Thermodynamics of hydrogen cyanide and hydrogen fluoride binding to cytochrome c peroxidase and its Asn-82-->Asp mutant.
The thermodynamics of binding of fluoride and cyanide to cytochrome c peroxidase (CCP) and its Asn-82-->Asp mutant (D82CCP) in phosphate and acetate buffer at an ionic strength of 0.15 mol.kg-1 from pH 5.0 to 7.1 were investigated by titration calorimetry at 289 and 297 K. The binding reactions are enthalpically driven. The fluoride-binding constants determined from the titration calorimetry results were in agreement with those determined from difference-spectroscopy measurements. For cyanide binding to CCP at 297.9 K, the binding constant decreased from 8.95 (+/- 0.83) x 10(5) M-1 at pH 7.0 to 4.04(+/- 0.23) x 10(5) M-1 at pH 5.0, and the binding enthalpy increased from -57.2 +/- 1.4 kJ.mol-1 at pH 7.0 to -48.6 +/- 1.8 kJ.mol-1 at pH 5.0. For fluoride binding to CCP, the binding constant increased from 8.41(+/- 0.54) x 10(3) M-1 at pH 7.0 to 3.11(+/- 0.09) x 10(5) M-1 at pH 5.0 and the binding enthalpy increased from -71.9 +/- 1.1 kJ.mol-1 at pH 7.0 to -67.0 +/- 1.9 kJ.mol-1 at pH 5.0. The binding enthalpies for D82CCP were about the same as those for CCP. However, the binding constants for cyanide and fluoride to D82CCP were respectively a factor of two less and at least an order of magnitude less than the corresponding binding constants of CCP. Decreased ligand-binding strength in the D82CCP mutant is thus entirely due to entropic effects.
DeLauder
S F
SF
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, MD 20850.
Mauro
J M
JM
Poulos
T L
TL
Williams
J C
JC
Schwarz
F P
FP
D001216:Asparagine / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D002021:Buffers; D002151:Calorimetry; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006858:Hydrofluoric Acid / Q000378:metabolism*; D006856:Hydrogen Cyanide / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D002021:Buffers; D006856:Hydrogen Cyanide; D001216:Asparagine; D003578:Cytochrome-c Peroxidase; D006858:Hydrofluoric Acid