The generation of superoixide radical during the autoxidation of ferredoxins.

Clostridial and spinach ferredoxins, reduced enzymatically by the action of ferredoxin-TPN+ oxidoreductase, have been shown to carry out the univalent reduction of oxygen. The superoxide radicals, so generated, were detected by their ability to cause the oxidation of epinephrine to adrenochrome. Superoxide dismutase prevented the production of adrenochrome in these reaction mixtures, while having no effect on the rate of oxidation of TPNH. The ratio of the univalent reduction of oxygen to the swn of the univalent plus divalent reductions of oxygen was computed as a function of pH and of the concentrations of oxygen. This percentage of univalent flux increased with rising pH and with increasing concentrations of oxygen as has previously been observed in the case of milk xanthine oxidase. Under comparable conditions of pH and of oxygen concentration, milk xanthine oxidase generated 02four times more rapidly than did clostridial ferredoxin.


I. FRIDOVICH
From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina W7706 SUMMARY Clostridial and spinach ferredoxins, reduced enzymatically by the action of ferredoxin-TPN+ oxidoreductase, have been shown to carry out the univalent reduction of oxygen. The superoxide radicals, so generated, were detected by their ability to cause the oxidation of epinephrine to adrenochrome. Superoxide dismutase prevented the production of adrenochrome in these reaction mixtures, while having no effect on the rate of oxidation of TPNH. The ratio of the univalent reduction of oxygen to the swn of the univalent plus divalent reductions of oxygen was computed as a function of pH and of the concentrations of oxygen. This percentage of univalent flux increased with rising pH and with increasing concentrations of oxygen as has previously been observed in the case of milk xanthine oxidase.
Under comparable conditions of pH and of oxygen concentration, milk xanthine oxidase generated 02-four times more rapidly than did clostridial ferredoxin.
Superoxide radicals have been shown to be produced during the aerobic action of milk xanthine oxidase (l-5).
Both the ironsulfur (6, 7) and the flavin (5) moieties of this enzyme have been proposed as the site responsible for the univalent reduction of oxygen.
It is difficult to design experiments which would unequivocally settle this point. It is, however, possible to compare the behavior of xanthine oxidase with that of simpler iron-sulfur proteins in order to detect parallelisms which may exist in their modes of reduction of oxygen.
That fraction of the total electron flux t.hrough xanthine oxidase which resulted in the univalent reduction of oxygen has been measured as a function of pH, oxygen tension, and concentration of xanthine (8). It appeared desirable to similarly investigate t,he reduction of oxygen by reduced ferredoxin, as a function of pH and of the concentration of oxygen.
The present report describes such measurements which reveal considerable similarity in the reduction of oxygen by milk xanthine oxidase and by the ferredoxins from Clostridium pasteurianum and from spinach.

MATERIALS AND METHODS
Ferredoxins-Spinach ferredoxin was prepared by a modification of the method of Petering and Palmer (9). The concen-* The work reported herein was supported in full by Grant GM-10287 from the National Institutes of Health, Bethesda, Maryland. trated solution of ferredoxin which they obtained by elution from DE-52 was treated with streptomycin to a final concentration of 5% and was then clarified by centrifugation at 9000 x g for 10 min. The supernatant solution (2 to 3 ml) was placed on a column (2.5 x 25 cm) of Sephadex G-75 and was eluted with 0.15 M Tris-HCl buffer at pH 7.8. Fractions for which the ratio of absorbance at 420 nm to that at 275 nm exceeded 0.45 were pooled and were concentrated by adsorption onto DE-32 and subsequent elution therefrom with 0.15 M Tris-HCl, 1.0 M NaCl, at pH 7.8 and at a slow flow rate. The resultant material gave a single band upon disc gel eletrophoresis when stained with Amido black (10). Its absorption spectrum, recorded with a Cary 15, agreed with the published spectrum for this ferredoxin (11) and exhibited peaks at 462 nm, 420 nm, 320 nm, and 275 nm. This ferredoxin was also active in mediating the photoreduction of TPNf by chloroplasts when tested by the method of San Pietro (12). The concentrations of solutions of spinach ferredoxin were determined on the basis of a molar extinction coefficient of 9400 M-I cm-l at 420 nm (13). Spinach ferredoxin was stored in the presence of 1.0 M NaCl and was diluted into reaction mixtures without prior desalting.
