J Biol Chem, Vol. 274, Issue 53, 37827-37833, December 31, 1999

Conversion of an Engineered Potassium-binding Site into a Calcium-selective Site in Cytochrome c Peroxidase^*

Christopher A. Bonagura, B. Bhaskar, M. Sundaramoorthy^§, and Thomas L. Poulos^¶

From the Departments of Molecular Biology and Biochemistry and Physiology and Biophysics and the Program in Macromolecular Structure, University of California, Irvine, California 92697-3900

We have previously shown that the K⁺ site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A., Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107-6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538-5545). All other peroxidases bind Ca²⁺ rather than K⁺. Using the K⁺-binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed that should bind Ca²⁺ selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na⁺, is bound instead of Ca²⁺. This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed Ca²⁺ binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands. Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp¹⁹¹ radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca²⁺ more effectively than K⁺, demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity.