Proton NMR study of model substrate binding in hemoproteins. Intercalation of mercuric triiodide in sperm whale met-aquo myoglobin.

1H-NMR spectra have been recorded for sperm whale met-aquo myoglobin intercalated with xenon, cyclopropane, mercuric triiodide and auric triiodide. All four agents are known to intercalate on the proximal side of the heme over pyrrole A. The complexes of xenon and cyclopropane exhibit insignificant shifts for all four heme methyls, suggesting that these species fit into an existing hole without causing significant perturbations on the heme cavity. Mercuric and auric triiodide, on the other hand, induce substantial changes in the hyperfine-shifts for the heme methyls. Based on the previously assigned methyl peaks in met-aquo myoglobin, we find that methyl-1, closest to the intercalating agent, is affected most, with the influence decreasing with distance from the binding site. These results indicate that determination of the relative perturbations on the assigned heme methyl shifts due to substrate binding can be utilized to ascertain the substrate-heme stereochemistry in high-spin ferric hemoproteins such as peroxidases.

Heme methyl hyperfine-shifted nuclear magnetic resonance peaks assigned by selective deuteration as indicators of heme-protein interactions in metmyoglobins.

The heme methyl resonances in a variety of high-spin, low-spin and spin-equilibrium forms of sperm whale metmyoglobin have been assigned by reconstituting myoglobin with selectively deuterated hemes. Two patterns for the heme methyl hyperfine shifts are observed, one characteristic of the low-spin state and the other typical of the high-spin state. The two protein forms which can change the position of their spin equilibrium significantly with changing temperature exhibit the pattern of the dominant spin state component at any temperature. The different hyperfine shift patterns for the low-spin and high-spin states are concluded to arise not from different heme-protein contacts in the two spin states, but from characteristic differential sensitivities of the dominant spin transfer mechanisms to the same rhombic perturbation.

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.

Proton nuclear magnetic resonance characterization of heme disorder in hemoproteins.

A proton NMR method is described for determining the orientation of a porphyrin within the heme pocket of a hemoprotein. The pattern of the hyperfine-shifted heme methyl resonances in low-spin ferric model compounds is demonstrated to characteristically reflect the position of a localized low-symmetry perturbation on the pi system. The specific assignments via deuteration of the two interconvertible sets of methyl resonances observed for deuteroporphyrin-reconstituted sperm whale metmyoglobin cyanide lead to the conclusion that the low-symmetry perturbations on the heme due to the apo-protein contacts differ for the two protein components by a 180 degrees rotation about the alpha-gamma meso axis. Hence the heme in the reconstituted myoglobin is "disordered" in solution, and the altered functional properties of the reconstituted protein cannot be simply attributed to the local effect of the heme substituent. This NMR technique has applicability for determining the relative heme orientation in related hemoproteins, and may clarify the origin of doubling of heme resonances observed in several native hemoproteins.

Proton NMR study of the influence on iron oxidation/ligation/spin state on the heme orientational preference in myoglobin.

Proton nuclear magnetic resonance spectroscopy has been utilized to demonstrate that the degree of heme orientational disorder within a given myoglobin protein matrix can be a sensitive function of the oxidation/ligation/spin state of the heme iron. For sperm whale deuterohemin-reconstituted myoglobin, the equilibrium was found to strongly favor (5.7 to 7.8 kJ/mol) the X-ray characterized heme orientation in all six-coordinate states, but with a considerable reduction in preference (to 1.6 kJ/mol) in the five-coordinate deoxy state. In native yellow fin tuna myoglobin, changes in heme orientational preferences of approximately 3 kJ/mol occur even between two six-coordinate ferric states differing solely in spin states.

Proton nuclear magnetic resonance investigation of the nature of solution conformational equilibria of monomeric insect deoxyhemoglobins.

The proton nuclear magnetic resonance spectra of the three monomeric deoxyhemoglobins of the insect larva Chironomus thummi thummi have been recorded, assigned, and analyzed. In the two allosteric hemoglobins, the heme methyls and vinyl protons were assigned by specific deuterium labeling. The hyperfine-shifted proximal histidyl imidazole exchangeable protons for the three native and two deuteroheme-reconstituted hemoglobins were assigned by comparison of spectra in H2O and 2H2O. Both native and reconstituted allosteric hemoglobins exhibit two sets of interconvertible resonances indicative of two heme orientations differing by a 180 degrees rotation about the alpha-gamma-meso axis, as previously found for the met-cyano analogues [La Mar, G. N., Smith, K. M., Gersonde, K., Sick, H., & Overkamp, M. (1980) J. Biol. Chem. 255, 66]. The relative pH sensitivities of the heme resonance hyperfine shifts for the two allosteric hemoglobins and the apparent pK approximately 8 indicate that the t in equilibrium r allosteric transition, as modulated by the Bohr proton, is being observed. For the native hemoglobins, the t in equilibrium r conformational transition was found to be centered at the heme periphery, with the proximal histidyl imidazole environment insensitive to both pH and the rotational position of the heme, consistent with the absence of a pH influence on the ligation on-rate. For the deuteroheme-reconstituted allosteric hemoglobins, both the heme and axial imidazole environments sense the t in equilibrium r transition, and the histidine environments for the two components for each hemoglobin can be clearly distinguished, suggesting that the ligation on-rates may depend on both pH and heme orientation.