Proton magnetic resonance investigation of the influence of quaternary structure on iron-histidine bonding in deoxyhemoglobins

Kiyoshi Nagai, Gerd N. La Mar, Thomas Jue, H. Franklin Bunn

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Abstract

The proton nuclear magnetic resonance (NMR) spectra of the separated deoxy chains of normal adult hemoglobin (Hb A), the deoxy form of chemically modified hem-oglobins, and Hb Kempsey [Asp-G1(99)β→Asn] have been investigated in H2O solution with emphasis on the hyperfine-shifted proximal histidyl imidazole exchangeable NδH as indicators of the iron-histidine interaction. While the NδH hyperfine shift was found to be sensitive to chain origin and subunit assembly, the likelihood of change in both electronic effects and various steric strains precludes interpretation of the NMR data for different proteins at this time. However, upon introduction of conformational perturbations for any given protein, correlations between changes in NMR spectral parameters and altered function are interpretable in terms of changes in iron-imidazole bonding. For the individual chains, the α NδH shift, as well as the oxygen affinity, is insensitive to forming the p-(hydroxymercuri)benzoate (pMB) adduct, while the β-chain NδH shift decreases by 10 ppm and its oxygen affinity is reduced 6-fold by the same reaction. Since the pMB binding site is much closer to the proximal histidine of the β chain than the α chain, the β NδH upfield shift reflects some strain in the iron-histidine interaction. In the R quaternary structure, the deoxy subunits of NES-des-Arg-(141α)-Hb, des-His(146β)-des-Arg(141α)-Hb, and des-Arg-(141α)-Hb exhibit NδH resonances at approximately 77 ppm for both α and β subunits. The R → T conversion is accompanied by a 10-14-ppm decrease in the hyperfine shift of the α subunit; the β subunit is essentially unaffected. The decreased NH hyperfine shift is consistent with the introduction of some strain into the iron-histidine bond of the α subunit during the R → T transition. The observed NδH shift change for the α subunit is comparable to that observed upon binding p-(hydroxymercuri)benzoate to the isolated β chain and hence can account for only a small change in the oxygen affinity. In light of the resonance Raman data on v(Fe-Nε), this study supports a dominant steric influence on iron-histidine bonding when Hb undergoes an R → T transition and argues against a major contribution of any electronic effect created by altered hydrogen bonding of NδH to a backbone carbonyl [Stein, P., Mitchell, M., & Spiro, T. G. (1980) J. Am. Chem. Soc. 102, 7795-7797]. In agreement also with the resonance Raman data [Nagai, K., Kitagawa, T., & Morimoto, H. (1980) J. Mol. Biol. 136, 271-289], the strain we observe is localized in the α subunit.

Original languageEnglish (US)
Pages (from-to)842-847
Number of pages6
JournalBiochemistry
Volume21
Issue number5
StatePublished - 1982

ASJC Scopus subject areas

  • Biochemistry

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