Title

Proton NMR Study of the Heme Rotational Mobility in Myoglobin: The Role of Propionate Salt Bridges in Anchoring the Heme

Document Type

Article

Publication Date

1-1-1991

Abstract

The 1H NMR spectra of the metcyano complexes of the reconstruction products with sperm whale apomyoglobin with the four coprohemin-type isomers, members of the series (propionate)n(methyl)8-n porphine-iron(III) with n = 4 with one propionate on each pyrrole, have been recorded and analyzed. Three of the coprophemins reconstituted cleanly at acidic pH to yield holoproteins with molecular/electronic structures minimally perturbed from those of the native protein; coprohemin IV failed to exhibit detectable incorporation into the heme pocket. Nuclear Overhauser effect spectra provided the unique orientation of the three coprohemins in the pocket, with the nonnative protonated propionates preferring to replace one or both vinyls and tolerating the replacement of one, but not two, interior methyl groups within the folded holoprotein. At alkaline pH, dcprotonation of one of the interior propionates leads to spontaneous dissociation into hemin and apomyoglobin. The heme methyls of centrosymmetric coprohemin I and II complexes of sperm whale metMbCN, as well as the sperm whale and horse myoglobin complexes of centrosymmetric dipropionate, hexamethyl hemin, exhibited temperature-dependent saturation transfer among symmetry-related hemin methyls in the holoprotein that can be traced to rotational “hopping” of the hemin about an intact iron-His F8 bond. Measurement of the saturation factors and selective relaxation rates yields hopping rates of 1−10 s−1 at ambient temperatures. Quantitative analysis of variable temperature data for the coprohemin II complex yielded Ea ~ 17 kcal/mol and A ~ 6 × 1012 or ΔH‡ ~ 17 kcal/mol, ΔS ~ 0, indicating that the reorientation takes place within the folded holoprotein. The rate of rotational hopping of the dipropionate hemin was found ~3 times faster in horse than sperm whale metMbCN, indicating that the propionate residue CD3 salt bridge is more stable for the Arg CD3 in sperm whale than the Lys CD3 in horse myoglobin, which supports previous conclusions reached on the basis of labile proton exchange and preferential formation of propionate salt bridges in the two proteins. © 1991, American Chemical Society. All rights reserved.

Publication Source (Journal or Book title)

Journal of the American Chemical Society

First Page

1544

Last Page

1550

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