Protein engineering of cytochrome c peroxidase in quest of novel activities in CcP

In an attempt to determine factors required for substrate specificity and formation of a porphyrin radical in ascorbate peroxidase (APX), our laboratory analyzed the structural homology between APX and cytochrome c peroxidase (CcP) and used protein engineering to design the ascorbate-binding loop responsible for native substrate binding in APX into CcP. Various mutants were created to contain the ascorbate-binding loop along with amino acid residues important in substrate binding and stabilization necessary for catalysis. High resolution data at 1.06 A revealed that when superimposed onto APX, the engineered loop region is virtually identical to that of APX with critical residues involved in ascorbate binding and stability, in the same orientation as in APX.;Initial transient-state kinetics failed to demonstrate formation of a porphyrin radical in the mutants, thereby, suggesting that there are other factors other than the substrate binding region necessary for porphyrin radical formation. Upon this discovery, we used site-directed-mutagenesis to add a W191F mutation onto the already mutated proteins since work in other labs had shown that the W191F mutant forms a porphyrin pi-cation radical. EPR results revealed a tyrosyl radical suggesting that the radical moves from the porphyrin to the tyrosine. Transient-state kinetics confirmed the EPR results by the presence of a porphyrin radical in the W191F mutants just past the instruments 2 millisecond dead time.;In an attempt to demonstrate ascorbate-binding and catalysis, we have used steady-state kinetics to show that our CcP mutants that contain the ascorbate-binding loop can indeed bind and catalyze ascorbate peroxidation. It was interesting to note, though, that the mutants with the added W191F mutation, which can generate a porphyrin radical, though short lived, exhibited lower levels of ascorbate catalysis than the CcP mutants that did not receive the added W191F mutation. We believe that the presence of a Phe instead of its native Trp results in delocalization of the radical to Tyr236 which is further from ascorbate than Trp191.