Properties and reactivity of myoglobin reconstituted with chemically modified protohemin complexes

The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and pro tohemin-6(7)L-histidine methyl ester (HM-H) were prepared by condensation o f suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yieldin g the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectra l, binding and catalytic properties of Mb-RA and Mb-H are significantly dif ferent from those of Mb. As shown by MM and MD calculations, these differen ces are determined by some local structural changes around the heme which a re 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 gro up is bound to the Arg-Ala or His residue and is no longer available for el ectrostatic interaction with Lys45. The mobility of the peptide segment nea r the active site allows the distal histidine to come to a closer contact w ith 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 NM R spectra, that also show that each of them consists of a mixture of the tw o most stable isomers resulting from cofactor reconstitution, as also antic ipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their af finity is reduced. On the other hand, mobilization of the peptide chain aro und the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a r igid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron trans fer of phenolic substrates in peroxidase-type oxidations catalyzed by the r econstituted proteins in the presence of hydrogen peroxide.