A SPECTROELECTROCHEMICAL METHOD FOR EVALUATING FACTORS WHICH REGULATETHE REDOX POTENTIAL OF HEMOGLOBINS

Spectroelectrochemical techniques were used to evaluate the redox pote ntial of various hemoglobins under various experimental conditions. We use Ru(NH3)(6)(3+) as a redox mediator, which exchanges electrons wit h heme iron through an outer-sphere mechanism. Use of this mediator of fers the advantage that it does not act as an allosteric effector. Stu dies of Hb stabilized in varied conformations confirm previous reports that Hb in the high oxygen affinity (R state) conformation is typical ly more easily oxidized than in the low oxygen affinity (T state) conf ormation. Accordingly, Nernst plots for Hb show evidence of cooperativ ity, with redox potentials that are sensitive to T state stabilization by anionic effecters. Alterations of the redox potential that are ind ependent of the protein's conformational equilibrium between R and T s tates are exemplified by imidazole interactions with Hb. Imidazole bin ds preferentially to metHb. Changes in the redox potential induced by imidazole are very different from the changes associated with binding of heterotropic anionic effecters. Effecters such as inositol hexaphos phate stabilize the T state, significantly lower cooperativity of the oxidation process, and reduce the ease of oxidation. Tn contrast, as t he imidazole concentration is increased, Hb is more readily oxidized, and cooperativity is maintained. These marked differences make it poss ible to use spectroelectrochemistry to differentiate between redox cha nges brought about by conformational shifts and changes brought about by other alterations in the active site.