Reaction of bovine erythrocyte green hemoprotein with oxygen and hydrogen peroxide.

Abstract

Bovine erythrocyte green hemoprotein was shown to undergo rapid and complete bleaching during aerobic reduction by dithionite. In order to explain this phenomenon, we have studied the reactions of the ferrihemoprotein with dithionite and H202 and the reactions of the ferrohemoprotein with HzOz and OZ. Bleaching caused by aerobic dithionite was shown not to involve either superoxide or bisulfite anions. Inhibition by the ligands CO and pyridine suggested that a free heme site was necessary for bleaching and that the ferrous hemoprotein may be involved. Catalase strongly inhibits this bleaching, implying that HzOz (produced in the aerobic dithionite solution) is involved. The ferrihemoprotein is bleached by HzOz at pH 7.2, 25”C, in a reaction which involves a complex with Hz02 (KD = 1.3 mM) that decays at a rate of 0.14 s-l. This reaction is inhibited by CN(Z& = K. = 0.083 mu). HzOz reacts more rapidly with ferrohemoprotein; within 5 s, 35 to 50% of the hemoprotein is bleached and the remainder is oxidized to ferrihemoprotein. The anaerobic reduction of ferrihemoprotein by dithionite is very fast. These findings lead to the conclusion that in the aerobic bleaching reaction dithionite both generates HzOz by reaction with O2 and maintains the heme in the more reactive ferrous state. The ferrohemoprotein reacts rapidly with O2 to form a transient Complex (& = 8.7 X Ioe5 M) which decays to the ferrihemoprotein at a rate of 501 S -’ at 5°C. This oxidation is accompanied by a loss of 13 to 20% of the original absorbance of the ferrihemoprotein. Like green hemoprotein, cytochrome P-450 is bleached by HzOz, but at a much slower rate; ferrocytochrome P-450 reacts more rapidly than does ferricytochrome P-450. The mechanism for the bleaching is envisioned to be similar to the peroxidatic cycle of Yokota and Yamazaki ((1965) Biochim Biophys. Actu 105, 301) with the important difference that the addition of reducing equivalents to Compounds I and II of the peroxidatic cycle has been replaced by the loss of HO l radicals. We postulate that HO* generated by such a mechanism attacks, and thereby bleaches, the porphyrin ring.