Oxygen infrared spectra of oxyhemoglobins and oxymyoglobins. Evidence of two major liganded O2 structures.

The dioxygen stretch bands in infrared spectra for solutions of oxy species of human hemoglobin A and its separated subunits, human mutant hemoglobin Zurich (beta 63His to Arg), rabbit hemoglobin, lamprey hemoglobin, sperm whale myoglobin, bovine myoglobin, and a sea worm chlorocruorin are examined. Each protein exhibits multiple isotope-sensitive bands between 1160 and 1060 cm-1 for liganded 16O2, 17O2, and 18O2. The O-O stretch bands for each of the mammalian myoglobins and hemoglobins are similar, with frequencies that differ between proteins by only 3-5 cm-1. The spectra for the lamprey and sea worm hemoglobins exhibit greater diversity. For all proteins an O-O stretch band expected to occur near 1125 cm-1 for 16O2 and 17O2, but not 18O2, appears split by approximately 25 cm-1 due to an unidentified perturbation. The spectrum for each dioxygen isotope, if unperturbed, would contain two strong bands for the mammalian myoglobins (1150 and 1120 cm-1) and hemoglobins (1155 and 1125 cm-1). Two strong bands separated by approximately 30 cm-1 for each oxy heme protein subunit indicate that two major protein conformations (structures) that differ substantially in O2 bonding are present. The two dioxygen structures can result from a combination of dynamic distal and proximal effects upon the O2 ligand bound in a bent-end-on stereochemistry.

Relation between variations in the phenotypic expression of an unstable hemoglobin disorder (hemoglobin Zürich) and carboxyhemoglobin levels.

Longitudinal studies in a group of 15 asymptomatic subjects with hemoglobin Zürich from two unrelated families indicated marked variation in the clinical and hematologic expression of the hemoglobinopathy, even within the same family. Carbon monoxide bound to normal hemoglobin stabilized the molecule when exposed to a variety of denaturing procedures. The marked increase in the affinity of hemoglobin Zürich for carbon monoxide, approximately 65 times that of normal hemoglobin, led to a study of carboxyhemoglobin levels in smoking and nonsmoking subjects with hemoglobin Zürich and their relation to the phenotypic expression of the hemoglobinopathy. The carboxyhemoglobin content in whole blood from persons with hemoglobin Zürich ranged from 3.9 to 6.7 percent in nine nonsmokers and from 9.8 to 19.7 percent in six smokers. Rates of hemolysis as determined by values for hematocrit, reticulocyte count, and haptoglobin and hemopexin levels were less in smokers than in nonsmokers. Also, rates of Heinz body formation in intact red cells and isopropanol-induced precipitation of the abnormal hemoglobin in hemolysates were less in smokers than in non-smokers. These observations suggest that variability in the phenotypic expression of hemoglobin Zürich is a post-translation event secondary to stabilization of the abnormal hemoglobin by carbon monoxide.

Mechanism of autooxidation for hemoglobins and myoglobins. Promotion of superoxide production by protons and anions.

Several hemoglobins and bovine myoglobin are shown to undergo autooxidation reactions promoted by anions. The reduced protein in the presence of oxygen and anion yields the anion complex of the oxidized (Met) species and a second product that is almost certainly superoxide. The second product can be detected by its reduction of cytochrome c3+ at the same rates and in the same amount as the Met species. Anions are increasingly effective as promoters in the same order as their strengths as nucleophiles, e.g. Cl- less than F- less than OCN- less than SCN- less than N3- less than CN-. Rates are directly proportional to anion concentrations. A linear dependence of rate upon [H+] is also observed and can be related to the protonation of a strongly acidic group. Globin from hemoglobin A and hemes with altered 2,4-substituents gave reconstituted hemoglobins that autooxidize at rates that decrease with the electron-withdrawing power of the substituent: acetyl greater than vinyl greater than hydrogen greater than ethyl. Changes in rate with globin structure can be interpreted in terms of steric access to the ligand binding site; the more sterically restricted is the site, the slower is the reaction. The effects of [O2] on the rate vary with the degree of saturation with O2. At high O2 levels (e.g. from saturation to the point where 5% deoxyHbA and 95% oxyHbA are present), a decrease in [O2] results in an increase in the rate of azide-promoted autooxidation. At O2 levels with from 5 to 75% deoxyHbA, the rate remains nearly constant. At still lower levels with oxyHbA less than 25%, the rate decreases as [O2] is lowered. Exposure to CO reduces the rate. The reaction mechanism for anion-induced autooxidation must provide for the stoichiometric formation of a cytochrome c3%-reducing species (presumably, superoxide), the anion acting as a nucleophile in the rate-determining step, the facilitation by protons, the sensitivity of the rate to the electronegativity of heme iron, and the varied effects of [O2] upon the rate. These findings can not be fully accommodated by mechanisms in which the Met species forms either via dissociative loss of superoxide from the oxy species followed by anion binding or via displacement of protonated dioxygen from oxyheme upon nucleophilic attack of the anion at heme iron. A consistent mechanism is the reaction of protonated deoxy species with the anion followed by the reaction of Fe2+-anion complex with O2 to give Fe3+-anion and (formula, see text). Here, the deoxy rather than oxy species is involved in Fe2+ oxidation to Fe3+; O2 can serve as one-electron acceptor but not while serving as an iron-bound ligand. A precise non-iron site for electron transfer from ferrous porphyrin to O2 remains unlocated but a process involving the porphyrin pi-system has analogy in simple heme, flavin, or other organic donor reactions with O2 that yield superoxide...

Carboxyhemoglobin levels in an unstable hemoglobin disorder (Hb Zürich): effect on phenotypic expression.

The affinity of Hb Zurich for carbon monoxide is approximately 65 times that of normal hemoglobin. The carboxyhemoglobin content in serum from individuals with Hb Zurich ranged from 3.9 to 6.7 percent in nine nonsmokers and from 9.8 to 19.7 percent in six smokers. Rates of hemolysis and hemoglobin denaturation were less in smokers than in nonsmokers, effects that may be secondary to the stabilization of Hb Zurich by carbon monoxide.

Structure of carboxymyoglobin in crystals and in solution.

The configuration of the heme-carbonyl group upon binding of carbon monoxide to sperm whale myoglobin (Mb) in crystals is evaluated on the basis of infrared spectroscopic methods. Multiplets of the totally symmetric C-O stretching mode are observed for the heme-bound ligand near 1933, 1944, and 1967 cm-1, corresponding to three different heme-carbonyl conformers. Variations in the relative proportions of these conformers can be induced by incorporation of small fractions of metMb or deoxyMb into MbCO crystals. The configuration of the iron-carbonyl with respect to the immediate coordination environment of the heme iron is assigned for each v(CO) stretching frequency on the basis of a detailed comparison of the three-dimensional structures of the heme environments of MbCO, metMb, and deoxyMb defined by crystallographic methods. The structures of the three heme-carbonyl conformers account for the v(CO) infrared absorption bands that can be observed for MbCO in solution.