Computational analysis of the tryptophan cation radical energetics in peroxidase Compound I

Thomas L. Poulos,Vidhi C. Murarka,Jenny S. Kim

doi:10.1007/s00775-022-01925-8

Thomas L. Poulos, Vidhi C. Murarka

Open Access

https://doi.org/10.1007/s00775-022-01925-8

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Abstract

Three well-characterized heme peroxidases (cytochrome c peroxidase = CCP, ascorbate peroxidase = APX, and Leishmania major peroxidase = LMP) all have a Trp residue tucked under the heme stacked against the proximal His heme ligand. The reaction of peroxidases with H2O2 to give Compound I results in the oxidation of this Trp to a cationic radical in CCP and LMP but not in APX. Considerable experimental data indicate that the local electrostatic environment controls whether this Trp or the porphyrin is oxidized in Compound I. Attempts have been made to place the differences between these peroxidases on a quantitative basis using computational methods. These efforts have been somewhat limited by the approximations required owing to the computational cost of using fully solvated atomistic models with well-developed forcefields. This now has changed with available GPU computing power and the associated development of software. Here we employ thermodynamic integration and multistate Bennett acceptance ratio methods to help fine-tune our understanding on the energetic differences in Trp radical stabilization in all three peroxidases. These results indicate that the local solvent structure near the redox active Trp plays a significant role in stabilization of the cationic Trp radical.Graphical abstract

Highlights

Heme peroxidases like cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) are monomeric proteins consisting of ≈ 300 amino acids and a single heme coordinated to a His residue
In HRP the residue corresponding to Trp191 in CCP is a Phe, and it was initially postulated that the reason CCP forms a Trp cationic radical rather than a porphyrin radical is that Trp is easier to oxidize than Phe [6]
The one missing structure for our computational studies is the mutant of ascorbate peroxidase (APX) that contains the three Met residues found in CCP that are thought to help stabilize the Trp191 radical

Summary

Introduction

Heme peroxidases like cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) are monomeric proteins consisting of ≈ 300 amino acids and a single heme coordinated to a His residue. H2O2 oxidizing equivalents to oxidize either small organic molecules, as is the case with HRP, or cytochrome c, as with CCP. This is achieved by storing both H 2O2 oxidizing equivalents in the active site (Fig. 1). The crystal structure of the closely related ascorbate peroxidase (APX) [7] showed that APX has the same active site Trp as CCP yet APX forms a porphyrin radical [8]. The crystal structure [12] shows that LMP has the same active site Trp as CCP, the same cation site as APX, yet LMP forms a very stable Trp radical [13]. LMP differs from both APX and CCP in having a Cys residue very near the active site Trp and mutating this Cys to the corresponding residue in CCP, Thr, destabilizes the Trp radical [13]

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