J. Biochem. 82, 1361-1367 (1977)
of Cytochrome Oxidase1 Shinya YOSHIKAWA, Tetsuji UENO, 1 and Tanhaku SAI Department of Biology, Faculty of Science, Konan University, Higashinada-ku, Kobe, Hyogo 658 Received for publication, May 7, 1977
The reaction of an oxygenated form of cytochrome oxidase [EC 1.9.3.1] with cyanide was examined under conditions where spontaneous decay was prevented. The equilibrium and kinetic constants for the reaction agreed well with those for the normally operating enzyme, indicating that the oxygenated form is one of the active intermediates of the cytochrome oxidase reaction.
Okunuki and co-workers discovered a new form of cytochrome oxidase with a Soret band at 428426 nm, and named it the oxygenated form, suggesting that it acts as an active intermediate of the cytochrome oxidase reaction (/, 2). In spite of many subsequent studies (3 and references cited therein), both its chemical structure and function remain to be elucidated. Recently Orii and King found that the so-called oxygenated form is not a single entity but involves at least three kinds of molecular species, Compounds I, II, and III, and they suggested that only Compound I is an active intermediate of the enzyme reaction (3, 4). On the other hand, Chance et al. reported that three types of intermediates, one of which readily dissociated O,, were formed in the reaction of membrane-bound cytochrome oxidase with Ot at low temperature (22, 23). The identity of these intermediates with Compounds I—III reported 1
This work was supported in part by a Research Grant from the Matsunaga Science Foundation. * Present address: School of Medicine, Kobe University, Ikuta-ku, Kobe, Hyogo 650. Vol. 82, No. 5, 1977
1361
by Orii and King has not been established. Extensive studies on the properties of these new species of cytochrome oxidase are clearly required to elucidate the mechanism of the cytochrome oxidase reaction. One of the difficulties in investigating the properties of Compound I is that it decays spontaneously to Compound II. In order to avoid this, we have taken advantage of our observation that the coexistence of ascorbic acid and O, effectively prevents spontaneous decay. Under these conditions, the reaction of cyanide with Compound I was examined and compared with that of the functioning enzyme, in order to investigate whether Compound I takes part in the enzyme reaction as an active intermediate. MATERIALS AND METHODS Cytochrome oxidase was isolated from beef heart muscle by the method of Okunuki et al. (5) with a slight modification (6). The final preparation was dissolved in 0.05 M sodium phosphate buffer, pH7.4, containing 0.25% (v/v) Tween 20 (see
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On the Reaction of Cyanide with an Oxygenated Form
S. YOSHIKAWA, T. UENO, and T. SAI
1362
RESULTS The oxygenated compound prepared as above showed a maximum at 605 nm in the a band region of the difference spectrum against oxidized cytochrome oxidase (Fig. 1-b). The absolute spectrum showed a Soret peak at 426 nm, as indicated by the spectrum in the absence of cyanide shown in Fig. 2. On the other hand, Orii and King reported that Compound I showed a maximum at 605 nm in the difference spectrum against the oxidized 1
Emasol 1130 could be used instead of Tween 20 without affecting the properties of the oxygenated compound, as far as was examined.
0.08
-
570
590
610
630
670
WAVELENGTH (nm)
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footnote 3) and stored at 0°C. Ascorbic acid and KCN were products of Nakarai Chemical Co., Kyoto. Ascorbic acid solution was adjusted to pH 7.4 with NaOH just before use, and KCN solution was adjusted with HC1. A Hitachi-356 spectrophotometer was used for spectrophotometric measurements. The rate of Ot consumption was measured with a Warburg apparatus or with a Yanagimoto PO-100 oxygen electrode. The concentration of cytochrome oxidase was determined using a millimolar extinction coefficient difference of 16.5 (^£605nm—630nm) for the fully reduced form. For preparation of the oxygenated cytochrome oxidase, 2.0 ml of 10-13 [IM cytochrome oxidase solution in 0.075 M sodium phosphate buffer, pH 7.4, containing 0.2 M ascorbic acid and 0.25 % (v/v) Tween 20 (see footnote 3) placed in a cuvette of 1 cm light path and left to stand at room temperature (23°Q for about 60min. After completion of reduction of the oxidase, which was monitored spectrally at 445 nm, the solution was aerated by shaking the cuvette gently 20 times in 10 s. The resulting spectrum hardly changed for about 20 min, until the system became anaerobic again. The absorbance change during this period was less than 0.005 at 445 nm, which corresponded to less than 2% of the absolute absorbance at this wavelength. The equilibrium constant of the reaction of the oxygenated compound with cyanide was estimated from the experimental data by nonlinear regression, as described in the previous paper (7). The numerical calculations were done on a NEAC 2200-700 digital computer at the Computation Center of Osaka University.