Ferredoxin from C. pasteuriunum was obtained from the Worthington Biochemical Corporation (Freehold, New Jersey).
Concentrations were determined on the basis of a molar extinction coefficient of 26.6 X lo3 M-I cm-' at 425 nm (14).
Other iMaterials-Superoxide dismutase was prepared from bovine erythrocytes and assayed as previously described (3). Ferredoxin-TPNf oxidoreductase was prepared according to Shin,Tagawa,and Arnon (15). Xanthine oxidase, prepared from unpasteurized cream by a procedure which avoided treatment with proteolytic agents (16), was kindly provided by Dr. K. V. Rajagopalan. Catalase was obtained as a suspension of crystals from the Sigma Chemical Company.
One unit of catalase was defined as that amount which decomposed 1 pmole of Hz02 per min when acting on 0.02 M H202 in phosphate-buffered solutions at pH 7.8 and at 25". TPN+ was obtained from Boehringer Mannheim and TPNH from P-L Biochemicals.
All other materials were purchased at the highest available purities.
Assays-All spectrophotometric assays were performed at 25" in a Gilford model 2000 recording spectrophotometer equipped with a thermostatted cell compartment. Reactions under controlled atmospheres were performed in cuvettes which allowed purging the reaction volume with the desired gas mixture. These cuvettes are similar to those described by Lazarow and Cooperstein (17) and were obtained from Pyrocell.
Oxygen consumption was measured with a Gilson Medical Electronics (Middleton, Wisconsin) oxygraph equipped with a Clark oxygen electrode. 2.5, 1971 H. P. Misra and I. Fridovich 6887

Issue of
Ferredoxin (M x IO71 FIG. 1. The ferredoxin dependence of TPNH oxidation. Reaction mixtures contained 3.3-X low5 M TPNH, 1.8 X 1O-8 M FTreductase, 10m4 M EDTA, 2.4 X 10Y4 M 02. the indicated concentrations of ferredoxin, and 6.05 M pot,assium phosphate at pH 7.8 and 25". A, data obtained with clostridial ferredoxin; 0, data obtained with clostridial ferredoxin in the presence of 1 rg per ml of superoxide dismutase; l , data obtained with spinach ferredoxin.
The transfer of electrons from TPNH through the ferredoxin-TPN+ oxidoreductase to oxygen was mediated by ferredoxin. The total flux of electrons from TPNH to oxygen was measured in terms of the oxidation of TPNH, which was followed at 340 nm. Since ferredoxin is readily reduced by the ferredoxin-TPNf oxidoreductase and TPNH oxidation was totally dependent upon the presence of ferredoxin and since O2 was the only electron acceptor present in greater than catalytic amounts, the net oxidation of TPNH is presumed to have been dependent upon the oxidation of reduced ferredoxin by 02. The extent of univalent reduction of 02 by reduced ferredoxin was measured with the use of epinephrine as an indicator.
It has been demonstrated that Oi-causes the oxidation of epinephrine to adrenochrome (3). Adrenochrome exhibits an absorption maximum at 480 nm with an extinction coefficient of 4020 M-~ cm-r (18). When using nearly saturating concentrations of epinephrine it was therefore possible to quantitatively determine the univalent reduction of oxygen in terms of the rate of accumulation of adrenochrome. Fig. 1, the rate of aerobic oxidation of TPNH by ferredoxin-TPN+ reductase was a linear function of the concentration of ferredoxin, in the range 0 + 2 X 10h6 M. It is also clear that superoxide dismutase had no effect on the ability of clostridial ferredoxin to mediate the oxidation of TPNH.

Ferredoxins as Mediators of TPNH Oxidation-As shown in
This was also shown to be the case with spinach ferredoxin.
These results follow directly from the ability of FT-reductaser to transfer electrons from TPNH to ferredoxins and from the autoxidizability of reduced ferredoxins.
The linear relationship between the concentration of ferredoxin and the rate of TPNH oxidation indicates that the dissociation constant of the FT-reductase-ferredoxin complex was greater than 2 X 10m6 M under these conditions. This is in accord with the result's of Faust, Mayhew,and Massey (19). If superoxide anions are generated during the oxidation of reduced ferredoxin, their rate of dismutation should have no influence on the rate of TPNH oxidation.
