Catalytic Activity, Stability, Unfolding, and Degradation Pathways of Engineered and Reconstituted Myoglobins

The structural and functional consequences of
engineering a positively charged Lys residue and
replacing the natural heme with a heme-L-His derivative
in the active site of sperm whale myoglobin (Mb) have
been investigated. The main structural change caused by
the distal T67K mutation appears to be mobilization of
the propionate-7 group. Reconstitution of wild-type and
T67K Mb with heme-L-His relaxes the protein fragment
around the heme because it involves the loss of the
interaction of one of the propionate groups which stabilize
heme binding to the protein. This modification
increases the accessibility of exogenous ligands or substrates
to the active site. The catalytic activity of the
reconstituted proteins in peroxidase-type reactions is
thus significantly increased, particularly with T67K Mb.
The T67K mutation slightly reduces the thermodynamic
stability and the chemical stability of Mb during catalysis,
but somewhat more marked effects are observed by
cofactor reconstitution. Hydrogen peroxide, in fact, induces
pseudo-peroxidase activity but also promotes
oxidative damage of the protein. The mechanism of
protein degradation involves two pathways, which depend
on the evolution of radical species generated on
protein residues by the Mb active species and on the
reactivity of phenoxy radicals produced during turnover.
Both protein oligomers and heme-protein cross-links
have been detected upon inactivation.