Magnetic resonance studies of the binding of 13C-labeled carbon monoxide to myoglobins and hemoglobins containing modified hemes

Abstract

The effects of changes in the groups attached to the periphery of the porphyrin ring of the heme of various hemoglobin and myoglobins on the environment experienced by the ligand, carbon monoxide, have been studied by observation of the chemical shift of the bound 13CO. The results indicate that the major interaction between bound ligands and substituents around the porphyrin is that transmitted electronically from substituent to ligand. The nature of the protein environment around the ligand and the interaction between the proximal histidine (F8) and the ligand (through the iron atom) impose differences between subunits of hemoglobin and between myoglobins and hemoglobins which are largely, but not entirely, independent of these substituent effects. To assess the influence of protein structure on the chemical shifts of bound ligand, the shifts of 13CO bound to myoglobin and hemoglobins from a wide range of species have also been measured.

Recent developments in the 13 C NMR spectroscopic analysis of paramagnetic hemes and heme proteins

“…In the 1970s and 1980s the most common application of 13 C NMR spectroscopy to the analysis of heme proteins involved the characterization of resonances originating from the distal carbonmonoxy (CO) ligand of heme proteins coordinated by a 13 C-enriched CO molecule [46,47]. An early attempt to overcome the problems imposed by the low natural abundance of 13 C nuclei was to develop synthetic methods to introduce 13 C labels into the vinyl groups of the heme macrocycle [39,48].…”

“…These studies have typically been aimed at understanding the nature of the distal pocket in globins and other heme proteins [46,47,132,133]. Recently, the 57 Fe chemical shift has been found to correlate well with the C m chemical shift of CO complexes of several Fe(II) metalloporphyrins.…”

13C nuclear magnetic resonance studies of the binding of isocyanides to various hemoglobins and myoglobins

“…For example, the chemical shifts of the resonances of [l 3C°]EIC bound to these myoglobins are dependent on pH over a range of pH 6 to 10. (For l3CO bound to dolphin myoglobin a pH dependence between pH 5 and 7 has been reported by Moon et al. 1977.…”

“…Where such l3CO-[i3C°]EIC exchange experiments were performed (opossum, rabbit, and human hemoglobins), the downfield resonance of the hemoglobin saturated with EIC represented the isocyanide bound to the ß subunit. The signals corresponding to l3CO bound to the a and ß subunits of these hemoglobins have been previously determined (Moon & Richards, 1974;Moon et al, 1977). In all these cases, the resonance corresponding to EIC bound to the a subunit did not decrease in intensity until all the EIC was displaced from the ß chain by l3CO.…”

“…Previous results of l3CO bound to various hemoglobins (Moon & Richards, 1972bMoon et al, 1977) show that l3CO bound to the ß subunits of various hemoglobins exhibits resonances which have essentially the same chemical shift; in contrast l3CO bound to the a chains of these hemoglobins exhibit resonances which fall over a wider range. With [l3C°]EIC, the chemical shift range is much greater than with l3CO.…”