (20, 21).

A Superoxide: Ferricyanide Oxidoreductase Activity of Cu,Zn-SOD-- O₂ is known to reduce Fe(III) with a rate constant of 3 × 10² M¹ s¹ (17), and as shown by line 1 in Fig. 2A, the flux of O₂ produced by the XO reaction (15 nmol/ml/min) caused the reduction of Fe(III). Cu,Zn-SOD added to 0.002 mg/ml inhibited the rate of Fe(III) reduction, but adding more did not inhibit further and indeed increased the rate of Fe(III) reduction. Line 2 presents a repetition of this experiment but at one-fifth the XO and thus at one-fifth the flux of O₂. Thus, at a low concentration, Cu,Zn-SOD inhibits the reduction of Fe(III) by O₂ by catalyzing the dismutative elimination of O₂, whereas higher [Cu,Zn-SOD] catalyzes the reduction of Fe(III) by O₂ (i.e. it acts as a superoxide oxidase). When the concentration of Fe(III) was decreased to 0.1 mM, its rate of reduction by the flux of O₂ was much lower than that flux due to the loss of O₂ to the spontaneous dismutation of O₂. Under these conditions, the SOO activity of Cu,Zn-SOD was evident at lower concentrations of the enzyme. This is shown by line 3 in Fig. 2.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Cu,Zn-SOD catalyzes the O2-dependent reduction of Fe(III). Reaction mixtures contained 20 mM acetaldehyde, 0.1 mM DETAPAC, 0.2 mg/ml Cu,Zn-SOD, 0.5 mM Fe(III), 50 mM MOPS, pH 7.8, at 25 °C. Line 1, XO was added at the first arrow, 0.002 mg/ml Cu,Zn-SOD (SOD) was added at the second arrow, 0.004 mg/ml Cu,Zn-SOD was added at the third arrow, and 0.2 mg/ml Cu,Zn-SOD was added at the fourth arrow. Line 2, one-fifth the usual amount of XO was added at the first arrow, 0.001 mg/ml Cu,Zn-SOD was added at the second arrow, and 0.2 mg/ml Cu,Zn-SOD was added at the third arrow. Line 3, 0.1 mM Fe(III) and XO were added at the first arrow, 0.04 mg/ml Cu,Zn-SOD was added at the second arrow, and 0.2 mg/ml Cu,Zn-SOD was added at the third arrow.

	DISCUSSION

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We have seen that Cu,Zn-SOD can catalyze the dismutation of O₂, the reduction of O₂ by Fe(II), or the oxidation of O₂ by Fe(III). That is to say that Cu,Zn-SOD can act as a SOD, SOR, or SOO. Although these activities were demonstrated using the decidedly unnatural Fe(II)/Fe(III) redox couple, it is possible that in the reducing environment of the cell, there is some natural redox pair that can interact with the active site of the Cu,Zn-SOD. Because a number of otherwise puzzling observations can be explained based on these multiple activities, it may be worthwhile to express the component reactions and their relationships more rigorously.

SOD activity depends upon the sum of Reactions 1 and 2 as noted earlier. The SOR activity involves Reactions 3 and 2, whereas the SOO activity involves Reactions 4 and 1. 

and

The rates of the component reactions can be written as follows.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

Under steady state conditions, the sum of the rates of the reduction of E  Cu(II) must equal the sum of the rates of the oxidation of E  Cu(I), hence, V_I + V_III = V_II + V_IV. In the presence of a constant flux of O₂ and when [Fe(II)] [Fe(III)]V_IV is negligible and then

(Eq. 5)

or equivalently

(Eq. 6)

When [Cu,Zn-SOD] is increased the V_III term increases in direct proportion because Fe(II) is in large excess and is effectively constant, but V_I and V_II will not increase proportionately because [O₂] will fall as [Cu,Zn-SOD] increases. It follows that increasing [Cu,Zn-SOD] will favor SOR activity over SOD activity. This effect was demonstrated in Fig. 1B. Similarly, raising Fe(II) will also increase the SOR:SOD ratio.

Anything that decreases [O₂] will favor the SOR reaction over the SOD reaction. This can be made obvious by rearranging Equation 6.