Fig. 1. Effect of cyanide on the difference spectrum of oxygenated cytochrome oxidase versus the fully oxidized form. The reaction mixture contained 10.3 ^M cytochrome oxidase and 0.2 M ascorbic acid in a cuvette of 1 cm light path, a, In the presence of 100 /JM cyanide; b, in the absence of cyanide. Other experimental conditions are described in the text. form and an absolute Soret spectrum at 425 to 426 nm (5, 4). The intensities of these peaks per heme a (estimated from Figs. 1 and 4 in Ref. 3) coincide closely with those of our compound. Thus, the identity of the absorption spectra of these compounds seems clear. Further, the functioning cytochrome oxidase in the aerobic steady state system containing cytochrome c and ascorbic acid as the electron donor system, shown in Fig. 6 of Ref. 16 and Figs. 2 and 3 of Ref. 7, also has a and Soret bands which are very similar to those of the oxygenated compound prepared as above. No significant change in the spectrum of the oxidase preparation was observed in the range of ascorbate concentration from 0.15 M to 0.4 M. This was also the case for O t concentration in the range from ca. 250 fit* (the concentration of O,-saturated aqueous solution) to nearly 0 /iM, since the spectrum scarcely changed with time until all the dissolved O t in the system was consumed by ascorbic acid. Therefore, it is evident that no
J. Biochem.
REACTION OF CYANIDE WITH OXYGENATED CYTOCHROME OXIDASE
2
A
6
8
10 100
(CYANIDE) uM
0.2
-
0.0 4 SO
470
WAVELENGTH ( nm)
Fig. 2. Effect of cyanide on the Soret band of the oxygenated cytochrome oxidase. The reaction mixture contained 12.0 /JM cytochrome oxidase, 0.2 M ascorbic acid and 0-100 /JM cyanide. Other conditions were similar to those in Fig. 1. a: Spectral change induced by cyanide. The final concentrations of cyanide in /JM were 0, 1, 2, 8, and 100 in increasing order, as indicated by the arrow in the figure, b: A set of absorbance changes with cyanide at 445 nm. The solid line is the theoretical curve based on a simple bimolecular reaction mechanism, E + 1=EI.
