Nuclear magnetic resonance studies of hemoprotein Proton hyperfine shifts and structural characterization of the different heme environments in methemoglobin and metmyoglobin

Abstract

The different features of the nature of heme iron-proximal histidine binding in methemoglobin and metmyoglobin were investigated by high resolution proton nuclear magnetic resonance spectroscopy at 220 MHz. From the comparison of the hyperfine-shifted heme methyl resonances of various methemoglobin and metmyoglobin derivatives it was revealed that the metHb derivatives exhibit smaller hyperfine shifts than the corresponding metMb derivatives. This observation was interpreted in terms of the stronger interaction between proximal histidine and ferric heme iron in methemoglobin than in metmyoglobin. The spectral contributions from the α and β subunits in methemoglobin in high spin state were successfully separated by the analysis of the temperature dependent heme methyl shifts of the methemoglobin azide complex which is in the high and low spin thermal spin equilibrium. It was shown that the β heme methyl resonances have smaller hyperfine shifts than the α heme ones in ferric high spin state. This tendency in methemoglobin derivatives was also interpreted in terms of the stronger iron-histidine binding in the β subunits than in the α subunits within the tetramer. From the comparisons between methemoglobin and metmyoglobin, and between the α and β subunits in methemoglobin, the order of the strength of the iron-histidine bond interaction was deduced as follows: metmyoglobin < α subunits in methemoglobin ⪅ β subunits in methemoglobin