Properties and Reactivity of Myoglobin Reconstituted with Chemically Modified Protohemin Complexes

Abstract

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.

Formation of reactive nitrogen species at biologic heme centers: a potential mechanism of nitric oxide-dependent toxicity.

“…In this case kinetic experiments carried out as for LPO and HRP show that the K M PhOH values are similar to those found for the corresponding catalytic oxidations of phenols in the presence of hydrogen peroxide (21). This indicates that the same binding site for the phenol is maintained in the two types of reactions.…”

Kinetic Analysis of Semisynthetic Peroxidase Enzymes Containing a Covalent DNA–Heme Adduct as the Cofactor

“…[53] This study also showed that the heme-binding pocket is not sufficiently large to entirely accommodate peptide-modified heme derivatives and the peptide-carrying propionate chains rather extend to the outside of the heme pocket. Therefore, we hypothesize that the increased reactivity of the MbD 2 , observed here, may result from steric constraints of the DNA strands leading to an opening of the active site, thereby facilitating an increased accessibility for substrate molecules.…”

“…Biphasic behavior of Mb catalysis was previously observed by Monzani and co-workers, and it was explained by two consecutive binding interactions between the enzyme and the substrate. [53] As shown in Scheme 1, both consecutive substrate binding interactions lead to the generation of product. The first reaction pathway (K D1 , k 1 ) is dominant at low sub- strate concentration.…”

pH-dependent redox and CO binding properties of chelated protoheme-l-histidine and protoheme-glycyl-l-histidine complexes

“…Complexes of this type have generally been used to investigate the ligand binding properties of the iron center, as peroxidase mimics as well as modified cofactors for reconstituted proteins [22][23][24]. To show the effect of strain in the axial ligand on the binding and catalytic properties of the iron center, a group of deuterohemins containing a histidine residue bound in a side arm with variable length were previously used [18].…”