Cyanide binding to cytochrome c peroxidase (H52L).
Cyanide binding to a cytochrome c peroxidase (CcP) variant in which the distal histidine has been replaced by a leucine residue, CcP(H52L), has been investigated as a function of pH using spectroscopic, equilibrium, and kinetic methods. Between pH 4 and 8, the apparent equilibrium dissociation constant for the CcP(H52L)/cyanide complex varies by a factor of 60, from 135 microM at pH 4.7 to 2.2 microM at pH 8.0. The binding kinetics are biphasic, involving bimolecular association of the two reactants, followed by an isomerization of the enzyme/cyanide complex. The association rate constant could be determined up to pH 8.9 using pH-jump techniques. The association rate constant increases by almost 4 orders of magnitude over the pH range investigated, from 1.8 x 10(2) M(-1) s(-1) at pH 4 to 9.2 x 10(5) M(-1) s(-1) at pH 8.6. In contrast to wild-type CcP, where the binding of HCN is the dominant binding pathway, CcP(H52L) preferentially binds the cyanide anion. Above pH 8, cyanide binding to CcP(H52L) is faster than cyanide binding to wild-type CcP. Cyanide dissociates 4 times slower from the mutant protein although the pH dependence of the dissociation rate constant is essentially identical for CcP(H52L) and CcP. Isomerization of the CcP(H52L)/cyanide complex is observed between pH 4 and 8 and stabilizes the complex. The isomerization rate constant has a similar magnitude and pH dependence as the cyanide dissociation rate constant, and the two reactions are coupled at low cyanide concentrations. This isomerization has no counterpart in the wild-type CcP/cyanide complex.
Bidwai
Anil
A
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, USA.
Witt
Misty
M
Foshay
Miriam
M
Vitello
Lidia B
LB
Satterlee
James D
JD
Erman
James E
JE
D019943:Amino Acid Substitution; D003486:Cyanides / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D017186:Titrimetry
D003486:Cyanides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D003578:Cytochrome-c Peroxidase; D007930:Leucine