Reaction of myoglobin with hydrogen peroxide forms a peroxyl radical which oxidizes substrates.

Evidence is presented that the radical observed upon reaction of myoglobin with hydrogen peroxide is a peroxyl radical. Simulation of this spectrum gives principal values for the g tensor of gx = 2.0357, gy = 2.0082, and gz = 2.0016, which are consistent with those of a peroxyl radical. Use of molecular oxygen isotopically labeled with 17O confirmed that the radical observed was a peroxyl radical. Removal of oxygen from the incubation by use of glucose and glucose oxidase revealed two radicals, one at giso = 2.0028 and the other at giso = 2.0073. Addition of various amounts of the spin trap 5,5-dimethyl-1-pyrroline N-oxide revealed that the spin trap and oxygen compete for the same radical site. Four model substrates, glutathione, styrene, arachidonic acid and linoleic acid, were individually added to both the aerobic and anoxic systems. Glutathione reacted with the peroxyl radical, reducing its intensity by 98%, and entirely eliminated the giso = 2.0028 line from the spectrum of the anoxic incubation. Styrene, arachidonic acid and linoleic acid reacted with the peroxyl radical, reducing its amplitude by 84, 57, and 35%, respectively, but did not decrease the amplitude of either radical species in the anoxic incubation. The giso = 2.0028 species detected in the anoxic incubation appears to be the original radical site to which molecular oxygen binds to form the peroxyl radical. This myoglobin-derived peroxyl radical species is responsible for the advent of lipid peroxidation as proposed in ischemia/reperfusion injury, as well as other reactions, as exemplified by the O2-dependent epoxidation of styrene.