Cl by the reduced and oxidized form, respectively. The Wr~ax for the reduction of cl 3+ is a b o u t 175 nmol/min per milligram of submitochondrial particles (the K e i l i n - H a r t r e e preparation) in 0.1 M phosphate buffer, p H 7.4, at 23 ° with succinate as the electron donor, whereas in the same CI conditions V~ax for the oxidation of the c y t o c h r o m e is about 150 nmol/ rain per milligram with oxygen as the electron acceptor. C y t o c h r o m e Cl reacts with c y t o c h r o m e c and other artificial reductants and oxidants. S o m e of the rate constants are s u m m a r i z e d in Table III. C y t o c h r o m e c, has been crystallized. The crystals are, h o w e v e r , too small for practical use as so far prepared.

Other Methods of Preparation C y t o c h r o m e cl was independently discovered by Yakushiji and Okunuki 25 and Keilin and H a r t r e e , 26 who originally named it c y t o c h r o m e e. Several m e t h o d s for isolation a p p e a r e d 25"27-3° prior to the procedure described above. The /3-mercaptoethanol method for the separation of c y t o c h r o m e ci f r o m b c y t o c h r o m e s and presumably other c o m p o n e n t s has been applied to yeast 31'~2 25E. Yakushiji and K. Okunuki, Proc. Imp. Acad. (Tokyo) 16, 299 (1940). 26D. Keilin and E. F. Hartree, Nature (London) 164, 254 (1949). 27R. Bomstein, R. Goldberger, and H. Tisdale, Biochim. Biophys. Acta 50, 527 (1961). 2~1. Sekuzu, Y. Orii, and K. Ohnishi, Tampakushitsu, Kakusan, Koso 10, 1610 (1965). 29S. Yamashita and E. Racker, J. Biol. Chem. 244, 1220 (1969). .~0D. E. Green, J. J/irnefelt, and H. D. Tisdale, Biochim. Biophys. Acta 31, 34 (1959). al See E. Ross and G. Schatz, this volume [24]. a2 M. L. Claisse and P. F. Pajot, Eur. J. Biochem. 49, 49 (1974).

[21] Ligands

of Cytochromo

c Oxidase


T h e r e are four oxidation-reduction c o m p o n e n t s in c y t o c h r o m e c oxidase (see p. 193) and most of the ligands discussed in this chapter bind to one or m o r e of these c o m p o n e n t s . Current knowledge of the reactivity of the four c o m p o n e n t s toward added ligands indicates that the " v i s i b l e " c o p p e r is unreactive, whereas c y t o c h r o m e a3 is readily accessible to and binds m a n y ligands. The reactivities of c y t o c h r o m e a and the " i n v i s i b l e " c o p p e r are m u c h less well known.



[21 ]

Specific C o m m e n t s

Interaction of Cyanide with Cytochrome c Oxidase The inhibition of mitochondrial respiration following cyanide addition is slow (minutes), 1,2 and the time required to reach the final steady state depends on the redox state of cytochrome a3; it is shorter when cytochrome a3 is more reduced (this is true also for the isolated oxidase). Although cyanide reacts with both, its affinity for the oxidized form is high but the reaction rate is slow, whereas the opposite is true for the reaction with the reduced enzyme. 2 Since the reduced cytochrome a3cyanide complex is rapidly oxidized by molecular oxygen, 3 the inhibition thus appears to occur through formation of the reduced cytochrome a3cyanide complex, which is then oxidized to the more stable oxidized cytochrome a3-cyanide complex. Cyanide reacts with the oxidized form of cytochrome c oxidase to form a spectrally distinct species. 4-6 The difference spectrum is characteristic of a ferric heme changing from high spin to low spin with troughs at 650 nm and 410 nm and maxima at 584 nm, 548 nm, and 434 nm. 4"5 The reaction is approximately first order for the isolated hemoprotein 4 and strictly first order for intact mitochondria. ~ The inhibition by cyanide is not completely reversed by dialysis unless the preparation is reduced, r presumably owing to a faster rate of cyanide dissociation from the reduced form of the hemoprotein than from the oxidized form of the hemoprotein. A general observation for the cyanide inhibition is that the cyanide inhibitor constant is greater than the dissociation constant for cyanide from the oxidized cytochrome c oxidase, but smaller than the cyanide dissociation constant from the reduced oxidase. In mitochondria, but not in the isolated hemoprotein, the cyanide reaction is strongly pH-dependent. The Km for cyanide in intact mitochondria ranges from approximately 4 ~ at pH 6.0 to approximately 200 /~4 at pH 8.0. The Vmax at pH 7.0 is 4 × 10-2 sec -1. The pH dependence appears to be associated with an ionizing group with a pK ' B. Chance, Nature (London) 169, 215 (1952). 2 T. Yonetani and G. S. Ray, J. Biol. Chem. 240, 3392 (1%5). 3 D. Keilin and E. F. Hartree, Proc. R. Soc. London Ser. B 127, 167 (1939). 4 K. J. H. van Buuren, P. Nicholls, and B. F. van Gelder, Biochim. Biophys. Acta 256, 258 (1972). 5 D. F. Wilson, M. Erecifiska, and E. S. Brocklehurst, Arch. Biochem. Biop/lys. 151, 180 (1972). 6 y . Orii and K. Okunuki, J. Biochem. (Tokyo) 55, 37 (1%4). r p. W, Camerino and T. E. King, J. Biol. Chem. 241,970 (1%6).




of 6.9 5 and was proposed to be the same group as that responsible for the pH dependence of the Em of cytochrome a3. 8 The cyanide binding may be schematically expressed as HCN

HCN K>20

a33+H - a , ~ + ~ a ~ + H ( H C N )

- a 3+


) a~+H - a S+

k = 4 × 10 - 2 s e e - 1

p K = 6.9

a~+ - a3+ + H÷ where cyanide forms a spectroscopically undetected complex with a protonated form of cytochrome c oxidase (pK 6.9) with a dissociation constant of approximately 4 ~ ' / . This complex then undergoes a strongly exergonic reaction, which gives rise to the described spectral change? The latter reaction is essentially irreversible under the experimental conditions (K > 20) and occurs with the first-order rate constant of 4 x 10.2 sec -~. The isolated hemoprotein does not show the pK that is observed in intact mitochondria; the Ka is 10 mM and k is 1.8 x 10.2 sec-1. 4 The activation energy for the cyanide reaction with the oxidized form of cytochrome c oxidase is approximately 15.6 kcal/mol in pigeon heart mitochondria, ~ and 14 kcal/mol in cytochrome oxidase isolated from beef heart. 4 In pigeon heart mitochondria, azide is kinetically competitive with respect to cyanide for the formation of the spectroscopically undetected complex, although probably not because of competition for a common site? These two inhibitors have been classified as kinetically competitive for the inhibitory site in isolated cytochrome oxidase based on kinetic "measurements of the inhibition of oxidation of cytochrome c. 9 The competition between cyanide and azide may be an expression of interaction between two different sites on the cytochrome oxidase. The rate of combination, the rate of dissociation, and the equilibrium constant for the reaction of cyanide with the reduced cytochrome oxidase are listed in the table. Cyanide and carbon monoxide are considered to combine with the same group on the reduced enzyme. Electron spin resonance measurements show that the addition of cyanide leads to the formation of a ferric cytochrome aa cyanide compound with a g value of 3.58. l° This signal is observed only with the partially reduced form of the hemoprotein. s D. F. W i l s o n , J. G. L i n d s a y , and E. S. B r o c k l e h u r s t , Biochim. Biophys. Acta 256, 277 (1972). S. Y o s h i k a w a a n d Y. Orii, J. Biochem. (Tokyo) 7 1 , 8 5 9 (1972). 10 D. V. D e r V a r t a n i a n , I. Y. L e e , E. C. S l a t e r , and B. F. van G e l d e r , Biochim. Biophys. Acta 347, 321 (1974).




8 6i .










'~ g



~ _ ~ = ~ ~


'~ E






? ---


I '~


I x~,



× I

I xx

I I~.xx

I Ix

I x Ix

I xx

I xx

I xx~


< e~


I x






0 e~ -r


0 >.




