The in Vitro Inhibition of Cytochrome Oxidase by Azide and Cyanide by J. N. Stannard

Differences in the action of the commonly employed respiratory inhibitors, cyanide and azide, have been described in studies on a number of tissues and microorganisms; e.g., resting and active frog muscle (1, 2)) mammalian salivary glands (3), embryonic fish hearts (4), and yeast (5). More recently differences in the action of these substances on the oxidation of various substrates by cell-free tissue preparations have been noted (6). Many of these findings and their interpretation were reviewed by Stotz (7) and Goddard (8) and specific examples were outlined in the preceding paper (9). As a possible mechanism to account for these differences in the action of cyanide and azide .on respiration, it has been postulated that a different pathway of metabolism, independent of the cytochrome-cytochrome oxidase system, may account for the azide-insensitive, cyanide-sensitive respiration. As discussed in the preceding paper, the possible nature of this pathway is still unclear, although it is not difficult to find examples of systems with these characteristics (6, 10). As an alternative to the separate pathway hypothesis, Ball (11) has proposed that the oxidation-reduction potentials of the azide and cyanide complexes formed with cytochrome oxidase may be sufficiently different to account for the results. Specifically it was postulated that azide. might combine with both the ferro and ferri forms of the enzyme, thus stabilizing the potential at a level sufficiently above that of cytochrome c to permit oxidation of the cytochromes at a diminished rate. In the presence of cyanide, on the other hand, the potential of the cytochrome oxidase system would be too low to allow any oxidation. Since cytochrome oxidase has not yet been isolated, direct measurement of the oxidation-reduction potentials is impossible and no experimental verification of Ball’s suggestion is possible. However, it is clear that more fundamental study of the mode of action of these inhibitors, especially on cell-free preparations, is required. The present study was initiated with this general objective in view, and specifically to observe the effects of azide and cyanide on the oxidation of ferrocytochrome c measured spectrophotometrically.