oxidase in this oxygenated oxidase preparation remains to be converted to Compound I by ascorbic acid and/or O,. The O, consumption rate of oxygenated oxidase solution containing 10.3 /JM cytochrome oxidase at 25°C, determined from polarographic traces at an O, concentration of ca. 150 /JM, was 22.7 /JMmin"1, which is 10% larger than that for oxidation of ascorbic acid contained in the reaction mixture. A similar result was obtained by manometric measurement under similar conditions. The increment of O, consumption rate due to the presence of the enzyme is small but not negligible. Therefore, the possibility cannot be ruled out that Compound I has cytochrome oxidase activity even in the absence of cytochrome c, though it could not be very high. This confirms the previous result of Ozawa et al. (8). The reaction between the oxygenated oxidase and cyanide was not instantaneous. For instance, it took 12 min to attain equilibrium between Vol. 82, No. 5, 1977
10.3 fiM oxygenated oxidase and 10.0 /JM cyanide. However, when cyanide was added to the enzyme solution prior to ascorbate, the absorption spectrum did not change with time after addition of O, to the anaerobic system by the method used for preparation of the oxygenated oxidase, until the system became anaerobic again due to the oxidation of ascorbate. The stability of the cyanide complex, once formed, is probably responsible for this phenomenon. The spectrum at equilibrium, obtained by the former method, agreed well with that obtained by the latter method within the experimental accuracy. The affinity of cyanide for the oxygenated oxidase was examined with cyanide complex prepared by the latter method for experimental convenience. As shown in Fig. 2-a, a set of clear isobestic points was observed at 433 nm and 463 nm indicating that there are only two spectrally distinguishable species in the system, namely, Compound I and its cyanide complex. No significant change in the final
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1361/771477 by Leiden University / LUMC user on 17 January 2019
0
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1364
where -4» F i, A.i0, and /4»jOo are the absorbance at w nm in the presence of cyanide at (/) t /*M, 0 /JM, and a sufficiently high concentration, respectively, K is the formation constant of the cyanide complex, e, is the total concentration of cyanide binding sites, and ( / ) t is the total concentration of cyanide added to the reaction mixture. From the experimental values of Am>i and (/) t , numerical values of A.t0, A.iOa, K, and o=A:1(E)o(I). The numerical value of (E)o can be estimated from the total heme a concentration in the system, multiplied by r. On the other hand, in the presence of high concentrations of cyanide, the initial velocity, v0,°o, must be independent of (I), namely, v0>oo=£0(E)0. Equation 2 clearly shows that A(I)/B corresponds to v0>0 and A to v0>Co. The numerical values of k0 and ki thus calculated from A, B, and (E)o, are 0.02 s"1 and
2.5X103M-1-S"1.
Vol. 82, No. 5, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1361/771477 by Leiden University / LUMC user on 17 January 2019
05 1/(CYANIDE)
The inhibitory effects of cyanide on the cytochrome oxidase reaction have been studied extensively under various conditions. However, the numerical values of the inhibition constant, Kt, are not consistent, ranging from 4.5X10~ 8 M to 8xl0~ 8 M (7, 9-13). The inconsistency seems to arise mainly from the rather slow rate of cyanide binding to the enzyme. Thus, it is reasonable to estimate the numerical value of Ki at about 10"' M. The effect of cyanide on the spectrum of the oxygenated cytochrome oxidase (Compound 1) thus coincides well with that on the enzyme activity in terms of the equilibrium constant. The numerical value of r for the oxygenated compound (0.417±0.017) is slightly larger than that obtained from the inhibitory effect of cyanide on the enzyme activity (0.58 ±0.049), reported in Table I of Ref. 7. This discrepancy is not significant, because the latter value was obtained by manometric measurement at 30°C, in which cyanide is more likely to escape from the system than in the present spectral assay at 23°C. The increasing cyanide inhibition of ferrocytochrome c oxidation catalyzed by cytochrome oxidase can be accounted for by the twostep mechanism cited above with k0 and kx values of 0.029 s"1 and 2.2 x 10s M^-S" 1 , respectively (9), in close agreement with the values for the cyanide binding reaction to the oxygenated compound. Thus the effect of cyanide on the spectrum of the oxygenated oxidase, at equilibrium as well as non equilibrium, is strictly proportional to that on the enzymic activity. Further, the spectral properties of the cytochrome oxidase in the aerobic steady state, including the spectral change induced by cyanide and the reactivity toward cyanide as estimated from it, coincide with those of this oxygenated oxidase, as described above. Therefore, it seems clear that Compound I participates in the enzyme reaction as an active intermediate. Van Buuren et al. concluded that cyanide was a noncompetitive inhibitor of the cytochrome oxidase reaction (13). Thus, any enzyme species in the steady state, the concentration of which depends on the substrate concentration, is very likely to have a K& value identical with K\ for cyanide (7). In other words, any form of cytochrome oxidase having a Kt value much different
1366
Although Compound I undoubtedly acts as an active intermediate, it does not exhibit high activity in the absence of cytochrome c, as indicated above. This may be interpreted as follows: ascorbic acid can substitute for ferrocytochrome c as an electron donor to the enzyme to form Compound I, while cytochrome c is indispensable for the next step, in which cytochrome c transfers an electron (or electrons) to Compound I for the enzymic reduction of Os to water. Another possibility for the latter step may be that cytochrome c reacts with Compound I, without any electron transfer, only as an activator for the enzyme activity which Compound I exhibits even in the absence of cytochrome c. Tn any case, this confirms the earlier suggestion of Okunuki et al. (20, 21) that cytochrome c functions not only as an electron donor but also as an essential activator interacting with oxygenated cytochrome oxidase. The binding of cyanide to Compound I was limited by a reaction step with a rate constant of 0.02 s"1. On the other hand, Van Buuren et al. have suggested that the binding of fully oxidized oxidase with cyanide was limited at 0.018 s"1,
though no such limitation was observed in fully reduced oxidase-cyanide complex formation (17, 18). Therefore, we suggest that the protein portion of Compound I near the cyanide binding chromophore (probably heme a) resembles that of fully oxidized cytochrome oxidase. The authors are grateful to Dr. T. Tsukihara for writing the computer programs for least-squares fitting. Thanks are also due to Prof. H. Matsubara and his collaborators for their support and interest during this work. REFERENCES
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from 10~ 7 M is unlikely to be a candidate for an active intermediate of the enzyme reaction. It has been established that Kt values for both reduced and oxidized cytochrome oxidase are much larger than K\ for cyanide {14-18). As regards other forms of cytochrome oxidase, Brittain and Greenwood recently examined the reaction of cyanide with an oxygenated oxidase prepared by photolyzing their ' mixed valence CO complex' in the presence of Ot. The effect of cyanide concentration on the rate of cyanide binding to their oxygenated oxidase, given in Fig. 1 in Ref. 19, gives Ki of about 10~2M. (This value, more than 104 times larger than that for Compound I, clearly indicates nonidentity between the two types of oxygenated oxidase. Compound I seems unable to form this type of cyanide complex, since no indication of its formation was observed even in the presence of 50 mM cyanide.) Therefore, among the various forms of cytochrome oxidase so far reported, Compound I is the only enzyme species which satisfies the above requirement for an active intermediate. Further studies are required to determine the nature of the enzyme species other than Compound I taking part in the cytochrome oxidase reaction.
S. YOSHIKAWA, T. UENO, and T. SAI
1. Okunuki, K. & Sekuzu, I. (1954) Seitai no Kagaku (in Japanese) 5, 265-272 2. Okunuki, K., Hagihara, B., Sekuzu, I., & Horio, T. (1958) in Proceedings of the International Symposium on Enzyme Chemistry (Ichihara, K., ed.) pp. 264-270, Academic Press, New York 3. Orii, Y. & King, T.E. (1976) /. Biol. Chem. 251, 7487-7493 4. Orii, Y. & King, T.E. (1972) FEBS Lett. 21, 199-202 5. Okunuki, K., Sekuzu, T., Yonetani, T., & Takemori, S. (1958) J. Biochem. 45, 847-854 6. Yoshikawa, S., Choc, M.G., O'Toole, M.C., & Caughey, W.S. (1977) J. Biol. Chem. 252, 5498-5508 7. Yoshikawa, S. & Orii, Y. (1974) J. Biochem. 76, 271-281 8. Ozawa, T., Takahashi, Y., Malviya, A.N., & Yagi, K. (1974) Biochem. Biophys. Res. Commun. 61, 601-606 9. Yoshikawa, S. & Orii, Y. (1972) /. Biochem. 71, 859-872 10. Stannard, J.N. & Horecker, B.L. (1948) /. Biol. Chem. 172, 599-608 11. Wainio, W.W. & Greenless, J. (1960) Arch. Biochem. Biophys. 90, 18-21 12. Yonetani, T. & Ray, G.S. (1965) J. Biol. Chem. 240, 3392-3398 13. Van Buuren, K.J.H., Zuurendonk, P.F., Van Gelder, B.F., & Muijsers, A.O. (1972) Biochim. Biophys. Ada 256, 243-257 14. Orii, Y. & Okunuki, K. (1964) J. Biochem. 55, 37^t8 15. Gibson, Q.H. & Greenwood, C. (1963) Biochem. J. 86, 541-554 16. Yoshikawa, S. & Orii, Y. (1973) J. Biochem. 73, 637-645 17. Antonini, E., Brunori, M., Greenwood, C , Malmstrom, B.G., & Rotilio, G.C. (1971) Eur. J. Biochem. 23,396-400 18. Van Buuren, K.J.H., Nichols, P., & Van Gelder, B.F. (1972) Biochim. Biophys. Ada 256, 258-276 /. Biochem.
