Title

1H NMR Study of the Molecular and Electronic Structure of Paramagnetic Iron Chlorin Complexes of Myoglobin: Dynamic Heterogeneity of the Heme Pocket

Document Type

Article

Publication Date

7-1-1992

Abstract

The iron complex of pyropheophorbide a methyl ester, 1, and 2-devinylpyropheophorbide a methyl ester, 2, have been reconstituted into equine myoglobin, Mb. The NMR spectra of the resulting protein complexes indicate that the chlorins are incorporated into the heme pocket in a manner similar to that of the native heme. The NMR spectra of the cyano-met Mb complex containing 1 and 2 are shown to be heterogeneous. In the complex of 1, three species could be identified and labeled A, B, and C with equilibrium ratios at 5 °C of 1.0:0.4:0.04, respectively. In the complex of 2, two species A, B in a ratio ~2:1 are detected. The equilibrium nature of the species was established by observing saturation transfer among the three species of 1 and dynamic collapse of the resonance from the two species of 2. Assignment of the resolved resonances of the cyano metMb complex of 1 on the basis of limited isotope labeling and steady-state NOEs reveals contact shift patterns for the chromophore that are essentially identical to those of the low-spin bis-cyano model complex for the two major species, while that of the minor species C reflects a significantly altered electronic structure. The unaltered contact shift pattern for species A and B upon incorporating 1 into the protein is attributed to the fact that the rhombic perturbations due to the protein and chromophore ring saturation are co-linear and support orientation for 1 in species A and B with the 7-propionate placed as in the native protein. Conversely, the strongly perturbed contact shift pattern of species C is interpreted as reflecting an orientation of 1 with the two rhombic perturbations at right angles, indicating that 1 in C is rotated by 90° from that of A or B. The rate of interconversion among the A, B, and C species is consistent with demonstrated cases of 90° “hopping” of the heme about intact His-Fe-CN bonds. The species A and B differ primarily in the vinyl contact shift, suggesting that the orientation of the vinyl is altered by differential interactions with the protein matrix. Comparison of the presently observed heterogeneity of iron chlorophyllide complexes of Mb is made with earlier reports on related Zn2+ or Mg2+ complexes. The high-spin ferrous deoxy Mb complexes of 1 and 2 reveal a significant decrease in the axial His ring NH contact shift relative to that for hemes. Partial assignments of resonances in the deoxy Mb complex of 1 reveal extensive ir spin delocalization into vacant π molecular orbitals of the chlorin. Thus a chlorin appears to serve as a better π acceptor in the high-spin ferrous states than a porphyrin. Moreover, the observations of both positive and negative delocalized π spin density can be used to rule out significant contributions to the orbital ground state from a doubly occupied dxy orbital. © 1992, American Chemical Society. All rights reserved.

Publication Source (Journal or Book title)

Journal of the American Chemical Society

First Page

6513

Last Page

6520

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