HYDROGEN ISOTOPE EFFECTS ON THE PROTON NUCLEAR-MAGNETIC-RESONANCE SPECTRUM OF BOVINE FERRICYTOCHROME B(5) - AXIAL HYDROGEN-BONDING INVOLVING THE AXIAL HIS-39 IMIDAZOLE LIGAND

The potential role of hydrogen bonding interactions in modulating the molecular and electronic structure of the active site of solubilized b ovine ferricytochrome b(5) has been investigated by monitoring solvent isotope effects on proton-NMR spectral parameters. It is observed tha t the hyperfine shifts of both the heme prosthetic group and one coord inated His are sensitive, while those for the other axial His and non- coordinated residues are insensitive, to H-2 for H-1 exchange. Two typ es of isotope influences are characterized; one whose chemical shift i nfluence is time-resolved on the NMR time scale, and involves a single proton on one axial ligand, and a second effect which involves multip le protons, is not time resolved, and influences primarily the heme. A large isotope effect on the hyperfine shift is identified for the Cbe taH signals of His-39 but not His-63. The exchangeable ring NH of His- 39 is assigned, and the pH influence on p the exchange properties of h eme pocket labile protons, when compared to the rate of base catalyzed averaging of the His-39 CbetaH isotope effect, lead to the conclusion that the axial hydrogen bond which is responsible for this isotope ef fect is that between His-39 ring NH and Gly-42 carbonyl. The more rapi d exchange of labile protons with solvent for His-63 than His-39 confi rms 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 i nvolving the ring NH leads to more extensive His-39 imidazolate charac ter and hence a stronger iron-His-39 than iron-His-63 bond. The much l arger hyperfine shifts for His-39 than His-63 imidazole ring non-labil e protons support the stronger bonding of the former ligand, and accou nt for the orientation of the rhombic magnetic axes by His-39 rather t han His-63. The solvent isotope effect on the heme leads to rotation o f the prosthetic group about the His-Fe-His bond by approximate to 0.5 degrees so as to shorten the 7-propionate link to Ser-64. This sugges ts that the hydrogen bonds between the 7-propionate group and Ser-64 a re responsible for the effect.