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High resolution proton nmr as indicator of a silent mutation in the heme cavity of a monomeric allo steric heme globin

High resolution proton nmr as indicator of a silent mutation in the heme cavity of a monomeric allo steric heme globin

European Journal of Biochemistry 136(1): 161-166

Comparison of the high-field 1H-NMR spectra of the met-cyano complexes of allosteric Hb III and IV of the insect larva Chironomus thummi thummi (CTT-III and CTT-IV) shows that, in addition to the molecular heterogeneity previously described for both Hb as due to heme orientational disorder, CTT-III but not CTT-IV exhibits a 2nd heterogeneity as evidenced by splitting of numerous signals. Reconstitution of CTT-III with modified hemes alters the populations of the 2 heme rotational isomers but leaves the secondary heterogeneity unaffected. This argues directly for locating this secondary heterogeneity solely within the polypeptide chain rather than the result of protein-heme interaction. This secondary heterogeneity found solely in Hb-III is assigned to a point mutation in the heme cavity, 57E6 (Ile/Thr); CTT-IV is chemically homogenous. The observation of significant hyperfine shift differences for the alternative substitution at 57E6, particularly for non-heme single-proton resonances thought to arise from distal residues, indicates some structural consequences in the heme cavity due to the point mutation. While a difference in the allosteric properties cannot be detected in that point mutation appears to leave the pK for the allosteric transition unaltered, subtle influences on the function of the protein in both affinity states cannot be ruled out at this time.

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