Three variants of horse heart myoglobin (Mb) in which the proximal His93 residue has been replaced with a Cys residue have been constructed and studied by NMR, EPR, and MCD spectroscopy to evaluate the contributions of proximal and distal residues to the coordination environment of the heme iron in these proteins. Although no experimental conditions were identified that allowed quantitative ligation of the cysteine residue to the heme iron in the His93Cys variant, all of the spectroscopic evidence collected for the His93Cys/His64Ile and His93Cys/His64Val double variants supports the assignment of thiolate as the ligand to iron in the oxidized forms of these variants. The double metMb variants exhibit Soret maxima that are considerably blue-shifted, 1H NMR spectra with decreased mean methyl resonances, and EPR spectra with highly rhombic g values. These spectroscopic data for the Fe(III) variants resemble the corresponding properties reported for ferricytochrome P-450. The decrease in the reduction potential of the double variants by 280 mV relative to wild-type protein is also consistent with the low midpoint potential of cytochrome P-450. MCD spectroscopy of these variants confirms that the proximal cysteine residue is not bound in the reduced forms of these proteins and, in the case of the His93Cys variant, that the distal histidine is coordinated to the iron. Similar coordination environments were created in the ferrimyoglobin variants by cyanogen bromide modification, which resulted in cyanation of the sulfur atom and prevented the ligation of Cys93 to the heme iron.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of azide with variants of horse heart myoglobin (Mb) has been characterized by Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Glu/Lys63Glu) were included in this study. All variants, with the exception of Val67Arg, have a lower azide affinity than the wild-type protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type protein and all variants exhibit a high-spin/low-spin equilibrium. Introduction of positively charged amino acid residues shifts nu max for the low-spin form to higher energy while negatively charged residues shifted this maximum to lower energy. The low azide binding affinity exhibited by the His64Thr and His64Ile 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 the corresponding half-bandwidths. This observation indicates greater mobility of the bound azide ligand in these variants. The His64Lys variant 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 histidine and bound azide, and in the other conformation no interaction occurs.

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