Enhancement of enzymatic activity for myoglobins by modification of heme-propionate side chains

Abstract

The modification of myoglobin is an attractive process not only for understanding its molecular mechanism but also for engineering the protein function. The strategy of myoglobin functionalization can be divided into at least two approaches: site-directed mutagenesis and reconstitution with a non-natural prosthetic group. The former method enables us to mainly modulate the physiological function, while the latter has the advantage of introducing a new function on the protein. Particularly, replacement of the native hemin with an artificially created hemin having hydrophobic moieties at the terminal of the heme-propionate side chains serves as an appropriate substrate-binding site near the heme pocket, and consequently enhances the peroxidase and peroxygenase activities for the reconstituted myoglobin. In addition, the incorporation of the synthetic hemin bearing modified heme-propionates into an appropriate apomyoglobin mutant drastically enhances the peroxidase activity. In contrast, to convert myoglobin into a cytochrome P450 enzyme, a flavin moiety as an electron transfer mediator was introduced at the terminal of the heme-propionate side chain. The flavomyoglobin catalyzes the deformylation of 2-phenylpropanal in the presence of NADH under aerobic conditions through the peroxoanion formation from the oxygenated species. In addition, modification of the heme-propionate side chains has an significant influence on regulating the reactivity of the horseradish peroxidase. Furthermore, the heme-propionate side chain can form a metal binding site with a carboxylate residue in the heme pocket. These studies indicate that modification of the heme-propionate side chains can be a new and effective way to engineer functions for the hemoproteins.

The role of the heme propionates in heme biochemistry

“…myoglobins. A propionate-flavin reconstituted hemin, for example, showed for the first time a ferric peroxoanion intermediate in myoglobin [36]. In a similar fashion, early work by Ghosh et al [37] showed a mixing of spin densities between porphyrin and aryl derivatives orbitals.These studies indicate that modification of the heme-propionate side chains with electron rich groups can be a new and effective way to engineer functions for the hemoproteins.…”

“…This interaction is analogous to that of Asp297 in CYP101 (further discussed below). Direct chemical modification of the propionate groups by Hayashi et al [34] has shown enhanced ligand dissociation and peroxidase (and peroxygenase) activities [35,36] for the reconstituted Fig. 1.…”

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

“…In view of the increasing focus on heme propionate modification as a means of achieving specific chemical derivatization of myoglobin in the development of new chemical properties [60][61][62][63][64][65][66] and the study of electron transfer reactions with other proteins [67,68], the structural and functional consequences of such modifications to the protein that may be induced at positions remote from the site of chemical modification may merit further consideration. Table 1 pK a values for His residues of native and DME-metheme substituted horse heart Mb determined by 1 (3) The assignments of the peaks labeled in Fig.…”