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Since FMNH-nitrate reductase activity is heat-stable, whereas NADH-cytochrome c reductase activity is heat labile, we consider that donation of electrons by F M N H does not involve the cc-subunit and that exogenous cytochrome c accepts electrons directly from the heat-labile a-subunit. The differential stability of FMNH-nitrate reductase and reduced methyl viologen-nitrate reductase suggests that F M N H and reduced methyl viologen donate electrons at ditferent points in the electron-transport chain. We consider that F M N H donates electrons directly to the b-subunit, whereas reduced methy viologen donates electrons to molybdenum-containing component carried on the y-subunit. The a-subunits are then responsible for NADH-cytochrome c reductase activity, the p- and y-subunits together with molybdenum-containing component are responsible for FMNH-nitrate reductase activity and the y-subunits together with molybdenum-containing component are responsible for reduced methyl viologen-nitrate reductase activity. We gratefully acknowledge the award of Science Research Council Research Studentships to 1. S. S. and J. B.
Hewitt, E. J. (1974) MTP Int. Reu. Sci.: Biocheni. Ser. One 11, 199-245 Hewitt, E. J., Notton, B. A. & Rucklidge, G. J. (1977)J. Less-Common Met. 54, 537-553 MacGregor, C. H. (1975)J. Bncteriol. 121, 11 11-1 116 Nason, A., Lee, K. Y . ,Pan, S. S., Ketchum, P. A.,Lamberti, A. & DeVries, J. (1971)Proc. Not/. Acad. Sci. U.S.A. 68, 3242-3246
Notton, B. A. & Hewitt, E. J . (1971) Plotit CellPhysiol. 12, 465-477 Notton, B. A,, Fido, R. J . & Hewitt, E. J. (1977)P/crt?t Sci.Lett. 8, 165-170 Rucklidge, G. J., Notton, B. A. & Hewitt, E. J . (1976) Biochem. Soc. Trans. 4, 77-80 Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. A c f n 112, 346-362 Small, I. S. & Wray, J. L. (1979) Biochem. SOC.Trans. 7, 737-739 Wray, J. L. & Filner, P. (1970) Biochem. J. 119,715-725
Azide Interaction with Cytochrome c Oxidase: Does it Bind to Cytochrome a or a,? J. GORDON LINDSAY Department of Biochemistry, University of Glasgow, Glasgow G12 8 Q Q , Scotland, U.K. The individual haem groups of the cytochrome c oxidase complex, namely cytochromes u and a3, may be distinguished by their differing standard electiode potentials (Eh), spectral properties and pH-dependencies (Wilson et al., 1972; Lindsay et al., 1975). Potentiometric determination of cytochromes a3 and n in pigeon breast submitochondrial particles gives Eh values of 360+10mV and 220+10mV respectively at pH7.0. In the Soret (y) and a-bands regions of their reduced spectra, cytochrome a3 exhibits values at 444 and 604nm compared with 446 and 606nm for slightly shorter A,,. cytochrome u. For cytochrome 0 3 , its E i decreases by59mVper pH unit in the p H range 7.0-9.0, but is essentially pH-independent below pH 7.0. Thus oxidation-reduction of this component involves addition of 1 H+ per electron t o a chemical group with a pK of 7.0, possibly a histidine residue. In contrast, cytochrome a shows a non-specific behaviour, its EA becoming 20-30mV more negative for each pH unit from 6.0 to 9.0. Ligands (inhibitors) such as CO and azide interact preferentially with specific oxidation-reduction states of the enzyme. For example, CO has a high affinity for reduced ~ + o r fully cytochrome a3, binding stoicheiometrically to the half-reduced ( u ~.a3+) reduced form (a3*+-a2+)of the oxidase complex (Greenwood era/., 1974). Reduction of one of the two copper atoms of the complex (the e.p.r.-undetectable copper) is also a prerequisite for CO binding (Lindsay et al., 1975). Since the early studies of Keilin & Hartree (1939), which suggested that azide forms a complex with oxidized cytochrome a3, little evidence has accumulated to indicate that
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Fig. 1. Efect of p H on the EA values of cytochromes a and a3 in the presence of azide Simultaneous oxidation-reduction-potential-against-absorbance measurements were performed by using the wavelength pair 610 minus 624nm on pigeon breast submitochondrial particles (Lindsay & Wilson, 1974) in the presence of 20m~-sodiumazide, essentially as described by Dutton et al. (1970). Oxidative ( 0 ) and reductive titrations ( 0 )are repeated at pH values of 6.2 (o,.), 7.2 (A, A), 8.2 (0, m) and 9.2 (0, +). azide has any affinity for reduced haem. However, potentiometric analysis of cytochromes a3 and a in the presence of azide, indicates that the EA value of the low potential species moves 59mV more negative for each 10-fold increase in azide concentration (Wilson et al., 1972). This finding would be consistent with the formation of a 1 : l stoicheiometric complex between azide and oxidized cytochrome a. Surprisingly, the EA