1H NMR Hyperfine Shift Pattern as a Probe for Ligation State in High-Spin Ferric Hemoproteins: Water Binding in Metmyoglobin Mutants

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The 1H NMR spectra of a series of high-spin ferric metmyoglobins (metMbs) have been analyzed to assess the validity of meso proton (meso-H) contact shift direction as a probe for H2O coordination and to develop a quantitative interpretative basis of the hyperfine shift pattern as a structural probe for the heme-iron ligation state. The quantitative analyses of the hyperfine shifts are based on a comparison of the structurally characterized six-coordinate sperm whale metMbH2O and the five-coordinate Aplysia metMb. The developed spectral probes are subsequently applied to elucidate the role of distal residues in modulating H2O coordination in a series of E7 and El 1 point mutants of sperm whale Mb. The elusive distal El 1 residue signals are assigned in the two reference proteins on the basis of their unique relaxation properties and from the spectral characteristics of El 1 sperm whale Mb point mutants. Quantitative analysis of the El 1 residue dipolar shifts demonstrates that the loss of coordinated H2O leads to a substantial increase in the zero-field splitting constant, D. The change from strongly low-field meso-H shifts in six-coordinate sperm whale metMbH2O to strongly upfield meso-H shifts in five-coordinate Aplysia metMb is accompanied by predicted changes in chemical shifts for heme methyl, F8 His CβH, and El 1 Val methyl protons. The most readily recognized change is the 5-ppm low-field bias of the mean methyl shift upon loss of water coordination. The consistency in the changes of all the NMR spectral parameters supports the use of the meso-H chemical shifts as probes for ligation state but suggests that all accompanying changes should be analyzed. Substitution of El 1 Val by lle, Phe, or Ala results in minimal perturbation with full retention of coordinated water. Substitution of E7 His by Val or Phe abolishes H2O coordination, while replacement by Gin or Gly leads to a fractional H2O coordination. The sensitivity of the hyperfine shifts of the heme methyl protons to solvent isotope composition supports the proposed changes in H2O ligation. The application of the heme mean methyl shifts as a probe for H2O coordination in a series of natural genetic variants differing in E7 residue confirms previous conclusions, except for elephant metMb (E7 Gin) which is concluded here to be primarily five-coordinate rather than six-coordinate. The occupation of the sixth position by water as a function of E7 residue is found to be very similar in sperm whale E7 point mutants and natural genetic variants, and it is concluded that H bonding by E7 residue is the strongest but not the only stabilizing influence on H2O coordination. © 1991, American Chemical Society. All rights reserved.

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Journal of the American Chemical Society

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