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-', 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 x(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 hemecarbonyl conformers account for the s(CO) infrared absorption bands that can be observed for MbCO in solution.The three-dimensional structures of carbon monoxide (CO)-liganded hemoglobins (Hbs) and myoglobins (Mbs) (1-5) exhibit a bent or tilted configuration of the CO ligand with respect to the porphyrin ring, whereas small-molecule model CO-liganded heme complexes (6, 7) exhibit a perpendicular linear (Fe-C-O) bonding structure. The origin of the different configuration in heme proteins is attributed primarily to nonbonding steric interactions of the axial ligand with nearby amino acid residues. The assumption is generally made that the heme-carbonyl configuration of heme proteins in crystals is identical to that in solution. It is well known, nonetheless, that the infrared (IR) absorption spectrum of sperm whale carboxymyoglobin (MbCO) in solution is indicative of two structurally distinguishable components (8, 9), although only one configuration is identified in the crystal by neutron diffraction dataIn recent studies of the polarized single crystal absorption spectra of Mb complexes (10, 11), we have detected differences in the configuration of the heme-carbonyl group induced by crystal environment and have demonstrated that the singlecrystal spectrum of MbCO formed under conditions comparable to those in neutron diffraction studies (3) is not quantitatively compatible with the spectrum of MbCO in solution. In this communication we report the results of investigations in which we have extended these observations by application of IR spectroscopic methods. We provide evidence on the basis of the IR absorption of MbCO in crystals that three structurally distinct heme-carbonyl conformers arising from changes in nearest neighbor intermolecular interactions in the crystal can be identified on the basis of the v(CO) stretching frequency.The structural origin of the v(CO) stretching frequencies indicative of each conformer is explained on the basis of the three-dimensional structure of sperm whale Mb (3, 12, 13) determined by crystallographic methods. The results suggest that structural changes in the immediate environment of the iron-carbonyl group ...

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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.

In hemoglobin Zürich the side chain of the distal arginine attaches itself to the propionate of the heme, leaving the heme pocket wide open, allowing sulfanilamides easy access to the iron, and doubling the partition coefficient between CO and O2. The replacement of the distal valine by alanine in hemoglobin Sydney leaves a large gap inside the heme pocket, which is partly filled by a water molecule bonded to the distal histidine. Hemoglobin Sydney has the same partition coefficient between CO and O2 as hemoglobin A.Replacement of the distal histidine increases the stretching frequency of CO linked to the beta heme by 6 cm-1, but replacement of the distal valine increases it by only 3 cm-1, but replacement of the distal histidine leaves the O-O stretching frequency unchanged.

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