(Eq. 7)

The left side of Equation 7 must fall as [O₂] falls, but the SOR:SOD ratio will rise because decreasing [O₂] will increase the [E  Cu(I)]:[E  Cu(II)] ratio. This is the case because O₂ is the only oxidant of E  Cu(I) under the conditions specified. We have seen that decreasing [O₂] by increasing [Cu,Zn-SOD], by adding Mn-SOD, or by lowering [XO] increased the SOR:SOD ratio in accordance with these deductions.

The Fe(II)/Fe(III) couple has been shown here to support the SOR and SOO activities of Cu,Zn-SOD but not the activities of Mn-SOD because this redox couple interacts with the active site copper but not with the active site manganese. If the cell contains redox couples competent to interact with both active sites, both Cu,Zn-SOD and Mn-SOD could act as SORs and SOOs. Given that cell cytosols are reducing environments, SOR activity is more likely to occur in vivo than SOO activity.

Whether the SOD enzymes act only in the SOD, SOR, or SOO modes, they have an effect on the amount of H₂O₂ produced from O₂. Thus, in the SOD reaction, 0.5 H₂O₂ is produced per O₂ consumed, whereas in the SOR mode, 1.0 H₂O₂ is the yield per O₂. In the SOO mode, no H₂O₂ would be made from O₂. Because we have seen that lowering [O₂] favors the SOR mode, we deduce that raising [Cu,Zn-SOD] would increase H₂O₂ production only in the presence of a reductant capable of reducing the active site Cu(II). If O₂ were acting to initiate oxidative chain reactions, then the yield of H₂O₂/O₂ could be significantly greater than 1.0 H₂O₂/O₂. In such a situation SODs would decrease H₂O₂ production.

If an endogenous reductant that can act as a SOR substrate is an essential molecule or if its oxidized form is toxic, we can understand the reports that overproduction (13, 14) or overadministration (12) of SOD has deleterious effects. Thus, increasing [SOD] increases the SOR:SOD ratio because it lowers [O₂] and at the same time increases the net SOR action. The neurotoxic effect of the mutant forms of Cu,Zn-SOD that have been associated with the familial amyotrophic lateral sclerosis (22) may be because of SOR activity. Thus, the mutated Cu,Zn-SODs may be able to catalyze the oxidation of essential reductants within motor neurons by O₂, a SOR activity that is not exerted by the wild-type enzyme.

O₂ is scavenged very effectively by desulfoferrodoxin (7, 8) and neelaredoxin (9, 10) acting as SORs. The SOD-mimic O-Mn(III)meso-tetrakis-(N-methylpyridinium-2-yl)porphyrin (MnTMPyP) has also been reported to act as a SOR within E. coli (11). Given that low molecular weight SOD-mimics are certain to be less discriminating than the SODs themselves with regard to interaction with reductants, it seems probable that the biological effects of most of these mimics are because of SOR rather than SOD activity. The results of Offer et al. (23) who have reported that the nitroxide-catalyzed oxidation of Fe(II) by a flux of O₂ could be inhibited by low [Cu,Zn-SOD] but not by high [Cu,Zn-SOD] can now be understood in terms of the increase in SOR activity with increasing [Cu,Zn-SOD] as described above. It should be stressed that any agent acting as a SOR could be beneficial or detrimental depending on the nature of the reductant consumed and the oxidized product generated from thereon, the availability of that reductant and the possibility of its regeneration, and the magnitude of the flux of O₂ and the availability of critical targets for O₂ attack. Thus, not only the toxic effects of the nitroxides as Offer et al. (23) suggest but also their beneficial actions are explicable on the basis of their SOR activity.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 919-684-5122; Fax: 919-684-8885; E-mail: fridovich@biochem.duke.edu.

Published, JBC Papers in Press, September 25, 2000, DOI 10.1074/jbc.M007891200

	ABBREVIATIONS

The abbreviations used are: SOD, superoxide dismutase; Cu, Zn-SOD, copper- and zinc-containing superoxide dismutase; SOO, superoxide oxidase; SOR, superoxide reductase; Mn-SOD, human manganese-containing superoxide dismutase; Fe(II), potassium ferrocyanide; Fe(III), potassium ferricyanide; MOPS, 4-morpholinepropanesulfonic acid; DETAPAC, diethylenetriaminepentaacetic acid; XO, xanthine oxidase.

	REFERENCES

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