| Summary: | Site-directed mutagenesis has been used to construct variants of horse heart myoglobin to
probe the structural contributions of specific amino acid residues in the active site of this protein. In
particular four groups of variants were examined: (i) The proximal histidine (H93) ligand to the heme
iron was substituted with tyrosine and cysteine as occurs in the coordination environments of catalase
and cytochrome P-450, respectively, (ii) The distal histidine (H64) was substituted with valine and
isoleucine to assess the effects of nonpolar residues on the coordination and ligand binding properties
of myoglobin, (iii) Double variants, H64V/H93C and H64I/H93C, were also constructed to study the
influence of these substitutions on the ability of the proximal cysteine ligand to ligate to the heme
iron, (iv) Insight concerning the functional consequences of increasing the polarity of the distal heme
binding pocket was sought through investigation of the V68H and V67A/V68S variants. The
influence of mutations on the proximal and distal side of the heme were examined by spectroscopic
and electrochemical methods.
The electronic, EPR and NMR spectra of the H93 Y ferriMb derivative suggest that the axial
ligation of the heme iron in this variant is pentacoordinate, with the heme iron coordinated by a
phenolate group provided by the proximal Y93 residue. Unlike the H93Y variant, however, no
experimental conditions were identified that allowed quantitative ligation of the proximal cysteine
residue to the heme iron in the H93C variant. All of the spectroscopic evidence for the H64V/H93C
and H64I/H93C variants, however, supports the assignment of thiolate as the ligand to the heme iron
in the oxidized forms of these proteins. With the exception of an additional high-spin component in
the electronic absorption spectrum, the V68H Mb variant exhibits absorption maxima similar to those
of ferricytochrome b5.
Electronic absorption, MCD and EPR spectroscopy of the double variant, V67A/V68S,
confirm that the coordination environment of this protein is the same as that of wild-type Mb.The increase in active site polarity may account for the enhancement in the rate of coupled oxidation of
heme exhibited by the variant upon aerobic exposure to ascorbate relative to that observed for wildtype
myoglobin. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate
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