THE REACTION OF CATALASE AND CYANIDE*

BY BRITTON CHANCEt

(From the Biochemical Department of the Medical Nobel Institute, Stockholm, Sweden, and the Johnson Research Foundation, University of Pennsylvania, Philadelphia)

(Received for publication, October 20, 1948)

Previous work has suggested that the inhibition of catalase activity by cyanide is of the non-competitive type. This is remarkable, because both cyanide and peroxide are considered to combine with the iron atom of a hemoprotein. In this paper, the mechanism of the cyanide inhibition of catalase is studied in detail. First, kinetic and equilibrium measure- ments show that the reaction of catalase and cyanide is in accordance with the law of mass action and that the three or four catalase hematins act independently. Second, an improved method for the determination of catalase activity (1) has been used to measure the inhibition of catalase activity by cyanide. The dissociation constants of catalase cyanide calcu- lated from the data obtained in these two cases are in agreement and verify non-competitive inhibition. This result is explained by the assump- tion that only a portion of the three or four catalase hematins is bound to peroxide during the destruction of hydrogen peroxide. This assumption is strongly supported by the recent finding of the intermediate compound of catalase and hydrogen peroxide, which probably has only one hematin bound to hydrogen peroxide (2).

The non-competitive inhibition of catalase activity by cyanide is in striking contrast to the analogous studies of the cyanide inhibition of peroxidase activity in which classical competitive inhibition was demon- strated in detail (3). Peroxidase, however, has only one hematin group and does not catalyze the decomposition of hydrogen peroxide into water and oxygen.

The reaction kinetics of the formation and dissociation of catalase cyanide have been studied spectrophotometrically by the rapid flow technique. The dissociation constant of catalase cyanide calculated from these kinetic data is in agreement with that obtained from titration and activity data. The velocity of combination of catalase and cyanide is not affected by the presence of hydrogen peroxide (4).

* Thii is Paper 1 of a series on catalases and peroxidases. t John Simon Guggenheim Memorial Fellow (1946-48). Present address, Johnson

Research Foundation, University of Pennsylvania, Philadelphia. 1299

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1300 REACTION OF CATALASE AND CYANIDE

The small effect of varying pH upon the dissociation constant of catalase cyanide indicates that the reaction studied under these conditions is

ks Cat (OH)4 + 4HCN \ ks \ Cst(CN)d + 4HzO (1)

in which the hydroxyl group found by Agner and Theorell (5) is replaced by the cyanide ion. Since it is found here that the four hematin groups of blood catalase act independently, the reaction is written,

FeOH + HCN \ k6 4

‘F&N + HnO (2)

For these purposes the concentration of water is omitted and the apparent dissociation constant is conveniently evaluated on a hematin iron basis as follows:

Kr = IHCNI[FeOHl [FeCN] (3)

The dimensions of KI are moles per liter. Preparations-The catalase preparations were purified by Dr. R. K.

Bonnichsen, to whom many thanks are due. Their optical densities were measured spectrophotometrically in the Beckman spectrophotometer, and their concentrations are calculated from the extinction coefficients, ~405

= 340 cm.-’ X rn& for the horse liver catalases, and EMS = 380 cm.-’ x n-~-l for the horse blood catalases (1). The cyanide solutions were freshly made up before the experiments, but were found to be reasonably stable for a few days as determined from titrations by Liebig’s method.

Soret Band of Catalase Cyanide-The Soret band of catalase cyanide can be measured in the Beckman spectrophotometer (6, 7) and has a peak at 425 rnp where E = 319 cm.+ X rn& for erythrocyte catalase and 274 cm.-l X no+ for a three-hematin liver catalase. Catalase and catalase cyanide are isosbestic (have the same extinction coefficient) at 417 rnp. Catalase and catalase hydrogen peroxide are isosbestic at 435 rnE.1 (2), and, because some measurements are made in the presence of hydrogen peroxide, this wave-length is used for kinetic studies.

