Diazene reacts rapidly with cytochrome c oxidase to reduce cytochrome a and CuA and to form a charge-transfer complex with ferric cytochrome a3; the diazene may serve to bridge the heme iron of this cytochrome and CuB. The complex is characterized by an intense, optically active absorbance located at 847 nm. A similar band had been observed previously upon reduction of cytochrome oxidase with hydrazine [Markossian, K. A., Paitian, N. A., and Nalbandyan, R. M. (1983) FEBS Lett. 156, 235-238], but it appears that this band is actually due to the diazene produced as a result of the oxidation of the hydrazine that occurs in this process. A similar diazene to iron charge-transfer band is found following the reaction of diazene with ferric horseradish peroxidase and with hemin chloride but not with met-myoglobin.