Interactions of solvent with the heme region of methemoglobin and fluoro-methemoglobin

Abstract

It is now more than 20 years since Davidson and collaborators (1957, Biochim. Biophys, Acta. 26:370-373; J. Mol. Biol. 1:190-191) applied the theoretical ideas of Bloembergen et al. (1948. Phys. Rev. 73:679-712) on outer sphere magnetic relaxation of solvent protons to studies of solutions of methemoglobin. From then on, there has been debate regarding the relative contributions to paramagnetic solvent proton relaxation by inner sphere (ligand-exchange) effects and by outer sphere (diffusional) effects in methemoglobin solutions. Gupta and Mildvan (1975. J. Biol. Chem 250:146-253) extended the early measurements, attributed the relatively small paramagnetic effects to exchange with solvent of the water ligand of the heme-Fe3+ ion, and interpreted their data to indicate cooperativity and an alkaline Bohr effect in the presence of inositol hexaphosphate. They neglected the earlier discussions entirely, and made no reference to outer sphere effects. We have measured the relaxation rate of solvent protons as a function of magnetic field for solutions of methemoglobin, under a variety of conditions of pH and temperature, and have given careful consideration to the relatively large diamagnetic corrections that are necessary by making analogous measurements on oxyhemoglobin, carbonmonoxyhemoglobin, and cyano- and azide-methemoglobin. (The latter two, because of their short electronic relaxation times, behave as though diamagnetic). We show that the paramagnetic contribution to solvent relaxation can be dominated by outer sphere effects, a result implying that many conclusions, including those of Gupta and Mildvan, require reexamination. Finally, we present data for fluoro-methemoglobin, which relaxes solvent protons an order of magnitude better than does methemoglobin. Here one has a startling breakdown of the dogma that has been the basis for interpreting many ligand-replacement studies; in contrast to the prevailing view that replacement of a water ligand of a protein-bound paramagnetic ion by another ligand should decrease relaxation rates, replacement of H2O by F- increases the relaxation rate drastically. The data can all be reconciled, however, with what is anticipated from knowledge of ligand interactions in the heme region.

Gd(DOTP)5‐ outer‐sphere relaxation enhancement promoted by nitrogen bases

“…Originally it was postulated for the case of fluorornethemoglobin characterized by the presence of strong hydrogen bonds with second-sphere water molecules (34) and later it was found to account for the relaxation behavior of some paramagnetic macromolecules (33,35). Nevertheless, with reference to Fig.…”

Relaxometry of ferritin solutions and the influence of the Fe3+ core ions

“…The serum protein transferrin takes iron from the gut (the mechanism of transport across the intestinal wall is not well understood) to the A to the iron cores of femtin, which are not magnetically ordered at room temperature (10, IZ), the outer sphere relaxation contribution to l/Tl can be readily estimated (cf. (12)); the effect is miniscule for reasonable conditions. One would expect a similarly small influence on 1/T2 ( 9 ) which is not in accord with the observations.…”

Magnetic field dependence of solvent proton relaxation in aqueous solutions of Fe3+ complexes

“…3. Perhaps the most interesting are the two hemoglobin complexes (12), which represent the extremes of the relaxivity range: methemoglobin (with the iron oxidized to Fe3+), which has a single H 2 0 ligand directly coordinated to each Fe3+ ion at pH 7; and fluoromethemoglobin, in which each of the H 2 0 ligands is displaced by F-, with the electronic properties of the Fe3+ relatively unchanged.…”

“…The behavior of the two methemoglobin complexes may appear counterintuitive, but the reasons are understood (12): the directly coordinated water molecules exchange with solution too slowly to contribute much to the relaxation rates of solvent protons, hence the low rates; displacing these waters by F-allows formation of a short-lived, second-coordination, complex of protein with a solvent water molecule, with one of its protons hydrogen bonded to the F and therefore relatively close to the Fe3+ ion, thereby producing the relatively high relaxivities. The Fe3+-EDTA sample was 1.03 mM Fe3+, and the Geritol sample, a -5-fold dilution of the commercial product, was 11.1 mM Fe3+.…”

“…The results for transferrin and the hemoglobin derivatives are from Refs. (21, IZ), respectively. analyzed rather thoroughly, and are understood quantitatively (12); that for transfenin is more complex, and relatively intractable computationally because of the highly anisotropic ligand field of the Fe3+ ions in transfemn (21). However, the best relaxing of these three Fe3+ protein complexes has solvent proton relaxivities that are an order of magnitude less than comparable complexes of Mn2+ and Gd3+ ions with protein; the latter have relaxivities in the range 50-300 mA4 s-' and, unlike Fe3+, have a characteristic maximum in their NMRD profiles in the 10-100 MHz decade (13), where imaging tends to be done.…”