I x x x x x x x x x x

I xxxx



0 < u~


~ . . ~









. ~=




Z •










I n t e r a c t i o n o f A z i d e with C y t o c h r o m e c O x i d a s e

The inhibition of respiration of particulate preparations from heart muscle by azide occurs with oxidation of c y t o c h r o m e a3 and reduction of c y t o c h r o m e a. 3 The a and T absorption m a x i m a of c y t o c h r o m e a reduced in the azide-inhibited steady state in mitochondria are shifted from their positions in the a b s e n c e of azideH-14; at the t e m p e r a t u r e of liquid nitrogen, this shift in the o~ region is from 602 nm to 596 nm, while the split Soret m a x i m a at 448 nm and 441 nm are shifted to 447 nm and 438 nm. The inhibition of respiration by azide is kinetically uncompetitive with substrate in yeast cells 15 and in mitochondria, 12"1~but n o n c o m p e t i t i v e in isolated c y t o c h r o m e c oxidase. 2 In mitochondrial suspensions the apparent inhibitor constant is d e p e n d e n t on the rate at which the substrate is oxidized and w h e t h e r the m i t o c h o n d r i a are coupled or uncoupled (see the table). The p H d e p e n d e n c e o f the inhibitor constant in both the isolated oxidase and in mitochondria arises f r o m the " o n " reaction (the rate of binding) as the reactive species is HN3 (pK = 4.6). An azide-induced spectral change is reported to o c c u r in the Soret band of the oxidized form of isolated c y t o c h r o m e oxidase (see the table). The rate of dissociation ( " o f f " constant) for azide is 0.1 sec -1 in wellcoupled rat liver mitochondria at p H 7.2, but the addition of uncouplers increases this rate to 0.5 sec -1.17 The " o f f " constant m e a s u r e d from the spectral change in the oxidized f o r m of isolated c y t o c h r o m e c oxidase is found to be 0.4 sec -1.18,19 Activation energies o f 1.0 kcal/mol and 14 kcal/ mol are reported for the " o n " and " o f f " reactions for azide binding as measured by the spectral change in isolated c y t o c h r o m e c oxidase. ~8"19 Although the relationship of the spectrally detectable azide c o m p l e x with fully oxidized c y t o c h r o m e c oxidase to the inhibition by azide is not completely established, the reaction rates and specificities are very similar. P o t e n t i o m e t r i c titrations of the o x i d a t i o n - r e d u c t i o n potential dependH D. F. Wilson, Biochim. Biophys. Acta 131,431 (1%7). 12D. F. Wilson and B. Chance, Biochem. Biophys. Res. Commun. 23, 751 (!%6). 13D. F. Wilson and M. V. Gilmour, Biochirn. Biophys. Acta 143, 52 (1%7). 14p. Nicholls and H. K. Kimelberg, Biochim. Biophys. Acta 162, 11 (1968). 15R. J. Winzler, J. Cell. Comp. Physiol. 21, 229 (1943). 16D. F. Wilson and B. Chance, Biochirn. Biophys. Acta 131,421 (1967). 17D. F. Wilson, in "Probes of Enzymes and Hemoproteins" (B. Chance, T. Yonetani, and A. S. Mildvan, eds.), Vol. 11, p. 593. Academic Press, New York, 1971. 18R. Wever, A. O. Muijsers, and B. F. van Gelder, Biochim. Biophys. Acta 325, 8 (1973). 19R. Wever, A. O. Muijsers, B. F. van Gelder, E. P. Bakker, and K. J. H. van Buuren, Biochim. Biophys. Acta 325, 1 (1973).




