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.

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The effect of strain in the axial coordination of imidazole to the heme has been studied in the chelate complexes deuterohemin-histidine (DH-His) and deuterohemin-alanylhistidine (DH-AlaHis). Molecular mechanics calculations indicate that three types of distortion of the axial ligand occur in DH-His, due to the relatively short length of the arm carrying the donor group: tilting off-axis, tipping, and inclination of the imidazole plane with respect to the axial Fe-N bond. The effects of tilting (Deltagamma approximately 10 degrees ) and inclination of the imidazole ring (Deltadelta approximately 17 degrees ) are dominant, while tipping is small and is probably of little importance here. By contrast, the axial imidazole coordination is normal in DH-AlaHis and other computed deuterohemin-dipeptide or -tripeptide complexes where histidine is the terminal residue, the only exception being DH-ProHis, where the rigidity of the proline ring reduces the flexibility of the chelating arm. The distortion in the axial iron-imidazole bond in DH-His has profound and negative influence on the binding and catalytic properties of this complex compared to DH-AlaHis. The former complex binds more weakly carbon monoxide, in its reduced form, and imidazole, in its oxidized form, than the latter. The catalytic efficiency in peroxidative oxidations is also reduced in DH-His with respect to DH-AlaHis. The activity of the latter complex is similar to that of microperoxidase-11, the peptide fragment incorporating the heme that results from hydrolytic cleavage of cytochrome c.

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