Velocity Constant for Combination of Catalase and Cyanide-The reac- tions represented by Equation 1 are fairly fast but can be measured by the rapid flow apparatus (8). Since later data show that ks of Equation 1 is about 3 sec.-l, ks can only be measured without correction for Jcg by using a large excess of cyanide, and this precaution has been observed, as shown by the data of Table I. Thus ks is found to be 9 X lo6 M-’ X sec.-I.

In the presence of hydrogen peroxide, ka is found to be 8 X lo6 I& X sec.?.

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B. CHANCE 1301

Velocity Constant for Dissociation of Catalase Cyanide-The reaction of silver ion with cyanide

Ag+ + 2CN- + Ag(CN)s (4)

has been utilized to reduce the cyanide concentration rapidly and hence to cause the dissociation of catalase cyanide. A buffered catalase solution is saturated with cyanide and then is mixed with less than an equivalent of unbuffered silver nitrate solution in the rapid flow apparatus. The de- crease in the concentration of catalase cyanide is followed spectrophoto- metrically at 425 rnp. Table II shows that the velocity constant, 3.2

TABLE I Velocity Constant for Formation of Catalase Cyanide in Presence or Absence of

Hydrogen Peroxide

1.1 PM of Fe horse liver catalase, 0.01 M phosphate buffer, pH 6.5; X = 435 rnp (Experiments 82a and 82b).

Initial cyanide concentration, ~tbi 50 200 20 50 200 “ hydrogen peroxide concentra- 0 0 10 10 10

tion, pM Reaction velocity constant, kb dhl X 9.0 9.0 9.0 8.0 7.5

sec.-l X IFS

TABLE II Velocity Constant for Dissociation of Catalase Cyanide

0.54 PM of catalase and 10 PM of cyanide; X = 425 rnp (Experiment 317).

0.01 M phosphate, pH 7.0 0.01 Y acetate, pH 4.6 -

Silver ion concentration, PM.. . . . 5 ks, sec.-l . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 4 10 20

I I 2.8 2.8 2.6 3.3

sec.-l, is very nearly independent of the silver ion concentration. Thus the limiting step in the reaction is the dissociation of catalase cyanide and the average values of the velocity constant, Jcg, are 3.2 sec.-’ at pH 7.0 and 2.9 sec.? at pH 4.6. This very large increase of hydrogen ion con- centration has a nearly negligible effect upon Ica, and the formation of water according to Equation 2 is thereby substantiated.

The dissociation constant of catalase cyanide is I&& = 3.2/(9 X 106) = 3.6 X lo+ M, in fair agreement with the values obtained from spectro- photometric and activity data in the following experiments.

Dissociation Constant of Catalase Cyanide-The changes of optical density at 425 rnp upon the addition of measured amounts of cyanide to catalase have been measured in the Beckman spectrophotometer. The

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1302 REACTION OF CATALASE AND CYANIDE

relation between the experimental values of free catalase (FeOH), catalase cyanide (FeCN), and hydrocyanic acid (HCN) is shown by the 45” straight line of Fig. 1 to be in accordance with the logarithmic form of Equation 3,

[FcOHJ log LFeCNJ

- + log JHCNJ = log K~ (5)

The dissociation constant (KI) is 4 X 10” M with one cyanide bound to each hematin group. These experiments have been carried out over as wide a range of values of FeOH/FeCN as the accuracy of the spectropho- tometer permits in order to obtain any evidence of interaction between the four catalase hematins. Over the range from about 7 to 98 per cent saturation of catalase with cyanide there is no change of the dissociation constant of catalase cyanide in excess of the experimental error, and thus there is no detectable heme-heme interaction.

TABLE III Eflect of Change from pH 7.0 to 4.6 upon Dissociation Constant of Catalase Cyanide

x = 425 q (Experiment 285).

