CYANIDE INHIBITION AS A MEANS OF ELUCIDATING THE MECHANISMS OF CELLULAR RESPIRATION

Summary The effect of cyanide on the respiration of living cells makes possible the differentiation between and the description of two types of respiratory systems. (1) The cyanide-sensitive respiration may be identified with the Warburg-Keilin respiratory system. The normal variation in Qo1 between different organs, organisms, developmental stages (in certain enumerated cases) and random variations among samples of the same tissue, seem largely to be accountable by differences in the activity of this system. The rate of oxygen consumption by this system varies over a wide range of values, and accounts for about 90% of the maximum possible respiratory rate of most actively aerobic tissues and organisms. It seems likely (on the basis of the available data) that the cyanide-sensitive system tends most actively to oxidize carbohydrates and other metabolites that have a similarly high water-solubility and O/C ratio. Thus, cyanide-sensitive respiration is characterized by a respiratory quotient of about 1.0. The Warburg-Keilin system also appears to be the more sensitive (of the two) to variations in temperature and po2. (2) The cyanide-stable respiration may be identified with the yellow enzyme or flavoprotein. Its activity is relatively small and constant from organ to organ and from organism to organism (within certain groups), as compared with the activity of the cyanide-sensitive system. It seems likely that the metabolic substrates of this respiratory system (in vivo) are restricted, in the main, to fatty compounds and other substances that have a similarly low O/C ratio. Thus, the cyanide-stable respiration is characterized by a respiratory quotient of 0.8 or less. Hence, since most variations in the total rate of respiration are mainly due to variations in the activity of the cyanide-sensitive system alone, percentage inhibition by cyanide (i.e. “cyanide-sensitivity”) increases with the normal rate of respiration. Therefore, this value is no index of the relative activity of the two systems unless the substrate environment and other influential conditions are specified. Certain intrinsic properties of the Warburg-Keilin system are elucidated by partial inhibition of this system. It is shown thereby that the inhibitory effect of cyanide is related to the extent of saturation of the dehydrogenase with its substrate. When the dehydrogenase is only partially saturated or “covered”, the respiratory rate is reduced only by high cyanide concentrations. Thus, under such circumstances, part of the oxidase may be inactivated by a low concentration of cyanide without affecting the rate of respiration. However, such a cyanide concentration prevents the rise in respiratory rate that normally follows the restoration of the dehydrogenase to complete saturation by the addition of substrate to the medium. Thus, in this case we again note that the percentage inhibition by cyanide increases with the original (i.e. cyanide-free) rate of respiration. It appears therefore that the respiratory systems that can be distinguished by means of cyanide-sensitivity are to a large degree independent of each other. It has frequently been suggested that the two systems present alternative paths for the oxidation of any given metabolite. The amount of oxidative activity of each system would then depend on their relative concentrations in the cell and on their relative oxidative tendencies, i.e. redox potentials. If this were so, we would expect that the ratio of their activities would be constant regardless of the nature or concentration of the metabolite present in the medium. Furthermore, if the activity of one of these systems be curtailed (e.g. by cyanide), it follows that the insensitive system would become more active since it would then be exposed to a greater portion of the “reduction potential”) which is produced by the metabolite and its dehydrogenase. It does not seem likely, -therefore, that this is a valid interpretation of the interrelationships of these processes as they obtain in the living cell. The ratio of the rates of activity of the two systems (i.e. the percentage inhibition by cyanide) is not constant, but varies directly with the total rate of respiration. Thus, in a living cell the cyanide-stable system seems to operate at a low and constant rate, to a large degree independent of the highly variable rate of activity of the cyanide-sensitive system. Furthermore, the systems appear to have qualitative specificities as to substrates, rather than sharing the oxidation of each metabolite. These facts seem to indicate that there is no direct thermodynamic equilibrium between the oxidative activity of the two systems. The internal relationships of the various parts of the cyanide-sensitive system appear to be equally discontinuous. Under certain conditions the saturation of the oxidase or the dehydrogenase may be altered without affecting the total rate of respiration. Finally, these specific relationships of the respiratory systems are intimately associated with the normal integrity of the cell. Destruction of the cellular protoplasm disorients the qualitative specificities that obtain in the living cell. It would appear that the ordering of the interrelated processes of cellular respiration is in some manner facilitated by the normal intracellular structure.