ence of the reduction of cytochromes a and a3 carried out using suspensions of pigeon heart mitochondria in the presence of azide revealed changes in the EmT.0 values of both cytochromes which were dependent on azide concentration. 8 For each 10-fold increase in azide concentration greater than approximately 1 mM, the Em value of cytochrome a became 0.060 V more negative, indicating a direct binding of azide to the oxidized form of this cytochrome. The measured Em of cytochrome a3 decreased from 0.385 V in the absence of azide to 0.350 V at Saturating azide concentrations. The dissociation constant for azide binding to the oxidized form of cytochrome a is approximately 250/xM at pH 7.2 based on total azide. Electron paramagnetic resonance (EPR) measurements of the isolated oxidase in the presence of azide show an EPR signal at g 2.9, which is seen only if the enzyme is partially reduced (i.e., either cytochrome a of a~ reduced and the other one oxidized). In the aerobic, inhibited steady state (cytochrome a3 oxidized and cytochrome a reduced), such a signal is attributed to a low-spin azide compound of ferric cytochrome a3. 2°'2' In potentiometric titrations of pigeon heart mitochondria and submitochondrial particles in the presence of azide, the appearance of the g 2.9 absorbance is parallel to the reduction of cytochrome a3 (E,, = 0.35 V) and the signal disappears parallel to the reduction of cytochrome a (E,, = 0.16 V in the presence of 1 mM azide, sal,2z The Reaction o f Carbon Monoxide with Cytochrome c Oxidase One molecule of carbon monoxide binds to the reduced form of cytochrome c oxidase per two heme a's 23'24 The dissociation constant was reported to be approximately 0.4 p~M in intact rat liver mitochondria 2~ and to be increased slightly (0.47 /xM to 0.91 /zM 2~) when ATP was added. The dissociation constant for isolated cytochrome c oxidase 2Gis very similar to that in intact mitochondria. As a respiratory inhibitor, CO is competitive with respect to oxygen over all concentrations that have been measured, and it forms a reduced cytochrome a3-CO compound with characteristic absorption maxima at 589 nm and 430 nm. 2o B. F. van Gelder and H. Beinert, Biochim. Biophys. Acta 189, 1 (1969). 2, D. F. Wilson and J. S. Leigh, Jr., Arch. Biochem. Biophys. 150, 154 (1972). z2 D. F. Wilson, J. S. Leigh Jr., J. G. Lindsay, and P. L. Dutton, in "'Oxidases and Related Redox S y s t e m s II'" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 715. Univ. Park Press, Baltimore, Maryland, 1973. 23 G. E. M a n s l e y , J. T. Stanbury, and R. Lemberg, Biochim. Biophys. Acta 113, 33 (1965). 24 D. C. W h a r t o n and Q. H. Gibson, J. Biol. Chem. 251, 2861 (1976). 2~ H. Wohlrab and G. B. O g u n m o l a , Biochemistry 10, 1103 (1971). 2~ G. Wald and D. W. Allen, J. Gen. Physiol. 40, 593 (1957).



[2 1]

The photodissociation of the cytochrome a3-CO compound occurs very rapidly (within 10-iz sec of absorption of the photon) and with a quantum efficiency near 1.0. The reassociation of the CO is slow [k,,o,,, = 7 × 104 M -1 s e c -1 at 25 ° with activation energy of 6.4 kcal/mol in isolated oxidase 27'2s and 1.2 × 105 M -1 sec -1 at 25 ° with an activation energy of 3.1 kcal/mol in pigeon heart mitochondria 29] relative to the oxygen reaction (approximately 1 × 10s M -1 sec-l). 3°-32 The CO dissociates slowly in the absence of light (k = approximately 2.5 × 10-2 sec-~), so that an anaerobic sample treated with CO can be mixed with 02 under conditions for which no significant reaction with oxygen occurs until a short but very bright light flash is used to photodissociate the CO. The oxygen reaction then occurs as if no CO were present in the mixture, and measurements can be made as soon as the optical interference of the photodissociation flash is over. The half-reduction potential of cytochrome a3 becomes more positive upon addition of CO as expected for a ligand having a higher affinity for the reduced form of the enzyme than for the oxidized form of the enzyme. The Em7.2 value of cytochrome a changes from 210 mV to 260 mV when CO is added, s'33 and this change in Era is proportional to the formation of the reduced cytochrome-CO complex, i.e., 50% change at 50% formation of the CO compound. The change in cytochrome a half-reduction potential is complete at CO concentrations of approximately 10 /zM, which corresponds to the saturation of the reaction between reduced cytochrome as and CO (Kd = 0.4 ttM). Potentiometric determinations of the reduction of cytochrome a3 in the presence of CO measured by the appearance of the reduced cytochrome a3-CO compound give titration curves with n values of 2 (a two-electron process); and in the absence of ATP, the Em of the cytochrome as-CO compound becomes 30 mV more positive with each 10-fold increase in CO concentration. 34 These results suggest that carbon monoxide binds only when two electron carriers are reduced (cytochrome as and a spectroscopically undetected component,

27 Q. H. Gibson, C. Greenwood, D. C. Wharton, and G. Palmer, J. Biol. Chem. 240, 888

(1%5). 28 Q. H. Gibson and C. Greenwood, Biochem. J. 86, 541 (1963). 29 M. Erecifiska and B. Chance, Arch. Biochem. Biophys. 151,304 (1972). 30 Q. H. Gibson and C. Greenwood, J. Biol. Chem. 239, 586 (1964). 31 Q. H. Gibson, C. Greenwood, D. C. Wharton, and G. Palmer, in "Oxidases and Related Redox Systems" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 591. Wiley, New York, 1965. 32 O. Warburg and F. Kubowitz, Biochern. Z. 203, 95 (1928). 33 T. Tsudzuki and D. F. Wilson, Are/1. Biochem. Biophys. 145, 149 (1971). 34 j. G. Lindsay and D. F. Wilson, f E B S Lett. 48, 45 (1974).

