Nuclear Magnetic Resonance Study of the Molecular and Electronic Structure of the Stable Green Sulfhemin Prosthetic Group Extracted from Sulfmyoglobin

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The 1H NMR spectral characteristics of the stable green heme extract sulfhemin C from the terminal alkaline equilibration product of sulfmyoglobin have been investigated in order to completely define its molecular structure and to shed light on the nature of metal-prosthetic group π-bonding as reflected in the contact shift pattern in paramagnetic ferric complexes. A complete stereospecific assignment of the 1H NMR spectrum of the low-spin dicyano complex was effected by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic dipolar relaxation, and nuclear Overhauser effect, which confirmed that all functional groups of the precursor hemin are retained with the exception of the saturation of pyrrole B to form a cyclic thiolene. The assignments depended upon the use of a viscous solvent to render NOEs detectable, but it is shown that the combination of NOEs and metal-centered relaxation is sufficient to yield assignment without recourse to isotope labeling, opening the possibility for assignment of the spectra of natural low-spin ferric chlorin complexes. The dicyanosulfhemin C contact shift pattern is shown to reflect primarily π-bonding with the highest filled π MO of the chlorin, as found previously for hemin, except that its metal spin is symmetry-restricted to interacting solely with the pyrroles cis to the saturated ring. The raising of the irond orbital degeneracy by the ring B saturation provides the explanation of why low-spin ferric sulfhemin C, in contrast to hemin, experiences a negligible perturbation of the contact shift pattern upon incorporation into a protein and why the contact shift pattern does not uniquely identify the saturated pyrrole in a chlorin. The assignment by isotope labeling of the peripheral methyl signals of the high-spin ferric bis(dimethyl sulfoxide) complex of sulfhemin C confirms an electronic structure very similar to that observed upon incorporation into a protein matrix. We also confirm that the unique and sharply attenuated 3-methyl contact shift identifies the saturated ring as pyrrole B and that the assignment of high-spin ferric chlorin peripheral methyl signals will yield the identity of the saturated pyrrole(s). © 1988, American Chemical Society. All rights reserved.

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Journal of the American Chemical Society

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