Inactivity of apoperoxidase in indoleacetic acid oxidation and in ethylene formation.

Peroxidase catalyzes oxidatic reactions (in which oxygen is the electron acceptor) as well as the classic peroxidatic reactions (in which hydrogen peroxide is the electron acceptor) (4, 10). There is considerable evidence that the enzyme which catalyzes the oxidatic degradation of the plant growth hormone, indole-3acetic acid, is a peroxidase (8). Recently Yang (12, 13) and Ku et al. (5) have demonstrated that ethylene, another plant growth hormone (7), is readily formed aerobically from methional ((3methylthiopropionaldehyde) or from a-keto--y-methylthiobutyric acid by peroxidase in the presence of Mns+, sulfite, and a monophenol. Siegel and Galston (9) reported that the apoprotein moiety of horseradish peroxidase, though devoid of peroxidase activity, retains its activity as an IAA oxidase when it is supplied with 10 ,uM Mn2+and 2,4-dichlorophenol. This report prompted us to examine whether the activity of peroxidase, as an oxidase catalyzing ethylene production from a-keto-y-methylthiobutyric acid, requires the heme prosthetic group or not. In contrast to the findings of Siegel and Galston (9) we found that the heme prosthetic group in peroxidase is essential for the oxidase activity of both IAA oxidation and ethylene production, as well as for its peroxidase activity. Cleavage of horseradish peroxidase into heme and apoenzyme by acid butanone and reconstitution of holoenzyme from heme and apoenzyme were performed as described by Yonetani (14), who has discussed the advantages of acid-butanone method over the conventional acid-acetone method for preparation of apoperoxidase. For reconstitution of holoenzyme, we used crystalline heme (equine hematin) purchased from Sigma. The reaction mixture and the method used here for the assay of IAA oxidase were essentially as described by Siegel and Galston (9), except that the amount of the enzyme employed was so adjusted that the increase in optical density per minute at 254 nm was no more than 0.10. Within this range, the rate of the reaction is proportional to the enzyme concentration. The kinetics of the reaction shows that a lag period usually precedes the steady state of the increase in absorbancy. Enzyme activity measurements were taken from the linear portion of the time course curve following the lag period. Table I clearly shows that the apoenzyme is nearly devoid of all the enzyme activities catalyzing o-dianisidine peroxidation, IAA oxidation (in the presence of Mn2+ and dichlorophenol), and ethylene production. The residual enzyme activity of the apoenzyme may be due to a slight contamination by unresolved holoenzyme in that fraction rather than to the apoenzyme itself. However, about 60% of the specific enzyme activities are restored