PRODUCTION OF SUPEROXIDE FROM HEMOGLOBIN-BOUND OXYGEN UNDER HYPOXIC CONDITIONS

By low temperature electron paramagnetic resonance we have detected th e formation of a free radical signal during incubation of partially ox ygenated hemoglobin at 235 K. The observed signal has g(parallel to) = 2.0565 and g(perpendicular to) = 2.0043, consistent with the previous ly reported values for superoxide. The presence of additional EPR sign als for oxygen-17 bound hemoglobin, with ((O17-O17))A(perpendicular to ) = 63 G and ((O17-O16))A(perpendicular to) = 94 G under identical con ditions, confirms the presence of a radical containing two nonequivale nt oxygens as required for a superoxide in magnetically inequivalent e nvironments, The superoxide radical has not previously been directly d etected during hemoglobin autoxidation because of its rapid dismutatio n. Our ability to follow the formation of superoxide for more than 15 min is attributed to its production in the hydrophobic heme pocket whe re dismutation is slow. The enhanced production of this free radical a t intermediate oxygen pressures is shown to coincide with enhanced rat es of hemoglobin autoxidation for partially oxygenated intermediates. The formation of superoxide in the heme pocket under these conditions is attributed to enhanced heme pocket flexibility, Greater flexibility facilitates distal histidine interactions which destabilize the iron- oxygen bond resulting in the release of superoxide radical into the he me pocket.