Energetics of Cation Radical Formation at the Proximal Active Site Tryptophan of Cytochrome-c-Peroxidase and Ascorbate Peroxidase

Despite very similar protein structures, ascorbate peroxidase (APX) and yeast cytochrome-cperoxidase (CCP) stabilize different radical species during enzyme turnover. Both enzymes contain similar active site residues, including the tryptophan that is oxidized to a stable cation radical in CCP. However, the analogous trytophan is not oxidized in APX, and the second oxidizing equivalent is retained as a porphyrin 7t-cation radical. In this study, we provide an improved computational approach to estimate the contribution of solvent and protein electrostatics to the energetics of tryptophan cation radical formation in the two enzyme environments. The Protein Dipoles Langevin Dipoles (PDLD) model is combined with molecular dynamics to estimate the role of discrete solvation, atomic polarizabilities, and dynamic motional averaging on the electrostatic potentials. The PDLD model shows that the protein environment of CCP stabilizes the tryptophan cation radical by 330 mV relative to that in APX. Analysis of the components contributing to this difference supports proposals that the cation binding site contributes to, but is not the sole cause of, the different sites of radical stabilization. The enzymes have thus evolved this distinction using several contributing interactions including the cation binding site, solvent access, and subtle differences in protein structure and dynamics.