REACTION OF CYANIDE WITH OXYGENATED CYTOCHROME OXTDASE
Vol. 82, No. 5, 1977
22. Chance B., Saronio, C , & Leigh, J.S., Jr. (1975) Proc. Nail. Acad. Sci. U.S. 72, 1635-1640 23. Chance, B., Saronio, C , & Leigh, J.S., Jr. (1975) /. Biol. Chem. 250, 9226-9237
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19. Brittain, T. & Greenwood, C. (1976) Biochem. J. 155, 453-455 20. Orii, Y., Sekuzu, I., & Okunuki, K. (1962) / . Biochem. 51, 204-215 21. Orii, Y. & Okunuki, K. (1963) /. Biochem. 53, 489-499
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of Cytochrome Oxidase1 Shinya YOSHIKAWA, Tetsuji UENO, 1 and Tanhaku SAI Department of Biology, Faculty of Science, Konan University, Higashinada-ku, Kobe, Hyogo 658 Received for publication, May 7, 1977
The reaction of an oxygenated form of cytochrome oxidase [EC 1.9.3.1] with cyanide was examined under conditions where spontaneous decay was prevented. The equilibrium and kinetic constants for the reaction agreed well with those for the normally operating enzyme, indicating that the oxygenated form is one of the active intermediates of the cytochrome oxidase reaction.
Okunuki and co-workers discovered a new form of cytochrome oxidase with a Soret band at 428426 nm, and named it the oxygenated form, suggesting that it acts as an active intermediate of the cytochrome oxidase reaction (/, 2). In spite of many subsequent studies (3 and references cited therein), both its chemical structure and function remain to be elucidated. Recently Orii and King found that the so-called oxygenated form is not a single entity but involves at least three kinds of molecular species, Compounds I, II, and III, and they suggested that only Compound I is an active intermediate of the enzyme reaction (3, 4). On the other hand, Chance et al. reported that three types of intermediates, one of which readily dissociated O,, were formed in the reaction of membrane-bound cytochrome oxidase with Ot at low temperature (22, 23). The identity of these intermediates with Compounds I—III reported 1
This work was supported in part by a Research Grant from the Matsunaga Science Foundation. * Present address: School of Medicine, Kobe University, Ikuta-ku, Kobe, Hyogo 650. Vol. 82, No. 5, 1977
1361
by Orii and King has not been established. Extensive studies on the properties of these new species of cytochrome oxidase are clearly required to elucidate the mechanism of the cytochrome oxidase reaction. One of the difficulties in investigating the properties of Compound I is that it decays spontaneously to Compound II. In order to avoid this, we have taken advantage of our observation that the coexistence of ascorbic acid and O, effectively prevents spontaneous decay. Under these conditions, the reaction of cyanide with Compound I was examined and compared with that of the functioning enzyme, in order to investigate whether Compound I takes part in the enzyme reaction as an active intermediate. MATERIALS AND METHODS Cytochrome oxidase was isolated from beef heart muscle by the method of Okunuki et al. (5) with a slight modification (6). The final preparation was dissolved in 0.05 M sodium phosphate buffer, pH7.4, containing 0.25% (v/v) Tween 20 (see
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1361/771477 by Leiden University / LUMC user on 17 January 2019
On the Reaction of Cyanide with an Oxygenated Form
S. YOSHIKAWA, T. UENO, and T. SAI
1362
RESULTS The oxygenated compound prepared as above showed a maximum at 605 nm in the a band region of the difference spectrum against oxidized cytochrome oxidase (Fig. 1-b). The absolute spectrum showed a Soret peak at 426 nm, as indicated by the spectrum in the absence of cyanide shown in Fig. 2. On the other hand, Orii and King reported that Compound I showed a maximum at 605 nm in the difference spectrum against the oxidized 1
Emasol 1130 could be used instead of Tween 20 without affecting the properties of the oxygenated compound, as far as was examined.
