Properties and Reactivity of Myoglobin Reconstituted with Chemically Modified Protohemin Complexes

The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-
L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His
residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from
horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding
and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by
MM and MD calculations, these differences are determined by some local structural changes around the
heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing
the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the
reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available
for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows
the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an
equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole
ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of
them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also
anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide
can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of
the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor
molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the
distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and
electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins
in the presence of hydrogen peroxide