I Corrected optical density of cat&se cyanide

Cyanide added

OIIM 3.3@ 6.1~~ 10 PM 20 ,uY ~--

pH 7.0, 0.01 M phosphate.. . . . . . . . . . . . 0.087 0.111 0.130 0.138 0.148 “ 4.6, 0.001 “ “ . . . . . . . . . . . . . . 0.082 0.112 0.131 0.137 0.144

In accordance with tests which showed that the dissociation constant of peroxidase cyanide is independent of pH from 4.2 to 6.2 (3), Table III shows that the dissociation constant of catalase cyanide is also independ- ent of pH in the region 4.6 to 7.0. In this experiment, cyanide was added to two catalase solutions of nearly identical concentration at pH 4.6 and 7.0. At pH 4.6, sufficiently dilute phosphoric acid was used to avoid forming the catalase-phosphate complex (5). In a similar experiment, K, is found to be about 3 times greater at pH 9.3 than at pH 7.0.

The competition between formate and cyanide for catalase hematin is clearly shown by the data of Table IV. At pH 4.0, formate has a high affinity for catalase (K = 10m5 M (5)) and decreases the amount of catalase cyanide. This competition shows that cyanide and formate attach to the same place on catalase hematin. At pH 7.0, the affiity of formate for catalase is much less (K = 7 X 10m3 M (5)) and the concentrations of formate employed cause no effect upon catalase cyanide.

EJect of Cyanide upon Destruction of Hydrogen Peroxide by Catalase-

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B. CHANCE 1303

A modified technique for measuring catalase activity with a minimum of inactivation has been used to redetermine the dissociation constant of catalase cyanide from activity data.

The extinction coefficient of hydrogen peroxide at 215 rnp is sufficiently large to permit the measurement of the disappearance of a few mM with good accuracy in the Beckman spectrophotometer. Dilute peroxide (- 1 mM) gives a few very small bubbles of oxygen at the end of the reaction, which cause no significant error. The optical density of the dilute cata- lase solution employed in the activity test (~1 X lo+ M) is so small that it is negligible. The spectrophotometer is allowed to run for about 15 minutes or more so that the light intensity and the “dark current” (ampli- fier plate current) are not drifting rapidly and do not require adjustment during the kinetic test.

TABLE IV Competition between Cyanide and Formute for Catalase Hem&in

0.86 paa of horse blood catalase and 130 pi of cyanide; X = 425 q (Experiment 321).

Corrected optical density

Formate added

ON 65 JU 195 PY 5lOrar 1120 J&* ---~-

pH 7.0, 0.01 M phosphate.. . . . . . . . . . . . 0.257 0.253 0.259 0.260 0.253 “ 4.0,0.005 “ acetate.. . . . . . . . . . . . . . . 0.267 0.258 0.228 0.202 0.190

In inhibition studies, the catalase and cyanide are added to the cuvette, and the spectrophotometer is adjusted. The peroxide is delivered onto a stirring rod and is rapidly stirred into the cuvette, and the stop-watch is started. Readings of the optical density of the hydrogen peroxide solu- tion are taken every 10 or 15 seconds for the 1st minute. It is desirable to “track” the density change continuously and to read off the density values at the appropriate intervals. The final value of density is measured and subtracted from each reading of optical density. The reaction velocity constant is calculated from the formula

kl - 2.3 -lo$

e(t2 - h) aa

where kl is the velocity constant for the destruction of hydrogen peroxide by catalase, e is the catalase concentration in moles per liter, x1 is the opti- cal density at tl, and x2 is the optical density at IS This form of the equa- tion is used because the value of optical density at t = 0 cannot be obtained when the hydrogen peroxide is added last.

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1304 REACTION OF CATALASE AND CYANIDE

This method has much to recommend it, since it is much more conve- nient and just about as accurate as the titrimetric method described pre- viously (1). The value of kI for horse blood catalase at 25” is the same according to both methods (3.5 X 10’ I& X sec.+).

