Production of superoxide from hemoglobin-bound oxygen under hypoxic conditions

Abstract

By low temperature electron paramagnetic resonance we have detected the formation of a free radical signal during incubation of partially oxygenated hemoglobin at 235 K. The observed signal has g|| = 2.0565 and g⊥ = 2.0043, consistent with the previously reported values for superoxide. The presence of additional EPR signals for oxygen-17 bound hemoglobin, with (O17-O17)A⊥ = 63 G and (O17-O16)A ⊥= 94 G under identical conditions, confirms the presence of a radical containing two nonequivalent oxygens as required for a superoxide in magnetically inequivalent environments. The superoxide radical has not previously been directly detected during hemoglobin autoxidation because of its rapid dismutation. Our ability to follow the formation of superoxide for more than 15 min is attributed to its production in the hydrophobic heme pocket where dismutation is slow. The enhanced production of this free radical at intermediate oxygen pressures is shown to coincide with enhanced rates 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 heme pocket.