value for the upper species (cytochrome a3 in the absence of azide) is unaffected by this inhibitor. To resolve this discrepancy, examination of the pH-dependencies of the two haem groups in the presence of 20m~-azideis carried out as shown in Fig. 1. It is clear that the Eh of the high-potential component (normally cytochrome as) now decreases by only 20-30mV per pH unit in the pH range 6.2-9.2, whereas the E; of the low-potential species is unaltered at 95+ lOmV under these conditions. It is possible to relate these pH effects on the haem groups of cytochrome c oxidase in the presence of azide to their intrinsic pH behaviour in the unliganded state. The 20-30 mV pH-dependence of the upper component would normally be characteristic of 1979
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cytochrome a in the absence of inhibitor. The apparent pH-independence of the lowpotential species mayrepresent a situation where theeffects of p H on theactive inhibitory species, in this case HN3 ( p K = 4.6), are exactly conterbalanced by the intrinsic pH behaviour of this component. Thus lowering of the HN3 concentration (10-fold per p H unit at alkaline pH) will result in a movement of the Eh value t o more positive potentials (59mV per p H unit), indicating that the intrinsic p H dependence of this component is 59mV per pH unit in the opposite direction, characteristic of cytochrome u3. This anomalous situation where cytochrome a titrates at higher electrode potentials than cytochrome u3 in the presence of azide can be explained by azide having a high affinity for the minor half-reduced species u33+.az+, generated during potentiometric titrations. This result is supported by analysis of the effects of azide on the spectral properties of cytochromes a and u3 and by direct measurement of azide affinity for the half-reduced and fully-oxidized states of cytochrome c oxidase. Dutton, P. L., Wilson, D. F. & Lee, C. P. (1970) Biochemistry 9, 5077-5082 Greenwood, C., Wilson, M. T. & Brunori, M. (1974) Biochem. J . 137,205-215 Keilin, D. & Hartree, E. F. (1939) Proc. Roy. SOC. Ser. B 127, 167-191 Lindsay, J. G. & Wilson, D. F. (1974) FEBS Lett. 48,45-49 Lindsay, J. G., Owen, C. S . &Wilson, D. F. (1975) Arch. Biochem. Biophys. 169,492-505 Wilson, D. F., Lindsay, J. G. & Brocklehurst, E. S. (1972) Biochim. Biophys. Acta 256,277-286
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Since FMNH-nitrate reductase activity is heat-stable, whereas NADH-cytochrome c reductase activity is heat labile, we consider that donation of electrons by F M N H does not involve the cc-subunit and that exogenous cytochrome c accepts electrons directly from the heat-labile a-subunit. The differential stability of FMNH-nitrate reductase and reduced methyl viologen-nitrate reductase suggests that F M N H and reduced methyl viologen donate electrons at ditferent points in the electron-transport chain. We consider that F M N H donates electrons directly to the b-subunit, whereas reduced methy viologen donates electrons to molybdenum-containing component carried on the y-subunit. The a-subunits are then responsible for NADH-cytochrome c reductase activity, the p- and y-subunits together with molybdenum-containing component are responsible for FMNH-nitrate reductase activity and the y-subunits together with molybdenum-containing component are responsible for reduced methyl viologen-nitrate reductase activity. We gratefully acknowledge the award of Science Research Council Research Studentships to 1. S. S. and J. B.
Hewitt, E. J. (1974) MTP Int. Reu. Sci.: Biocheni. Ser. One 11, 199-245 Hewitt, E. J., Notton, B. A. & Rucklidge, G. J. (1977)J. Less-Common Met. 54, 537-553 MacGregor, C. H. (1975)J. Bncteriol. 121, 11 11-1 116 Nason, A., Lee, K. Y . ,Pan, S. S., Ketchum, P. A.,Lamberti, A. & DeVries, J. (1971)Proc. Not/. Acad. Sci. U.S.A. 68, 3242-3246
Notton, B. A. & Hewitt, E. J . (1971) Plotit CellPhysiol. 12, 465-477 Notton, B. A,, Fido, R. J . & Hewitt, E. J. (1977)P/crt?t Sci.Lett. 8, 165-170 Rucklidge, G. J., Notton, B. A. & Hewitt, E. J . (1976) Biochem. Soc. Trans. 4, 77-80 Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. A c f n 112, 346-362 Small, I. S. & Wray, J. L. (1979) Biochem. SOC.Trans. 7, 737-739 Wray, J. L. & Filner, P. (1970) Biochem. J. 119,715-725
