Role of Electrostatics and Salt Bridges in Stabilizing the Compound I Radical in Ascorbate Peroxidase

Abstract

Cytochrome c (CcP) and ascorbate peroxidase (APX) are heme peroxidases which have very similar active site structures yet differ substantially in the properties of compound I, the intermediate formed upon reaction with peroxides. Although both peroxidases have a tryptophan in the proximal heme pocket, Trp191 in CcP and Trp179 in APX, only Trp191 in CcP forms a stable cation radical while APX forms the more traditional porphyrin pi-cation radical. Previous work [Barrows, T. P., et al. (2004)Biochemistry 43, 8826-8834] has shown that converting three methionine residues in the cytochrome c peroxidase (CcP) proximal heme pocket to the corresponding residues in APX dramatically decreased the stability of the Trp191 radical in CcP compound I. On the basis of these results, we reasoned that replacing the analogous residues at positions 160, 203, and 204 in APX with methionine should stabilize a Trp179 radical in APX compound I. Steady- and transient-state kinetics of this mutant (designated APX3M) show a significant destabilization of the native porphyrin pi-radical, while electron paramagnetic resonance (EPR) studies show an increase in the intensity of the signal at g = 2.006 with characteristics consistent with formation of a Trp radical. This hypothesis was tested by replacing Trp179 with Phe in the APX3M background. The EPR spectrum of this mutant was very similar to that of the CcP W191G mutant which is known to form a tyrosine radical. Previously published theoretical studies [Guallar, V., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 6998-7002] suggest that electrostatic shielding of the heme propionates also plays a role in the stability of the porphyrin radical. Arg172 in APX hydrogen bonds with one of the heme propionates. Replacing Arg172 with an asparagine residue in the APX3M background generates a mutant which no longer forms the full complement of the compound I porphyrin pi-radical. These results suggest that the electrostatics of the proximal pocket and the shielding of propionate groups by salt bridges are critical factors controlling the location of a stable compound I radical in heme peroxidases.

QM/MM methods: Looking inside heme proteins biochemisty

“…In the APX enzyme the unpaired electron is more delocalized, with a main contribution in the porphyrin ring but with a small component in the proximal Trp and a minimal one in the distal Trp. The theoretical spin densities obtained largely agree with the experimental knowledge of the spin distribution for these three systems [77,79].…”

Crystal Structure of Leishmania major Peroxidase and Characterization of the Compound I Tryptophan Radical

“…Our laboratory has engineered the APX K ϩ binding site into CCP to give the mutant termed CCPK1. The presence of this (11). Like CCP, changing the proximal Trp to Phe in LmP greatly decreased the cytochrome c peroxidase activity (31), suggesting that LmP might also form a Trp radical.…”

“…Where the radical is situated in the mutants remains unknown. However, mutagenesis studies of CCP where the Trp 191 Compound I radical no longer forms indicates that tyrosine(s) serves as electron donor(s) in forming Compound I and that the weak EPR signal in the CCP mutants is due to residual partial spin remaining on an unstable Tyr radical (5,11,34). The similarity in the EPR signals of the LmP mutants suggests that Tyr is the most likely alternate electron donor which is much less stable than the Trp radical in wild type LmP.…”

“…Our group has shown that engineering the APX K ϩ site into CCP destabilizes the Trp 191 radical in a cationic concentration-dependent manner and decreases activity to about ϳ0.02% of wild type levels (5-7). Other studies illustrated that the electronegative amino acids unique to CCP, Met 230 and Met 231 , near Trp 191 are also important for stabilization of the Trp radical (5,11,12).…”

Mapping protein electron transfer pathways with QM/MM methods

“…Additional evidence of the importance of the propionates in APX comes from mutation studies (Barrows & Poulos 2005).…”