0.08
-
570
590
610
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WAVELENGTH (nm)
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footnote 3) and stored at 0°C. Ascorbic acid and KCN were products of Nakarai Chemical Co., Kyoto. Ascorbic acid solution was adjusted to pH 7.4 with NaOH just before use, and KCN solution was adjusted with HC1. A Hitachi-356 spectrophotometer was used for spectrophotometric measurements. The rate of Ot consumption was measured with a Warburg apparatus or with a Yanagimoto PO-100 oxygen electrode. The concentration of cytochrome oxidase was determined using a millimolar extinction coefficient difference of 16.5 (^£605nm—630nm) for the fully reduced form. For preparation of the oxygenated cytochrome oxidase, 2.0 ml of 10-13 [IM cytochrome oxidase solution in 0.075 M sodium phosphate buffer, pH 7.4, containing 0.2 M ascorbic acid and 0.25 % (v/v) Tween 20 (see footnote 3) placed in a cuvette of 1 cm light path and left to stand at room temperature (23°Q for about 60min. After completion of reduction of the oxidase, which was monitored spectrally at 445 nm, the solution was aerated by shaking the cuvette gently 20 times in 10 s. The resulting spectrum hardly changed for about 20 min, until the system became anaerobic again. The absorbance change during this period was less than 0.005 at 445 nm, which corresponded to less than 2% of the absolute absorbance at this wavelength. The equilibrium constant of the reaction of the oxygenated compound with cyanide was estimated from the experimental data by nonlinear regression, as described in the previous paper (7). The numerical calculations were done on a NEAC 2200-700 digital computer at the Computation Center of Osaka University.
Fig. 1. Effect of cyanide on the difference spectrum of oxygenated cytochrome oxidase versus the fully oxidized form. The reaction mixture contained 10.3 ^M cytochrome oxidase and 0.2 M ascorbic acid in a cuvette of 1 cm light path, a, In the presence of 100 /JM cyanide; b, in the absence of cyanide. Other experimental conditions are described in the text. form and an absolute Soret spectrum at 425 to 426 nm (5, 4). The intensities of these peaks per heme a (estimated from Figs. 1 and 4 in Ref. 3) coincide closely with those of our compound. Thus, the identity of the absorption spectra of these compounds seems clear. Further, the functioning cytochrome oxidase in the aerobic steady state system containing cytochrome c and ascorbic acid as the electron donor system, shown in Fig. 6 of Ref. 16 and Figs. 2 and 3 of Ref. 7, also has a and Soret bands which are very similar to those of the oxygenated compound prepared as above. No significant change in the spectrum of the oxidase preparation was observed in the range of ascorbate concentration from 0.15 M to 0.4 M. This was also the case for O t concentration in the range from ca. 250 fit* (the concentration of O,-saturated aqueous solution) to nearly 0 /iM, since the spectrum scarcely changed with time until all the dissolved O t in the system was consumed by ascorbic acid. Therefore, it is evident that no
J. Biochem.
REACTION OF CYANIDE WITH OXYGENATED CYTOCHROME OXIDASE
2
A
6
8
10 100
(CYANIDE) uM
0.2
-
0.0 4 SO
470
WAVELENGTH ( nm)
Fig. 2. Effect of cyanide on the Soret band of the oxygenated cytochrome oxidase. The reaction mixture contained 12.0 /JM cytochrome oxidase, 0.2 M ascorbic acid and 0-100 /JM cyanide. Other conditions were similar to those in Fig. 1. a: Spectral change induced by cyanide. The final concentrations of cyanide in /JM were 0, 1, 2, 8, and 100 in increasing order, as indicated by the arrow in the figure, b: A set of absorbance changes with cyanide at 445 nm. The solid line is the theoretical curve based on a simple bimolecular reaction mechanism, E + 1=EI.