The inability of superoxide dismutase to inhibit this assay was therefore anticipated. It does, however, establish that superoxide dismutase does not 1 The abbreviation used is: FT-reductase, ferredoxin-TPN+ oxidoreductase. interact directly with and thus influence the activity of FT-reductase. The single measurement made with the spinach ferredoxin under these conditions demonstrated that it was approximately half as active as an equimolar amount of the clostridial ferredoxin.
Epinephrine as Detector of OS-The action of FT-reductase upon TPNH and ferredoxin in the presence of epinephrine and oxygen resulted in the production of adrenochrome.
As shown in Fig. 2, the rate of accumulation of adrenochrome was a function of the concentration of ferredoxin, and clostridial ferredoxin was a more effective mediator of adrenochrome formation than was spinach ferredoxin.
The effect of varying the concentration of epinephrine was also investigated and these results are shown in Fig. 3. Elimination of oxygen from these reaction mixtures completely prevented the formation of adrenochrome.
Superoxide dismutase was used to verify the role of 0, in the oxidation of epinephrine to adrenochrome.
Thus, 02, generated by the oxidation of reduced ferredoxin, could either dismute to Hz02 + 02 or could react with epinephrine.
Superoxide dismutase, by catalyzing the former reaction, should inhibit the formation of adrenochrome. Fig. 4 demonstrates that this was the case. Superoxide dismutase inhibited the production of adrenochrome when either spinach ferredoxin or clostridial ferredoxin was used as the electron carrier.
It follows that both of these ferredoxins are capable of the univalent reduction of oxygen. Since superoxide dismutase was in competition with epinephrine for the available 01, the sensitivity of this reaction to inhibition by superoxide dismutase could be enhanced or diminished at will by lowering or raising the concentration of epinephrine. Superoxide dismutase must also be considered to be in competition with the spontaneous dismutation reaction (1). Therefore, the greater the rate of generation of 0, in any system, the greater the amount of superoxide dismutase which will be required to effect a given reduction in the steady state concentration of 0,. This reasoning provides an explanation for the apparently greater sensitivity toward superoxide dismutase exhibited in Fig. 4   reaction mixtures containing clostridial ferredoxin and eliminates the need to seek an explanation based upon direct interactions between superoxide dismutase and these ferredoxins. E$ects of Epinephrine and of Superoxide Dismutase on Oxygen Uptake-If 01 reacts with epinephrine, in a manner which leads to the formation of adrenochrome, then adding epinephrine to reaction mixtures which generate 01 should result in an enhancement of oxygen consumption due to the co-oxidation of epinephrine.
Superoxide dismutase should prevent this co-oxidation and should thus inhibit the extra oxygen uptake.
The results shown in Fig. 5 demonstrate that this was the case. Line S represents the oxygen consumption by 1 x 10e8 M xanthine oxidase J Seconds FIG. 5. The effect of epinephrine on oxygen consumption by a xanthine oxidase system. Reaction mixtures contained 1.67 X lo+ M xanthine, 1 X 10-S M xanthine oxidase, 1 X 10-4 M EDTA, 2.4 X 10V4 M 02, and 0.05 M potassium phosphate at pH 7.8 and 25". In addition, reaction mixture 1 contained 1.11 X 10-4 M epinephrine and reaction mixture 2 contained 1.11 X lo+ M epinephrine plus 2.22 P,D per ml of superoxide dismutase.
Reactions were initiated at the jirst arrow by the addition of xanthine oxidase.
After oxygen consumption had ceased, catalase was added to a final concentration of 55 units per ml.
acting on 1.67 X 10V5 M xanthine in 0.05 M potassium phosphate, 1 X 10e4 M EDTA at pH 7.8 and 25". Line 1 demonstrates that adding 1.11 X 1O-4 M epinephrine to this reaction mixture enhanced the rate and the extent of oxygen uptake by a factor of 1.6. Line 2 shows that 2.22 pg per ml of superoxide dismutase completely prevented the effect of epinephrine on oxygen utilization, In all cases, the addition of catalase after oxygen uptake had ceased resulted in an evolution of oxygen in an amount equal to half of that which had been consumed. This is the expected result when the final product of oxygen reduction is HrOz.
Proportion of Univalent Reduction of Oxygen by Ferredoxin-The oxidation of TPNH in these reaction mixtures was totally dependent upon the presence of the FT-reductase, oxygen, and ferredoxin.
We assume that the only available pathway for electrons from TPNH to oxygen traversed both the osidoreductase and the ferredoxin.
