Engineering Peroxidase Activity in Myoglobin: the Haem Cavity Structure and Peroxide Activation in the T67R/S92D Mutant and its Derivativere Constituted with Protohaemin-L-Histidine

Protein engineering and cofactor replacement have been
employed as tools to introduce/modulate peroxidase activity in
sperm whale Mb (myoglobin). Based on the rationale that haem
peroxidase active sites are characterized by specific charged
residues, the Mb haem crevice has been modified to host a haemdistal
Arg residue and a proximal Asp, yielding the T67R/S92D
Mb mutant. To code extra conformational mobility around
the haem, and to increase the peroxidase catalytic efficiency, the
T67R/S92D Mb mutant has been subsequently reconstituted with
protohaem-L-histidine methyl ester, yielding a stable derivative,
T67R/S92D Mb-H. The crystal structure of T67R/S92D cyanometMb
(1.4 Å resolution; R factor, 0.12) highlights a regular
haem-cyanide binding mode, and the role for themutated residues
in affecting the haem propionates as well as the neighbouring
water structure. The conformational disorder of the haem propionate-
7 is evidenced by the NMR spectrum of the mutant. Ligandbinding
studies show that the iron(III) centres of T67R/S92D Mb,
and especially of T67R/S92D Mb-H, exhibit higher affinity
for azide and imidazole than wild-type Mb. In addition, both
protein derivatives react faster than wild-type Mb with hydrogen
peroxide, showing higher peroxidase-like activity towards phenolic
substrates. The catalytic efficiency of T67R/S92D Mb-H
in these reactions is the highest so far reported for Mb derivatives.
A model for the protein–substrate interaction is deduced based on
the crystal structure and on the NMR spectra of protein–phenol
complexes.