The effects of an engineered cation site on the structure, activity, and EPR properties of cytochrome c peroxidase

Earlier work [Bonagura et al. (1996) Biochemistry 35, 6107] allowed that th e K+ site found in the proximal pocket of ascorbate peroxidase (APX) could be engineered into cytochrome c peroxidase (CCP). Binding of K+ at the engi neered site results in a loss in activity and destabilization of the CCP co mpound I Trp191 cationic radical owing to long-range electrostatic effects. The engineered CCP mutant crystal structure has been refined to 1.5 Angstr om using data obtained at cryogenic temperatures which provides a more deta iled basis for comparison with the naturally occurring K+ site in APX, The characteristic EPR signal associated with the Trp191 radical becomes progre ssively weaker as K+ is added, which correlates well with the loss in enzym e activity as [K+] is increased. These results coupled with stopped-flow st udies support our earlier conclusions that the loss in activity and EPR sig nal is due to destabilization of the Trp191 cationic radical.