Heme-Spin Label Studies of Hemoglobin

Heme spin-labeled oxyferrohemoglobin was prepared by the recombination of mono-spin-labeled protoheme with human apohemoglobin and subsequent reduction with a ferredoxin reductase system. The spin-labeled oxyhemoglobin exhibited optical and oxygen-binding properties indistinguishable from those of native hemoglobin. The electron paramagnetic resonance spectrum of the spin label attached to the heme-moiety showed that the labels were moderately immobilized in the protein. Upon deoxygenation, the resonance amplitude was decreased to less than 50% of the original amplitude and there were small changes in the EPR line shape. Comparison between the optical and EPR measurements during the deoxygenation process clearly showed that the EPR changes are not proportional to the optical changes. This result is contrary to the model of oxygen binding to hemoglobin provided by the induced fit theory. Since the EPR change has no isosbestic point, it was concluded that the microscopic conformation in the vicinity of the heme can take more than two states during the oxygenation process. It was also shown that the EPR line shape of the heme spin-labeled oxyhemoglobin is different from that of the met form. This result suggests a conformational difference between these two hemoglobins in the dissolved state.