EPR parameters of amino acid radicals in P. eryngii versatile peroxidase and its W164Y variant computed at the QM/MM level

Abstract

Quantum mechanics/molecular mechanics (QM/MM) methods, employing density functional theory (DFT), have been used to compute the electron paramagnetic resonance (EPR) parameters of tryptophan and tyrosyl radical intermediates involved in the catalytic cycle of Pleurotus eryngii versatile peroxidase (VP) and its W164Y variant, respectively. These radicals have been previously experimentally detected and characterized both in the two-electron and one-electron activated forms of the enzymes. In this work, the well-studied W164 radical in VP has been chosen for calibration purposes because its spectroscopic properties have been extensively studied by multifrequency EPR and ENDOR spectroscopies. Using a B3LYP/CHARMM procedure, appropriately accounting for electrostatic, such as hydrogen bonding, and steric environmental interactions, a good agreement between the calculated and measured EPR parameters for both radicals has been achieved; g-tensors, hyperfine coupling constants (hfcc) and Mulliken spin densities have been correlated to changes in geometries, hydrogen bond networks and electrostatic environment, with the aim of understanding the influence of the protein surroundings on EPR properties. In addition, the present calculations demonstrate, for VP, the formation of a neutral tryptophan radical, hydrogen bonded to the nearby E243, via a stepwise electron and proton transfer with earlier involvement of a short-lived tryptophan cationic species. Instead, for W164Y, the QM/MM dynamics simulation shows that the tyrosine oxidation proceeds via a concerted electron and proton transfer and is accompanied by a significant reorganization of residues and water molecules surrounding the tyrosyl radical.

Redox-Active Sites in Auricularia auricula-judae Dye-Decolorizing Peroxidase and Several Directed Variants: A Multifrequency EPR Study

“…The role of such hydrogen bonds in lowering the g x value is in agreement with previous works on tyrosine radicals: in particular, this effect has been evidenced also in the catalytic cycle of the P. eryngii VP variant following the Tyr164 protein radical formation 46,60 . On the other hand, Tyr147 in AauDyP is involved in a weaker hydrogen bond with solvent water molecules (1.890Å) (Fig.…”

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

“…A similar g-tensor anisotropy effect would be expected for a hydroxylated Trp radical because the radical spin density is typically delocalized over the entire indole ring (22)(23)(24)(25)(26)(27). Therefore, βTrp57-OH of preMADH would be expected to have a Δg value greater than that of Trp radicals and closer to that of Tyr radicals.…”

Crystallographic, Kinetic, and Spectroscopic Study of the First Ligninolytic Peroxidase Presenting a Catalytic Tyrosine

“…The rationale for this proposal is the higher redox potential of the tryptophanyl cation radical (ϳ1.15 V) as compared with its neutral form (ϳ1.01 V) (55) that would facilitate lignin oxidation. Proton abstraction during phenol oxidation is generally simultaneous to electron abstraction, and this has been shown for tyrosyl radical formation in the W164Y VP variant (53). However, spectroscopic studies suggested that under acidic conditions, a cation-like radical (or H-bonded neutral radical) is formed at the high redox potential short-lived Tyr-161 of photosystem II (56,57).…”

“…In the EPR spectrum of the tyrosyl radical from peroxideactivated T. cervina LiP*, the g x value with the acceptable variation (g x ϭ 2.0078 -2.0082) suggests the existence of a hydrogen bond with a length of 1.8 -2.0 Å (51) because there is a clear relationship between the g x value and the hydrogen bond strength (52). The recent quantum mechanics and molecular mechanics calculations on the VP W164Y variant have demonstrated that the proton transfer to the nearby Glu-243, during tyrosine oxidation, proceeds barrierless, directly yielding the Tyr-164 neutral radical, but the variant is inactive because reorganization of hydrogen interactions (by water molecules and neighbor residues) precludes an easy recovery of the proton by the tyrosine residue (53). In the crystal structure of T. cervina LiP*, the phenolic hydroxyl group of Tyr-181 forms hydrogen bonds with the backbone carbonyl of Val-336 (2.72 Å) and with Wat-26 (2.74 Å) that forms two other H-bonds to His-39 (2.65 Å) and heme internal propionate (2.82 Å).…”