| The enzyme cytochrome c peroxidase (CcP) catalyzes the reduction of peroxides by cytochrome c. As part of this process, the oxidized form of CcP, known as compound I, stores two oxidizing equivalents: one in the form of an oxyferryl (Fe4+=O) heme, and the other as a protein-based cation radical located on the indole sidechain of tryptophan 191. The electron transfer pathway between the indole and the Fe center is unknown and has been the focus of this research project. Electron transfer may occur via the hydrogen-bonded network that links Trp191 to the axial Fe ligand His175 through a bridging aspartate (Asp235). Alternatively, electron transfer may occur via the π orbitals of the Trp191 indole and the His 175 imidazole; these aromatic rings are parallel and in van der Waals contact, allowing excellent π-orbital overlap.; In a collaboration with the Thomas L. Poulos lab, the site-directed suppression mutagenesis system has been employed to introduce noncoded amino acid analogs of tryptophan at position 191 of CcP. These analogs include isotryptophan, in which the indole attaches to the β carbon at the ring nitrogen, 2-methylisotryptophan, and 1(N)-methyltryptophan. In all three cases, the hydrogen bond between Asp235 and the indole is essentially eliminated with minimal steric change. The π-stacking arrangement between the indole and the His175 imidazole is believed to be retained, based upon analogy to a Trp191 → Phe mutant (Wang, et al., Biochemistry 1990, 29, 7160).; The CcP mutants containing tryptophan analogs are unable to catalyze the steady-state oxidation of hydrogen peroxide by cytochrome c even though they are produced in sufficient quantities that any such activity should be detected in the functional assay. This result is consistent with the idea that the hydrogen-bonded pathway is necessary for electron transfer from Trp191 to the Fe center. The small differences in the oxidation portentials of the analogs and tryptophan (<200 mV) are unlikely to affect the overall thermodynamic driving force of the reaction. However, it is possible that the stability of the π cation radical is delicately balanced and is compromised sufficiently by small changes in redox potential or geometry to eliminate the cytochrome c oxidation measured in the assay. |
