DISCRIMINATION BETWEEN O-16 AND O-18 IN OXYGEN-BINDING TO THE REVERSIBLE OXYGEN CARRIERS HEMOGLOBIN, MYOGLOBIN, HEMERYTHRIN, AND HEMOCYANIN- A NEW PROBE FOR OXYGEN-BINDING AND REDUCTIVE ACTIVATION BY PROTEINS

A method for the measurement of discrimination between O-16 and O-18 i n the reversible interaction of dioxygen with proteins is described. A ccording to this method, total dioxygen (+/- protein) in reaction vess els is converted to carbon dioxide and analyzed by isotope ratio mass spectrometry. We see a trend in equilibrium isotope effects in proceed ing from ancestral oxygen carriers, hemocyanin and hemerythrin, 18(K)o bs = 1.0184 and 1.0113, respectively, to myoglobin and hemoglobin, 18( K)obs = 1.0054 and 1.0039, respectively. Oxygen isotope effects have a lso been calculated by using available frequencies and force constants for dioxygen and various forms of superoxide and peroxide. These comp utations indicate a discrimination between O-16 and O-18 of ca. 3% for each 0.5 reduction in the bond order of the dioxygen bond. Protonatio n and concomitant reduction of dioxygen to form superoxide (HO2.) and peroxide (H2O2) lead to small normal isotope effects of ca. 1%. Compar ison of computed and measured isotope effects to known structures for bound dioxygen in hemocyanin, hemerythrin, hemoglobin, and myoglobin p rovides a frame of reference for further investigations of O-18 isotop e effects in oxygen-dependent reactions.