FTIR ANALYSIS OF THE INTERACTION OF AZIDE WITH HORSE HEART MYOGLOBIN VARIANTS

The interaction of azide with variants of horse heart myoglobin (Mb) h as been characterized by Fourier transform infrared (FTIR), electron p aramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Clu/Lys63Glu) were included in this study. All variants, with th e exception of Val67Arg, have a lower azide affinity than the wild-typ e protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type protein and all variants exhib it a high-spin/low-spin equilibrium. Introduction of positively charge d amino acid residues shifts nu(max) for the low-spin form to higher e nergy while negatively charged residues shifted this maximum to lower energy. The low azide binding affinity exhibited by the His64Thr and H is64Ile variants is accompanied by a shift of the nu(max) for the low- spin infrared band to lower energy and by a significant increase in th e corresponding half-bandwidths. This observation indicates greater mo bility of the bound azide ligand in these variants. The His64Lys varia nt exhibits two infrared bands attributable to low-spin forms that are assigned to two different conformations of the lysyl residue. In one conformation, the lysine is proposed to form a hydrogen bond with the bound azide similar to that proposed to occur between the distal histi dine and bound azide, and in the other conformation no interaction occ urs.