Hydrogen isotope effects on the proton nuclear magnetic resonance spectrum of bovine ferricytochrome b5: Axial hydrogen bonding involving the axial His-39 imidazole ligand

Abstract

The potential role of hydrogen bonding interactions in modulating the molecular and electronic structure of the active site of solubilized bovine ferricytochrome b 5 has been investigated by monitoring solvent isotope effects on proton-NMR spectral parameters. It is observed that the hyperffine shifts of both the heme prosthetic group and one coordinated His are sensitive, while those for the other axial His and non-coordinated residues are insensitive, to 2H for 1H exchange. Two types of isotope influences are characterized; one whose chemical shift influence is time-resolved on the NMR rime scale, and involves a single proton on one axial ligand, and a second effect which involves multiple protons, is not time resolved, and influences primarily the heme. A large isotope effect on the hyperfine shift is identified for the C β H signals of His-39 but not His-63. The exchangeable ring NH of His-39 is assigned, and the pH influence on the exchange properties of heme pocket labile protons, when compared to the rate of base catalyzed averaging of the His-39 C β H isotope effect, lead to the conclusion that the axial hydrogen bond which is responsible for this isotope effect is that between His-39 ring NH and Gly-42 carbonyl. The more rapid exchange of labile protons with solvent for His-63 than His-39 confirms a less solvent accessible and stronger hydrogen bonded His-39 than His-63. The stronger His-39-Gly-42 than His-63-Phe-58 hydrogen bond involving the ring NH leads to more extensive His-39 imidazolate character and hence a stronger iron-His-39 than iron-His-63 bond. The much larger hyperfine shifts for His-39 than His-63 imidazole ring non-labile protons support the stronger bonding of the former ligand, and account for the orientation of the rhombic magnetic axes by His-39 rather than His-63. The solvent isotope effect on the heme leads to rotation of the prosthetic group about the His-Fe-His bond by ≈ 0.5° so as to shorten the 7-propionate link to Ser-64. This suggests that the hydrogen bonds between the 7-propionate group and Ser-64 are responsible for the effect.