PROBING THE ROLE OF THE BURIED CHARGE, ASP235, IN CYTOCHROME C PEROXIDASE BY ENGINEERING IN COUNTER CHARGES OR H-BONDS. Yi Cao, David B. Goodin and Duncan E. McRee, Dept. of Molecular Biology, The Scripps Research Institute

Cytochrome c peroxidase (CCP) contains a buried charge, Asp 235, that is thought to control the reduction potential of cytochrome c peroxidase and coupling of the W191 free radical to the heme (D. Goodin & D. McRee, 1993, Biochemistry 32:3313). In this study, the role of this buried D235 is probed by introducing a counter-ion to it (with mutants M172K, M231H and F202K), or by introducing one more H-bond into the H175- D235-W191 H-bond network (with mutant F202Y). The X-ray crystal structures of these four mutant proteins show that: 1) in the M172K mutant, K172 surprisingly does not form a salt bridge with D235-Od1 as designed, but instead H-bonds are formed between these two groups with H2O as the bridge; 2) in the M231H mutant, H231 forms either a salt bridge or a H-bond with D235-Od2; 3) In the F202K mutant, K202 forms a salt bridge with D235-Od2, and 4) In the F202Y mutant, Y202 does form a H-bond with D235-Od1 as designed (with distance 2.5A between Y202- O and D235-Od1). For M231H and F202K, the H-bond D235(Od2)- W191 breaks and the indole ring of W191 is found to be reoriented in a similar way to that observed in the D235A and D235N mutants. No other major changes are observed in the protein structures of these mutants. This result reveals that the H-bond between the buried D235 and H175 in CCP is more stable than a potential salt bridge between D235 and K172. The result of the activity assay further shows that introducing a H-bond between D235 and Y202 in F202Y mutant (possibly weakening the D235- H175 H-bond) decreases the activity for cytochrome c oxidation and increases the activity for oxidation of some small molecule substrates.