Measurement of Heme Accessibility in Soybean Ferric Leghemoglobin a and Its Complexes by Proton Magnetic Relaxation

Abstract

The accessibility of the heme and solvent-exchange dynamics in solutions of soybean ferric leghemoglobin a and several of its complexes have been determined by measurement of the relaxation rates of water, acetone, and methanol protons. Acetone and methanol were used at low concentrations as probes of outer-sphere and inner-sphere exchange processes, respectively. Water proton relaxation in solutions of ferric leghemoglobin is exchanged limited. The rate of exchange of water molecules or protons from the environment of the heme is greater than that for ferric myoglobin and ferric hemoglobin. Methanol binds to ferric leghemoglobin in a homogeneous first-order reaction and in competition with fluoride, a known iron ligand. Both optical and NMR measurements indicate binding of methanol to the sixth coordination position of the iron atom. Methanol proton relaxation by both ferric leghemoglobin and whale ferric myoglobin is exchange limited. Exchange is more rapid for leghemoglobin (4.0 X 103 s1 at 25 °C) than for myoglobin (9.5 X 102 s”1 at 25 °C). Relaxation of acetone protons occurs by fast outersphere exchange from the heme pocket. Acetone is thus a useful probe of heme accessibility. Acetone proton relaxation rates show the heme to be more accessible in ferric leghemoglobin than in ferric myoglobin. Conformational changes occur on binding of fluoride or acetate which decreases the accessibility of the heme in leghemoglobin. In myoglobin, binding of fluoride or formate appears to make the heme more accessible to acetone molecules. These results indicate that the heme in soybean ferric leghemoglobin is in a flexible region of the protein and is more accessible to solvent than in vertebrate myoglobins and hemoglobins. The relevance of this open, flexible heme pocket to the physiological 02-binding reaction of ferrous leghemoglobin is discussed. Show more