pH Dependence of heme iron coordination, hydrogen peroxide reactivity, and cyanide binding in cytochrome c peroxidase(H52K).
Replacement of the distal histidine, His-52, in cytochrome c peroxidase (CcP) with a lysine residue produces a mutant cytochrome c peroxidase, CcP(H52K), with spectral and kinetic properties significantly altered compared to those of the wild-type enzyme. Three spectroscopically distinct forms of the enzyme are observed between pH 4.0 and 8.0 with two additional forms, thought to be partially denatured forms, making contributions to the observed spectra at the pH extremes. CcP(H52K) exists in at least three, slowly interconverting conformational states over most of the pH range that was investigated. The side chain epsilon-amino group of Lys-52 has an apparent pK(a) of 6.4 +/- 0.2, and the protonation state of Lys-52 affects the spectral properties of the enzyme and the reactions with both hydrogen peroxide and HCN. In its unprotonated form, Lys-52 acts as a base catalyst facilitating the reactions of both hydrogen peroxide and HCN with CcP(H52K). The major form of CcP(H52K) reacts with hydrogen peroxide with a rate approximately 50 times slower than that of wild-type CcP but reacts with HCN approximately 3 times faster than does the wild-type enzyme. The major form of the mutant enzyme has a higher affinity for HCN than does native CcP.
Foshay
Miriam C
MC
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, USA.
Vitello
Lidia B
LB
Erman
James E
JE
D019943:Amino Acid Substitution; D003486:Cyanides / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011522:Protons; D013057:Spectrum Analysis
D003486:Cyanides; D011522:Protons; D006418:Heme; D006861:Hydrogen Peroxide; D007501:Iron; D003578:Cytochrome-c Peroxidase; D008239:Lysine