3.0 -

Log I* e

OR 2.0 -

RI Log IO2 - RI-RI

1.0 -

01 I I I I

TO 0 I.0 2.0

Log 106[HCN]

FIG. 1. The dissociation constant of catalase cyanide, KI = IFeOEn IHCNl/ (FeCN], determined from spectrophotometric data (0) and inhibition data (a). The spectrophotometric data were obtained in the Beckman spectrophotometer with a wave-length of 425 rnp and 0.9 PM of horse blood catalase. The inhibition data were determined from the kinetics of hydrogen peroxide disappearance meas- ured at 215 w in a solution containing 1 mM of hydrogen peroxide and 1 X 10-O M horse blood catalase. Ru, the uninhibited rate, = 3.5 X 10’ ah1 sec.-l. RZ is the inhibited rate. All experiments were made in 0.01 M phosphate buffer, pH 7.0 at 25” (Experiment 315).

The dissociation constant of catalase cyanide is calculated from the inhibition data by the formula

which has been used previously in studies of peroxidase inhibition (3). R. is the uninhibited rate (3.5 X 10’ M? X sec.+) and Rr is the inhibited

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B. CHANCE 1305

rate. The data are plotted by using the logarithmic form of Equation 7, and are represented in Fig. 1. These points agree very well with the spectrophotometric data: the dissociation constant of catalase cyanide obtained from spectrophotometric data (4 X lo+ M) is very nearly identi- cal with the dissociation constant obtained from the inhibition data (4.7 x lo*M).

DISCUSSION

The velocity constant for the combination of catalase and cyanide (9 X lo5 I@ X sec.-‘) and the dissociation constant of catalase cyanide (4 X 10” M) are similar to the corresponding values for peroxidase cyanide (1 X lo5 M+ X sec.-l, 4 X 10” M (3)), although cyanide combines with and dissociates from peroxidase more slowly than from catalase.

The measured values of the dissociation constant of catalase cyanide are not altered over the range from 7 to 98 per cent saturation of catalase with cyanide, and therefore there is no detectable heme-heme interaction in the binding of cyanide to the three hematins of liver catalase. This is in accord with the results of later tests with methyl hydrogen peroxide (9).

In their “magnetic titrations” of catalase with cyanide, Theorell and Agner (10) noted that the curves of magnetic susceptibility versus cyanide concentration (cf. (lo), Figs. 1 and 2) did not follow a straight line but were bent before saturation of catalase with cyanide ion occurs and attributed this effect to heme-heme interaction: cyanide ion bound to 1 or 2 of the 3 hematin iron atoms alters the magnetic susceptibility of the free hematin iron atoms. But the curvature they found is caused by the dissociation of catalase cyanide. Their data follow the theoretical titration curve expected for GO0 PM of hematin iron catalase and KI = 3 X 10V6 M. There- fore, their magnetic data support the conclusion that there is no detectable heme-heme interaction in catalase cyanide.

The nature of the reaction of catalase and cyanide as given in Equation 1 is verified by these experiments. In the equilibria for catalase hydroxide and hydrogen cyanide,

FeOH -Fe+ + OH-, K = 10-lo*a w

HCN + H+ + CN-, K = 10-a’ (9)’

the product Fe] [CN-] is nearly constant over the range pH 8.7 to 3.8 (14 - 10.2); over this pH region [Fe+] is increasing as rapidly as [CN-] is decreasing. Both Fe+], and [CN-] are so small that [FeOH] and [HCN] are nearly constant. This has been verified by the experimental data which show no increase of Kr at pH 4.6 and some increase at pH 9.3 com- pared with the values found at pH 7.0.