The total flux of electrons from ferredoxin to oxygen was computed in terms of the oxidation of TPNH.
Since the conversion of epinephrine to adrenochrome was totally inhibitable by superoxide dismutase, we conclude that 02-was the agent responsible for causing this oxidation.
Increasing the concentration of epinephrine should result in t'he interception of an ever increasing proportion of the 02 generated and should therefore yield a saturation curve. This was shown to be the case in Fig. 3. It follows that saturating concentrations of epinephrine will trap virtually all of the 0, generated and that it should therefore be possible to quantitatively determine the rate of univalent transfer of electrons from ferredoxin to oxygen, in terms of the rate of conversion of epinephrine to adrenochrome. The saturation of the rate of adrenochrome formation by raising the concentration of epinephrine (Fig. 3) also demonstrates that chain mechanisms, involving epinephrine, were not quantitatively significant under these conditions.
If the stoichiometry of the reaction of epinephrine with 02-to yield adrenochrome were known, it would then be possible to calculate what fraction of the total electron flux, from ferredoxin to oxygen, was accomplished by univalent electron transfers. 6. Percentage of univalent reduction of oxygen as a function of oxygen concentration.
That percentage of the total electron flux through ferredoxin to oxygen which resulted in the univalent reduction of oxygen is here presented as a function of the percentage of oxygen in the gas phase. Cuvettes contained 3.3 X 10-J M epinephrine, 3.3 X 10-s M TPNH, 1.4 X 10U6 M ferredoxin, and 1.8 X 10-S M ferredoxin-TPN+ oxidoreductase in a total volume of 3.0 ml buffered at pH 7.8 or at pH 6.8 by 0.05 M potassium phosphate containing 1 X 10m4 M EDTA.
The reaction mixtures were bubbled with mixtures of nitrogen and oxygen for 10 min at 25" prior to sealing the cuvettes and starting the reaction by tipping the enzyme in from a side arm. l , data obtained with spinach ferredoxin; A, represents a measurement made with clostridial ferredoxin.
The percentage of univalent flux was calculated as described in the text.
oxidation of epinephrine to adrenochrome requires the removal of 4 hydrogen atoms or 4 electrons and 4 protons.
The stoichiometric relationship between 02 and adrenochrome cannot, however, be calculated on this basis, since the univalent oxidation of epinephrine by one 02may well yield an epinephrine radical, the subsequent conversion to adrenochrome of which could occur by a complex, oxygen-dependent reaction without consumption of additional 02. This stoichiometry, between 0, and adrenochrome, was therefore determined empirically by comparing the rate of reduction of ferricytochrome c with the rate of accumulation of adrenochrome, when saturating levels of ferricytochrome c or of epinephrine were used to intercept the 02 generated during the aerobic action of xanthine oxidase on xanthine. These measurements were made at pH 7.8 and at 6.8 and at 1 and 2 X 10e5 M cytochrome c and at 2 and 4 X 10m4 M epinephrine. The results of these measurements, expressed in terms of the molecules of adrenochrome accumulated per 02 generated, are given in Table I. These results are based on the assumption that 1 molecule of cytochrome c is reduced per 0,.
That fraction of the total electron flux from ferredoxin to oxygen, which occurred by univalent transfers, was determined as a function of oxygen concentration.
This was done with spinach ferredoxin at pH 6.8 and 7.8. The results of these measurements are shown in Table I and in Fig. 6 and indicate that the percentage of univalent flux increased with oxygen concentration and with pH.
Clostridial ferredoxin was studied only at pH 7.8 in air and the result illustrated by the solid triangle in Fig. 6 shows that its behavior was not significantly different from that of the spinach ferredoxin under the same conditions.

DISCUSSION
The univalent fraction of the total flux of electrons between xanthine oxidase and oxygen has been seen (8) to increase with increasing concentration of oxygen and with rising pH. The ferredoxins from spinach and from C. pasteurianum have now been shown to behave similarly. Xanthine oxidase and the ferredoxins share a number of properties among which are low oxidation-reduction potential (6, 14), G = 1.94, low temperature electron paramagnetic resonance spectrum when in the reduced state (20, 21), equimolar amounts of nonheme iron and acid-labile sulfide (14, 21), and a characteristic absorption spectrum in the visible region (14). This constellation of properties has been associated with an iron-sulfur chromophore which contains two iron atoms close enough to share one electron (22,23) or to engage in antiferromagnetic coupling (24). This common structural feature coupled with similar changes in percentage of univalent flux, in response to changes in pH and oxygen tension, does not prove that the site of oxygen reduction in intact xanthine oxidase is at the iron-sulfur chromophore, but it does make that proposal increasingly attractive.
