The Kinetics and Inhibition of Cytochrome Components of the Succinic Oxidase System

In the course of studies by Keilin and Hartree (1) on succinic oxidase preparation of heart muscle particles, it was observed that the kinetics of reduction of cytochrome b were not in accord with some rather fundamental criteria that must be met for an electron-transferring component in the respiratory chain (2). On the other hand, studies of the kinetics of cytochrome b in intact yeast cells showed its behavior to be in reasonably good agreement with that expected of an electron-transferring component (3). Studies of cytochrome b in isolated rat liver and heart mitochondria afforded an explanation for this apparent inconsistency, since in these efficiently phosphorylating particles cytochrome b has an active function (4, 5). At some point in the disruption of the heart sarcosomes occasioned by the Keilin and Hartree procedure, the function of cytochrome b is altered so that most of the electrons bypass it in their path from substrate to oxygen. Furthermore, at some stages in the disruption, phosphorylative activity is diminished and then is lost completely. To the extent that the kinetic response of cytochrome b of the KeiIin and Hartree preparation does not represent the behavior of the same component when it is a part of the electron-transfer chain of the phosphorylating particle, its behavior in this preparation is that of an artifact. Nevertheless, many useful kinetic data (2) can be obtained with the above mentioned preparation, which is, incidentally, very convenient for spectroscopic studies because of the degree of light scattering, which is relatively small as compared to that of mitochondria. Such kinetic data now seem to have a real value in our understanding of what has occurred to the reactivity of cytochrome b in the preparation of the Keilin-Hartree particles. The results are also useful in evaluating the relationship of cytochrome b to the succinic-cytochrome c reductase activity of portions of the respiratory chain that have been isolated by various preparative procedures. Such studies may shed light on the mechanism of electron transfer in interactions between flavoproteins and cytochromes, even though the actual reactions involved are not the same as those that occur in the mitochondria during oxidative phosphorylation. This paper describes methods for the spectroscopic study of cytochrome b, the kinetics of its reduction and oxidation by succinate and fumarate, and an unexpected activation of the succinate-cytochrome b reaction by antimycin A treatment.