1 See Agner and Theorell (5). a See Coryell, Stitt, and Pauling (11).

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1306 REACTION OF CATALASE AND CYANIDE

This reaction of catalase hydroxide and hydrocyanic acid differs from that found by Coryell, Stitt, and Pauling (11) in their studies of ferri- hemoglobin cyanide. First, the dissociation constants of the heme- linked hydroxyl groups differ (lo- *efi for ferrihemoglobin and 1O-1o.2 for catalase). Second, the effect of the hydroxyl group upon the spectra of these hemoproteins is quite different,; no shift has yet been detected in the catalase spectrum due solely to the formation of catalase hydroxide, while the shift from acid to alkaline ferrihemoglobin gives an obvious color change. Nevertheless, the data of Agner and Theorell clearly show that the hydroxyl group in catalase can be displaced by anions which do give a spectrophotometrically detectable effect, and which inhibit catalase activity. Therefore catalase has a heme-linked hydroxyl group (5).

Further proof that the hydroxyl group is attached to catalase hematin is given by the competition between formate and cyanide at pH 4.0. Thus cyanide and formate appear to attach to the same place on catalase hematin, which, in the case of cyanide, is recognized to be at the iron atom of catalase (10). Therefore the hydroxyl and formate ions also attach to the iron atom.

The excellent, agreement between the determinations of the dissociation constant of catalase cyanide from spectrophotometric and from activity data, typical of non-competitive inhibition, confirms the relatively crude tests of Zeile and Hellstriim (12). This result, is a distinct contrast to the results obtained in the cyanide inhibition of peroxidase activity (3).

The non-competitive inhibition of the activity of catalase by cyanide has been recognized for some time, and the only suitable explanation that could be offered previously in view of the then accepted “Michaelis con- stant” (No.025 M) for catalase activity was that peroxide and cyanide did not combine at, the same place on catalase hematin (13). This “Michaelis constant” for catalase activity has recently been shown to be an artifact due to catalase inactivation during the kinetic test (1).

An alternative explanation is that catalase hematins are not, all bound to hydrogen peroxide during the destruction of hydrogen peroxide. The free catalase hematins can then combine with cyanide in a “non-compet- itive” manner.3 This explanation is in accord with these experiments

8 The usual concepts of competitive and non-competitive inhibition have been evolved from considerations of the simple Michaelis theory. However, it has been suggested that the mechanism by which catalase decomposes hydrogen peroxide into oxygen and water involves consecutive reactions of hydrogen peroxide with catalase and with the catalase-hydrogen peroxide complex (14). In such a reaction, the steady state concentration of the catalase-hydrogen peroxide complex may not reach a value corresponding to all hematin groups bound to peroxide and may be quite independent of the hydrogen peroxide concentration. For thii reason, the inhibition catalase activity by cyanide will be independent of peroxide concentra-

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B. CHANCE 1307

and with the properties of the intermediate compound of catalase and hy- drogen peroxide which has recently been discovered (2). Both titration and spectrophotometric data (2) suggest that not all catalase hematinsare bound to peroxide in this complex. Inhibition of catalase activity may be

TABLE v Summary of Determinations of Dissociation Constant of Catalass Cyanide on Basis of

Activity Data

Temperature, v.

Initial hydro- gen peroxide aoncentra- tion, rnM

Dissociation con&ant, Y x 10

Reference

TM- metric

0

4

0

1.8-9.0

0

5

1.0 1.0 0.8

Wie- land (15)

Van Euler Iiele and and Hell- Joseph- etriim son (13) (12)

T

Titri- Mano- met&Z metric

--

0 18

10 4.5

6.3 4.6

[tern Keilin m> and

Har- tree (17)

0 25

5.0 1.0

3.4 4.7

Lemberg and Foulkee (18)

rhis Paper

Rapid Qx.ctro- photo- UI&iC

caused by the combination of cyanide with these free catalase hematins according to Equation 10.

HOFe-FeOOH NCFe-FeOOH

I I + 3HCN c-s

I I + 360 (10)

HOFe-FeOH NCFe-FeCN

These catalase hematins which become blocked by cyanide in this manner must have been active in the destruction of hydrogen peroxide; otherwise, no inhibition would be caused by the reaction of Equation 10.