oxidase in this oxygenated oxidase preparation remains to be converted to Compound I by ascorbic acid and/or O,. The O, consumption rate of oxygenated oxidase solution containing 10.3 /JM cytochrome oxidase at 25°C, determined from polarographic traces at an O, concentration of ca. 150 /JM, was 22.7 /JMmin"1, which is 10% larger than that for oxidation of ascorbic acid contained in the reaction mixture. A similar result was obtained by manometric measurement under similar conditions. The increment of O, consumption rate due to the presence of the enzyme is small but not negligible. Therefore, the possibility cannot be ruled out that Compound I has cytochrome oxidase activity even in the absence of cytochrome c, though it could not be very high. This confirms the previous result of Ozawa et al. (8). The reaction between the oxygenated oxidase and cyanide was not instantaneous. For instance, it took 12 min to attain equilibrium between Vol. 82, No. 5, 1977
10.3 fiM oxygenated oxidase and 10.0 /JM cyanide. However, when cyanide was added to the enzyme solution prior to ascorbate, the absorption spectrum did not change with time after addition of O, to the anaerobic system by the method used for preparation of the oxygenated oxidase, until the system became anaerobic again due to the oxidation of ascorbate. The stability of the cyanide complex, once formed, is probably responsible for this phenomenon. The spectrum at equilibrium, obtained by the former method, agreed well with that obtained by the latter method within the experimental accuracy. The affinity of cyanide for the oxygenated oxidase was examined with cyanide complex prepared by the latter method for experimental convenience. As shown in Fig. 2-a, a set of clear isobestic points was observed at 433 nm and 463 nm indicating that there are only two spectrally distinguishable species in the system, namely, Compound I and its cyanide complex. No significant change in the final
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1361/771477 by Leiden University / LUMC user on 17 January 2019
0
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where -4» F i, A.i0, and /4»jOo are the absorbance at w nm in the presence of cyanide at (/) t /*M, 0 /JM, and a sufficiently high concentration, respectively, K is the formation constant of the cyanide complex, e, is the total concentration of cyanide binding sites, and ( / ) t is the total concentration of cyanide added to the reaction mixture. From the experimental values of Am>i and (/) t , numerical values of A.t0, A.iOa, K, and o=A:1(E)o(I). The numerical value of (E)o can be estimated from the total heme a concentration in the system, multiplied by r. On the other hand, in the presence of high concentrations of cyanide, the initial velocity, v0,°o, must be independent of (I), namely, v0>oo=£0(E)0. Equation 2 clearly shows that A(I)/B corresponds to v0>0 and A to v0>Co. The numerical values of k0 and ki thus calculated from A, B, and (E)o, are 0.02 s"1 and
2.5X103M-1-S"1.