It is of interest to compute the rate at which ferredoxin can effect the univalent reduction of oxygen.
This can conveniently be expressed in terms of the molecules of 02 generated per molecule of ferredoxin per min under conditions of limiting ferredoxin. In reaction mixtures containing 1.12 X 10M6 M FT-reductase, 6 X lo-* M clostridial ferredoxin, 3.33 X lop5 M TPNH, 3.33 X 10-d M epinephrine, 2.4 X 10e4 M oxygen, 1 X 10m4 M EDTA, and 0.05 M potassium phosphate at pH 7.8 and at 25", the rate of adrenochrome formation was 47 molecules of adrenochrome per min per molecule of ferredoxin.
Since this formation of adrenochrome was inhibited 96% by 1.33 pg per ml of superoxide dismutase and since each adrenochrome corresponded to 1.39 02 (Table I), we may calculate that the rate of generation of 01 was 65 02 per min per ferredoxin.
This rate of generation of 02. by clostridial ferredoxin may be compared with that found with milk xanthine oxidase. Thus, at pH 7.0 and at 25", milk xanthine oxidase generated 242 molecules of 01 per min per molecule of enzyme, when operating at Vm,, in air. At pH 10.2 the corresponding number was 1060. Clostridial ferredoxin is thus one-fourth as effective a source of 02 as is xanthine oxidase under comparable conditions.
In contrast, the flavoenzymes (5) were less effective than xanthine oxidase by factors of 50 or more. This similarity between milk xanthine oxidase and the ferredoxins, in terms of the rate of univalent electron transfer to oxygen, coupled with the grossly dissimilar behavior of a variety of flavoenzymes, provides yet another reason for suspecting that univalent reduction of oxygen by milk xanthine oxidase occurs at its iron prosthetic groups.
Electron paramagnetic resonance spectrometry has been used to detect OS-generated during the oxidation of reduced clostridial Superoxide Radicals Generated by Ferredoxins Vol. 246,No. 22 ferredoxin but failed to detect 0~~ when similarly applied to the spinach ferredoxin (25, 26). Chemical and enzymatic methods have now allowed the demonstration that spinach and clostridial ferredoxins are both capable of the univalent reduction of oxygen. This result is not surprising in view of the reported similarities between these ferredoxins (14). Quick freeze electron paramagnetic resonance methods can detect the steady state level of 02 present in a reaction mixture at the instant of freezing, whereas chemical trapping methods can detect all of the 02 generated in a reaction mixture over a period of minutes.
The electron paramagnetic resonance methods of detecting OZ-are thus inherently less sensitive and more difficult to apply than the chemical methods. In view of the difficulties and limitations of the methods, it appears likely that the reported (25, 26) inability to demonstrate 02 during the reoxidation of reduced spinach ferredoxin was due to technical problems.
Since spinach ferredoxin has been found capable of transferring only 1 electron per molecule (14, 27, 28) we must rather consider how it can bring about any divalent reduction of oxygen.
One would expect it to be capable of only univalent reductions of oxygen, in which case the percentage of univalent flux would have to be 100% under all conditions. As was shown in Table I and in Fig. 6, this was not the case. The reason for this is not known, but two possible explanations may be advanced. Thus, ferredoxin is known to associate with 29). It is possible that part of the reduction of oxygen was accomplished by the FT-reductase-ferredoxin complex.
In such a situation, elect,rons could flow in sequential univalent steps from FT-reductase to ferredoxin to oxygen.
Univalent reduction of oxygen would then occur when oxygen separated from the FT-reductase-ferredoxin complex after the transfer of a single electron, whereas divalent reduction would depend upon oxygen remaining associated with the complex until the second electron had been transferred.
Another possibility is that association with ferredoxin so modifies FT-reductase that it becomes capable of directly transferring electrons to oxygen by a route not traversing ferredoxin. In this ca,se ferredoxin-dependent, divalent reduction of oxygen by FT-reductase plus TPNH could be observed. The actual mechanism of the divalent reduction of oxygen by the FT-reductase plus ferredoxin system remains to be ascertained.