Stabilizing a radical at Trp 191


























How can one create a stable indolyl radical within a protein? - Place a Trp residue in a cation binding site!

The structure of CcP is designed to focus a substantial negative electrostatic potential upon the indole ring of Trp 191. Calculations based on Delphi (Honig) show that the indole ring of Trp 191 (blue) exists in a region of extreme negative electrostatic potential. The picture shows electrostatic potential contours at 15 (yellow), 20, 25, 27, and 30 (red) kcal/mol. When the indole ring is removed by a Trp 191 -> Gly mutation, not only does indole refuse to occupy this cavity, but a potassium ion (yellow sphere) occupies the cavity instead. A tris cation can also occupy this space. Melissa Fitzgerald, along with Dave Goodin, found that an imidazole cation would readily bind in this cavity as well. Closer inspection shows that the structure in this region corresponds to that observed at alkalai cation binding sites of other proteins. Placing Trp 191 in this structure will destabilize ground state indole, and stabilize a cation radical on the indole ring.

Several features contribute to the negative electrostatic potential in this region. Part of the potential is contributed by the carboxylate side chain of Asp 235 (green) which interacts with NE of Trp 191 (Poulos et al., 1980). This interaction is crucial for formation of the Trp 191 radical (Fishel et al., 1990). Although necessary, this interaction is not sufficient. A very similar Asp - Trp interaction is found in ascorbate peroxidase, which does not form a stable indolyl radical. Additional structural features that contribute to local negative electrostatic potential include: dipole interactions from the carbonyl oxygen atoms of residues 175 and 177 (white), the negative dipole at the C- terminus of the helix that terminates at His 175 (not shown), and the carboxylate of heme pyrrole IV.


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