Vol. 82, No. 5, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1361/771477 by Leiden University / LUMC user on 17 January 2019
05 1/(CYANIDE)
The inhibitory effects of cyanide on the cytochrome oxidase reaction have been studied extensively under various conditions. However, the numerical values of the inhibition constant, Kt, are not consistent, ranging from 4.5X10~ 8 M to 8xl0~ 8 M (7, 9-13). The inconsistency seems to arise mainly from the rather slow rate of cyanide binding to the enzyme. Thus, it is reasonable to estimate the numerical value of Ki at about 10"' M. The effect of cyanide on the spectrum of the oxygenated cytochrome oxidase (Compound 1) thus coincides well with that on the enzyme activity in terms of the equilibrium constant. The numerical value of r for the oxygenated compound (0.417±0.017) is slightly larger than that obtained from the inhibitory effect of cyanide on the enzyme activity (0.58 ±0.049), reported in Table I of Ref. 7. This discrepancy is not significant, because the latter value was obtained by manometric measurement at 30°C, in which cyanide is more likely to escape from the system than in the present spectral assay at 23°C. The increasing cyanide inhibition of ferrocytochrome c oxidation catalyzed by cytochrome oxidase can be accounted for by the twostep mechanism cited above with k0 and kx values of 0.029 s"1 and 2.2 x 10s M^-S" 1 , respectively (9), in close agreement with the values for the cyanide binding reaction to the oxygenated compound. Thus the effect of cyanide on the spectrum of the oxygenated oxidase, at equilibrium as well as non equilibrium, is strictly proportional to that on the enzymic activity. Further, the spectral properties of the cytochrome oxidase in the aerobic steady state, including the spectral change induced by cyanide and the reactivity toward cyanide as estimated from it, coincide with those of this oxygenated oxidase, as described above. Therefore, it seems clear that Compound I participates in the enzyme reaction as an active intermediate. Van Buuren et al. concluded that cyanide was a noncompetitive inhibitor of the cytochrome oxidase reaction (13). Thus, any enzyme species in the steady state, the concentration of which depends on the substrate concentration, is very likely to have a K& value identical with K\ for cyanide (7). In other words, any form of cytochrome oxidase having a Kt value much different
1366
Although Compound I undoubtedly acts as an active intermediate, it does not exhibit high activity in the absence of cytochrome c, as indicated above. This may be interpreted as follows: ascorbic acid can substitute for ferrocytochrome c as an electron donor to the enzyme to form Compound I, while cytochrome c is indispensable for the next step, in which cytochrome c transfers an electron (or electrons) to Compound I for the enzymic reduction of Os to water. Another possibility for the latter step may be that cytochrome c reacts with Compound I, without any electron transfer, only as an activator for the enzyme activity which Compound I exhibits even in the absence of cytochrome c. Tn any case, this confirms the earlier suggestion of Okunuki et al. (20, 21) that cytochrome c functions not only as an electron donor but also as an essential activator interacting with oxygenated cytochrome oxidase. The binding of cyanide to Compound I was limited by a reaction step with a rate constant of 0.02 s"1. On the other hand, Van Buuren et al. have suggested that the binding of fully oxidized oxidase with cyanide was limited at 0.018 s"1,
though no such limitation was observed in fully reduced oxidase-cyanide complex formation (17, 18). Therefore, we suggest that the protein portion of Compound I near the cyanide binding chromophore (probably heme a) resembles that of fully oxidized cytochrome oxidase. The authors are grateful to Dr. T. Tsukihara for writing the computer programs for least-squares fitting. Thanks are also due to Prof. H. Matsubara and his collaborators for their support and interest during this work. REFERENCES
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from 10~ 7 M is unlikely to be a candidate for an active intermediate of the enzyme reaction. It has been established that Kt values for both reduced and oxidized cytochrome oxidase are much larger than K\ for cyanide {14-18). As regards other forms of cytochrome oxidase, Brittain and Greenwood recently examined the reaction of cyanide with an oxygenated oxidase prepared by photolyzing their ' mixed valence CO complex' in the presence of Ot. The effect of cyanide concentration on the rate of cyanide binding to their oxygenated oxidase, given in Fig. 1 in Ref. 19, gives Ki of about 10~2M. (This value, more than 104 times larger than that for Compound I, clearly indicates nonidentity between the two types of oxygenated oxidase. Compound I seems unable to form this type of cyanide complex, since no indication of its formation was observed even in the presence of 50 mM cyanide.) Therefore, among the various forms of cytochrome oxidase so far reported, Compound I is the only enzyme species which satisfies the above requirement for an active intermediate. Further studies are required to determine the nature of the enzyme species other than Compound I taking part in the cytochrome oxidase reaction.
S. YOSHIKAWA, T. UENO, and T. SAI
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REACTION OF CYANIDE WITH OXYGENATED CYTOCHROME OXTDASE
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