[21 ]



the "invisible" copper) to form reduced cytochrome a3-reduced copper complex. It was calculated from the experimental results that this copper species has an Emr.0 of 0.35 V in mitochondria in the absence of CO. Direct titrations of the cytochrome a3-CO compound with reducing equivalents (NADH) or oxidizing equivalents (O2) give two equivalents per cytochrome a3, in agreement with the stoichiometry observed in potentiometric titrations 3~ (see, however, Anderson et al.36). Although CO reacts with reduced cytochrome aa, in preparations in which cytochrome a3 is reduced and cytochrome a oxidized, addition of CO causes a change in the heme of cytochrome a from a high-spin state to a low-spin state. 21 Photodissociation of CO from such samples at near 6 °K results in a small change in the EPR spectrum of the low-spin ferric heme, which occurs in less than 10 msec. Although CO does not rebind in any measurable time period at this temperature, 37''~sthe appearance of the high-spin heine signal normally observed in the half-reduced oxidase does not occur at this temperature either in mitochondria or submitochondrial particles and is incomplete in isolated cytochrome oxidase. 39'4°

Hydroxylamine and NO Reactions with Cytochrome c Oxidase The inhibition of mitochondrial respiration by hydroxylamine is of the uncompetitive type with respect to N,N,N',N'-tetramethyl-p-phenylenediamine oxidation (ascorbate as reductant) by rat liver mitochondria and partially released by uncouplers or oxidative phosphorylation. 41-44 The metabolism of hydroxylamine generates products which are responsible for the inhibition rather than hydroxylamine itself. 41"4~The reactive product was found to be NO by identifying the NO compound of reduced a5 D. F. Wilson and Y. Miyata, Arch. Biochem. Biophys. (1976). :~ J. L. Anderson, T. Kuwana, and C. R. Hartzell, Biochemistry 17, 3847 (1976). :~7T. Yonetani, in "Oxidases and Related Redox Systems" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 614. Wiley, New York, 1%5. 38 B. Chance, B. Schoener, and T. Yonetani, in "Oxidases and Related Redox Systems" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 609. Wiley, New York, 1965. :~9j. S. Leigh, Jr., D. F. Wilson, C. S. Owen, and T. E. King, Arch. Biochem. Biophys. 160, 476 (1974). 40 j. S. Leigh, Jr. and D. F. Wilson, Biochem. Biophys. Res. Commun. 48, 1266 (1973). 4~ D. F. Wilson and E. Brooks, Biochemistry 9, 1090 (1970). 42 M. K. F. Wikstr6m and N. E. L. Saris, E,r. J. Biochem. 9, 160 (1969). 4:~ M. K. F. Wikstr6m and N. E. L. Saris, "'Electron Transport and Energy Conservation" (J. M. Tager, S. Papa, E. Quagliariello, and E. C. Slater, eds.), p. 77. Adriatica Editrice, Bari, 1970. 44 K. Utsumi and T. Oda, Arch. Biochem. Biophys. 131, 67 (1969). 4.~ M. F. J. Blokzijl-Homan and B. F. van Gelder, Biochim. Biophys. Acta 234, 493 (1971).




cytochrome a345 in hydroxylamine-inhibited cytochrome c oxidase. The hyperfine splittings of the EPR signal permit the conclusion that the fifth ligand of the iron of reduced cytochrome a3 (NO being the sixth) is a nitrogen atom, almost certainly that of a histidine (for a discussion of the method, see Kon 46 and Yonetani and Yamamoto47). Reduced cytochrome oxidase exhibits high affinity for NO and reacts with it rapidly 2s (see the table). Although NO is a powerful inhibitor of cytochrome oxidase, it also reacts rapidly with molecular oxygen and/or is readily metabolized. Thus, the inhibition is transient unless a NO-generating system (such as hydroxylamine, substrate, and oxygen) is present. For simple binding studies, NO can be readily generated by sodium nitrite and a reducing agent such as dithionite.

