David L. Budd

Assignment of proximal histidine proton nmr peaks in myoglobin and hemoglobin

Summary The proton nmr spectra of model compounds of deoxy myoglobin and hemoglobin have yielded the assignment of the axial imidazole resonances. The information leads directly to an assignment of all three proximal histidyl imidazole proton resonances in sperm whale deoxy myoglobin, and the two non-equivalent proximal histidyl imidazole NH signals of the α and β chains in deoxy hemoglobin A. The NH peaks are expected to serve as valuable probes for the T ⇌ R transition in hemoglobins.

Assignment of proximal histidyl imidazole exchangeable proton NMR resonances to individual subunits in hemoglobins A, Boston, Iwate and Milwaukee☆

Abstract The proton nmr spectra of the synthetic valency hybrids, α 2 (β + CN) 2 , (α + CN) 2 β 2 of hemoglobin A and the natural valency hybrids of the mutant hemoglobins Boston, Iwate and Milwaukee have led to the unambiguous assignment of the two proximal histidyl imidazole exchangeable proton signals at 64 and 76 ppm to individual α and β subunits, respectively. New single non-exchangeable proton resonances detected in the extreme downfield region of the spectra of Hbs Boston and Iwate are tentatively assigned to the coordinated tyrosine of the mutated α chains.

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.

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.

Carbon-13-iron-57 spin coupling as a new structural probe on hemoproteins. Carbon-13 NMR spectrum of iron-57-enriched carbonyl myoglobin.

Abstract Carbon-13 Fourier transform nmr has been used to make the first observation of a carbon-13-iron-57 spin-spin coupling constant in a protein, sperm whale carbonyl myoglobin enriched to 90% in both iron-57 and carbon-13. The coupling constant, 27.1±0.2 Hz, is found to be essentially identical to that of a model compound, supporting the view that the carbonyl is not tilted with respect to the heme plane in solution. Such carbon-13-iron-57 couplings, and the resultant iron-57 chemical shifts obtained from decoupling experiments, should provide valuable new tools for studying the different affinity states of tetrameric hemoglobins.

13C nuclear magnetic resonance spectra of aflatoxin B1 derived from acetate

Abstract The labeling distribution of the fungal metabolite, aflatoxin B 1 , produced from [1- or 2- 13 C] acetate was determined by 13 C FT NMR. The results support the polyketide hypothesis for aflatoxin biogenesis.

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 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.