The results of several determinations of the dissociation constant of

tion, “non-competitive;” in spite of the fact that both cyanide and peroxide can combine with cat&se hematin, they need not compete for the same cat&se hem- atin group in order to inhibit catalase activity. On the other hand, direct com- petition between peroxide and cyanide for the same hem&in group will occur at cyanide concentrations greater then those required to inhibit the activity, and thii reaction is studied spectrophotometrically in detail (see Chance (4)).

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1308 REACTION OF CATALASE AND CYANIDE

catalase cyanide based on activity studies are summarized in Table V. It is clear that the more recent determinations with pure catalase give consistently higher values than those obtained by the earlier workers who used cruder preparations (with the exception of Stern). This effect is not due to temperature alone, because Lemberg and Foulkes’ data taken at 0” agree with Keilin and Hartree’s and these data. Apparently the purer catalases are less sensitive to cyanide than are the cruder solutions.

SUMMARY

1. The velocity constant for the combination of catalase and cyanide is 9 X lo5 X sec.-l. The velocity constant for the dissociation of catalase cyanide is 3.2 sec.-’ at pH 7.0 and 2.9 sec.-’ at pH 4.6. The dissociation constant of catalase cyanide (&) calculated from kinetic data is 3.6 X lo+ M.

2. On the basis of spectrophotometric data, KI = 4 X lo4 M. 3. By an improved method for determining catalase activity, it is found

that Kr = 4.7 X lo* M, on the basis of the cyanide inhibition of catalase activity.

4. The non-competitive inhibition of catalase by cyanide is due to the fact that not all catalase hematins are bound to peroxide in the catalase- hydrogen peroxide complex.

5. Over the range 3.8 < pH < 8.7, the reaction of catalase with cyanide is represented by the equation

PeOH + HCN I ks k6

’ FeCN + Ha0

The four hematins of an erythrocyte catalase act independently. The apparent dissociation constant is calculated according to the relation

RI = [FeOHlIHCNl [F~CN]

BIBLIOGRAPHY

1. Bonnichsen, R. K., Chance, B., and Theorell, H., Acta Chem. Scan&, 1, 687 (1947).

2. Chance, B., Acta Chem. Scan&, 1, 236 (1947). 3. Chance, B., J. Cell. and Comp. Physiol., 22.33 (1943). 4. Chance, B., J. Biol. Chem., 179, 1311 (1949). 5. Agner, K., and Theorell, H., Arch. Biochem., 10, 321 (1946). 6. Bonnichsen, R. K., Dissertation, Karolinska Institutets, Stockholm (1948). 7. Chance, B., J. Biol. Chem., 179, 1331 (1949). 8, Chance, B., Rev. Scient. Znslrumenls, 18, 601 (1947). 9. Chance, B., J. Biol. Chem., 179, 1341 (1949).

10. Theorell, ET., and Agner, K., Ark. Kemi, Mineral. o. Geol., 16 A, No. 7 (1942).

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B. CHANCE 1309

11. Coryell, C. D., Stitt, F., and Pauling, L., J. Am. Chem. Sot., 69,633 (1937). 12. Zeile, K., and Hellstrom, H., 2. physiol. Chem., 192,171 (1930). 13. von Euler, H., and Josephson, B., Ann. Chem., 466,1 (1927). 14. Chance, B., Nature, 161, 914 (1948). 16. Wieland, H., Ann. Chem., 446, 193 (1925). 16. Stern, K. G., 2. physiol. Chem., 209, 176 (1932). 17. Keilin, D., and Hartree, E. F., Biochem. J., 39, 293 (1946). 18. Lemberg, R., and Foulkes, E. C., Nature, 161, 131 (1948).

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Britton ChanceCYANIDE

THE REACTION OF CATALASE AND

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