Inhibition of Cytochrome c Oxidase by Fluoride Fluoride has not been extensively used as an inhibitor of cytochrome c oxidase. Because fluoride did not appear to inhibit the rate of reduction of cytochrome a3 at the onset of anaerobiosis, 3 it was proposed that fluoride acts by inhibiting the reduction of cytochrome a and a3 (by cytochrome c) rather than by binding to the oxidized cytochrome a3. No effect of F- is observed in potentiometric titrations of cytochrome a and a3 in submitochondrial particles as measured by EPR, and no fluoride hyperfine structure is observed on the high-spin heme signal. 2°'2' Fluoride exhibits a complex inhibitory pattern with two inhibitor constants of 7 mM and 21 /.tM when added to isolated cytochrome oxidase oxidizing cytochrome c 48 and has a Ki value of 10 mM, which increases to 35 mM when the data were extrapolated to saturation with cytochrome a.49 A shift is reported to occur in the Soret band maximum of oxidized cytochrome c oxidase from 423 nm to 421 nm and the c~ band from 597 nm to 596 nm upon addition of F-. The spectral changes are very small, but measurements give a k,,,,,,, of 4 M -1 sec-' and a k,,off,, of 2.9 × 10-2 sec-' at 25 °, pH 7.4. 49 The measured dissociation constant is approximately 10 raM. The steady-state reduction of the components during Finhibition have not been measured for the more recent experiments, and 46 H. Kon, Biochem. Biophys. Res. Commun. 35, 423 (1969). 47 T. Yonetani and H. Yamamoto, in "'Oxidases and Related Redox Systems II" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 1, p. 279. Wiley, New York, 1973. 48 y . Orii and S. Yoshikawa, in "Oxidases and Related Redox Systems II" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 649. Univ. Park Press, Baltimore, Maryland, 1973. 49 A. O. Muijsers, K. J. H. van Buuren, and B. F. van Gelder, Biochim. Biophys. Acta 333, 430 (1974).




there remains questions as to whether F- inhibits the oxidation or the reduction of cytochromes a and a3. Reaction of Cytochrome c Oxidase with Isonitriles Binding of the isonitriles causes a shift in the a band of reduced cytochrome c oxidase from 605 nm to 600 nm, and in the Soret band from 444 nm to 439 nm. Addition of isonitriles to oxidized cytochrome oxidase leads first to reduction of the heme and then to binding of the isonitriles. Reaction of Cytochrome c Oxidase with Hydrogen Sulfide Hydrogen sulfide readily inhibits cytochrome oxidase under aerobic conditions? In the steady-state, cytochrome a is reduced and cytochrome a3 is oxidized. The sulfide is directly bound to the iron atom of the heme as shown by the presence of a typical low-spin heme EPR spectrum? ° In mitochondria and submitochondrial particles the spectrum is actually the composite of two spectra, one with g values of approximately 2.57 and 2.26 and the other with values of 2.54 and 2.22. The high field g value of near 1.9 is also double, but the splitting is less accurately measured. Isolated cytochrome oxidase reacts with sulfide to form a low-spin ferric heine compound with g values of 2.54, 2.23, and 1.87. ~ The reaction of sulfide with isolated cytochrome oxidase occurs at a rate comparable to that of cyanide and with high affinity.52 H2S is a strong reductant and can directly reduce cytochrome c and a. Reaction of Formate with Cytochrome c Oxidase Formate is reported to inhibit cytochrome-oxidase in suspensions of mitochondria and detergent-treated submitochondrial particles.5'~ The K~ values are 20-50/zM for suspensions of mitochondria in state 3, 100/.~M for suspensions of mitochondria treated with uncoupler, and 18-40/aM for suspensions of the submitochondrial particles. The onset of inhibition is relatively slow, giving a lag period of approximately 17 sec. A secondary inhibition site occurs in the inhibition of glutamate plus malate oxidation. Spectral evidence indicates that formate forms a high-spin complex of oxidized cytochrome a:~. s0 D. F. Wilson, M. Erecifiska, and C. ~ R. W e v e r , B. F. van Gelder, and D. (1975). :~" P. Nicholls, Biochim. Biophys. Acta 5:~ p. Nicholls, Biochim. Biophys. Acta

S. O w e n , Arch. Biochem. Biophys. 175, 160 (1976). V. DerVartanian, Bloc/lira. Biophys. Acta 387, 189 396, 24 (1975). 430, 13 (1976).

Ligands of cytochrome c oxidase.

[21] L I G A N D S OF C Y T O C H R O M E C OX1DASE 191 Cl by the reduced and oxidized form, respectively. The Wr~ax for the reduction of cl 3+ is...
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