Azide Interaction with Cytochrome c Oxidase: Does it Bind to Cytochrome a or a,? J. GORDON LINDSAY Department of Biochemistry, University of Glasgow, Glasgow G12 8 Q Q , Scotland, U.K. The individual haem groups of the cytochrome c oxidase complex, namely cytochromes u and a3, may be distinguished by their differing standard electiode potentials (Eh), spectral properties and pH-dependencies (Wilson et al., 1972; Lindsay et al., 1975). Potentiometric determination of cytochromes a3 and n in pigeon breast submitochondrial particles gives Eh values of 360+10mV and 220+10mV respectively at pH7.0. In the Soret (y) and a-bands regions of their reduced spectra, cytochrome a3 exhibits values at 444 and 604nm compared with 446 and 606nm for slightly shorter A,,. cytochrome u. For cytochrome 0 3 , its E i decreases by59mVper pH unit in the p H range 7.0-9.0, but is essentially pH-independent below pH 7.0. Thus oxidation-reduction of this component involves addition of 1 H+ per electron t o a chemical group with a pK of 7.0, possibly a histidine residue. In contrast, cytochrome a shows a non-specific behaviour, its EA becoming 20-30mV more negative for each pH unit from 6.0 to 9.0. Ligands (inhibitors) such as CO and azide interact preferentially with specific oxidation-reduction states of the enzyme. For example, CO has a high affinity for reduced ~ + o r fully cytochrome a3, binding stoicheiometrically to the half-reduced ( u ~.a3+) reduced form (a3*+-a2+)of the oxidase complex (Greenwood era/., 1974). Reduction of one of the two copper atoms of the complex (the e.p.r.-undetectable copper) is also a prerequisite for CO binding (Lindsay et al., 1975). Since the early studies of Keilin & Hartree (1939), which suggested that azide forms a complex with oxidized cytochrome a3, little evidence has accumulated to indicate that
Vol. 7
742
BIOCHEMICAL SOCIETY TRANSACTIONS
Fig. 1. Efect of p H on the EA values of cytochromes a and a3 in the presence of azide Simultaneous oxidation-reduction-potential-against-absorbance measurements were performed by using the wavelength pair 610 minus 624nm on pigeon breast submitochondrial particles (Lindsay & Wilson, 1974) in the presence of 20m~-sodiumazide, essentially as described by Dutton et al. (1970). Oxidative ( 0 ) and reductive titrations ( 0 )are repeated at pH values of 6.2 (o,.), 7.2 (A, A), 8.2 (0, m) and 9.2 (0, +). azide has any affinity for reduced haem. However, potentiometric analysis of cytochromes a3 and a in the presence of azide, indicates that the EA value of the low potential species moves 59mV more negative for each 10-fold increase in azide concentration (Wilson et al., 1972). This finding would be consistent with the formation of a 1 : l stoicheiometric complex between azide and oxidized cytochrome a. Surprisingly, the EA value for the upper species (cytochrome a3 in the absence of azide) is unaffected by this inhibitor. To resolve this discrepancy, examination of the pH-dependencies of the two haem groups in the presence of 20m~-azideis carried out as shown in Fig. 1. It is clear that the Eh of the high-potential component (normally cytochrome as) now decreases by only 20-30mV per pH unit in the pH range 6.2-9.2, whereas the E; of the low-potential species is unaltered at 95+ lOmV under these conditions. It is possible to relate these pH effects on the haem groups of cytochrome c oxidase in the presence of azide to their intrinsic pH behaviour in the unliganded state. The 20-30 mV pH-dependence of the upper component would normally be characteristic of 1979
582nd MEETING, ST. ANDREWS
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cytochrome a in the absence of inhibitor. The apparent pH-independence of the lowpotential species mayrepresent a situation where theeffects of p H on theactive inhibitory species, in this case HN3 ( p K = 4.6), are exactly conterbalanced by the intrinsic pH behaviour of this component. Thus lowering of the HN3 concentration (10-fold per p H unit at alkaline pH) will result in a movement of the Eh value t o more positive potentials (59mV per p H unit), indicating that the intrinsic p H dependence of this component is 59mV per pH unit in the opposite direction, characteristic of cytochrome u3. This anomalous situation where cytochrome a titrates at higher electrode potentials than cytochrome u3 in the presence of azide can be explained by azide having a high affinity for the minor half-reduced species u33+.az+, generated during potentiometric titrations. This result is supported by analysis of the effects of azide on the spectral properties of cytochromes a and u3 and by direct measurement of azide affinity for the half-reduced and fully-oxidized states of cytochrome c oxidase. Dutton, P. L., Wilson, D. F. & Lee, C. P. (1970) Biochemistry 9, 5077-5082 Greenwood, C., Wilson, M. T. & Brunori, M. (1974) Biochem. J . 137,205-215 Keilin, D. & Hartree, E. F. (1939) Proc. Roy. SOC. Ser. B 127, 167-191 Lindsay, J. G. & Wilson, D. F. (1974) FEBS Lett. 48,45-49 Lindsay, J. G., Owen, C. S . &Wilson, D. F. (1975) Arch. Biochem. Biophys. 169,492-505 Wilson, D. F., Lindsay, J. G. & Brocklehurst, E. S. (1972) Biochim. Biophys. Acta 256,277-286
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