Theoretical investigation of a proposed [HIS 175-ASP 235-TRP 191] proton transfer system in cytochrome c peroxidase

Abstract

Peroxidases are oxidative metabolizing heme proteins, whereas myoglobin (Mb) with the same heme active site, a protoporphyrin IX with a proximal histidine and a water as axial ligands, belongs to the “globin” heme protein family that functions as reversible oxygen transfer agents. Thus, differences in function of these two heme proteins seems to be modulated by the amino acids surrounding the heme unit, specifically the presence of key polar residues on both the proximal and distal side in CCP that are absent in Mb. In the study presented here, we have investigated the plausibility of a key postulated transformation of the proximal imidazole ligand (His 175) to an imidazolate by proton transfer to a nearby aspartate (Asp 235) absent in Mb. The proton relay system studied included not only models for the His 175 and Asp 235 residues but also for a nearby Trp 191 residue that could also interact with Asp 235 through hydrogen bonding and polarization. Two semiempirical quantum mechanical methods, AM 1 and MNDO/H, with improved capabilities of describing H-bonded systems, were used to calculate the enthalpies of the three tautomeric forms of the proton relay system corresponding to the proton on the His, Asp, and Trp, respectively. These calculations were made for several models of the effect of the iron. Relative tautomeric enthalpies were calculated both with H-atom only optimization, keeping the heavy atoms fixed in their X-ray positions, and additional optimization that allowed the model Asp residue to relax. Transition-state enthalpies for the proton transfer from His to Asp were also calculated. The results of these studies suggest that the crucial postulated proton transfer from His to Asp is energetically favored, but only in the presence of the interaction of the iron with the imidazole ligand. Another stable form of the cluster, with competing proton transfer from the Trp to the Asp, was found only when the Asp position was allowed to optimize. Although no attempt was made in these studies to emulate the protein environment, the results obtained strongly support the hypothesis that an imidazolate could be the predominant species of the peroxidase heme unit.