Properties of the copper associated with cytochrome oxidase as studied by paramagnetic resonance spectroscopy.

There appears to be general agreement that cytochrome oxidase preparations obtained by various procedures contain amounts of copper in the range of the heme content of this enzyme (l-9). Various workers disagree, however, on the significance of this finding (6-10). Whereas several investigators hold the view that the copper associated with cytochrome oxidase has properties that would qualify it as a functional oxidation-reduction component of the oxidase (2,4,8, 9), others tend to consider this copper as an impurity (6, 7, 10). Experiments have been reported that support both points of view. A final decision, however, cannot be made on the basis of presently available information. Experiments that would demonstrate that copper undergoes oxidation-reduction at a rate commensurate with the activity of the oxidase are technically rather difficult. This paper presents the results of work on reactions and properties of the copper of cytochrome oxidase, which may be studied by the technique of paramagnetic resonance spectroscopy. With this technique, it is possible to probe into the immediate environment of the copper atom and obtain information on its valency and binding state. Although we must admit that we are unable to determine the exact environment of the copper from paramagnetic resonance spectra obtained from materials as complex as cytochrome oxidase, it is possible, as will be shown below, to observe distinct changes of paramagnetic resonance spectra under certain conditions, which can be evaluated on a largely empirical basis. We have studied the interaction of the copper of cytochrome oxidase with reducing substrates, chemical reducing agents, oxygen, inhibitors, and chelating agents for copper. This work forms a basis for future studies on the early kinetics of oxidationreduction of the components of the oxidase under various conditions, which, we hope, will provide an answer to the question of the function of the copper in the enzyme.