Role of electrostatics and salt bridges in stabilizing the compound I radical in ascorbate peroxidase.
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.
Barrows
Tiffany P
TP
Department of Molecular Biology and Biochemistry, Center in Chemical and Structural Biology, University of California, Irvine, California 92697, USA.
Poulos
Thomas L
TL
D001120:Arginine / Q000737:chemistry; D060387:Ascorbate Peroxidases; D001665:Binding Sites; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D009154:Mutation; D010544:Peroxidases / Q000737:chemistry*; D011166:Porphyrins / Q000737:chemistry*; D012492:Salts / Q000737:chemistry*; D055672:Static Electricity; D014364:Tryptophan / Q000737:chemistry
D005609:Free Radicals; D011166:Porphyrins; D012492:Salts; D014364:Tryptophan; D001120:Arginine; D008715:Methionine; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D003578:Cytochrome-c Peroxidase