Characterization of the reaction between cytochrome c peroxidase and potassium fluoride
Pokropinski, Sharon L.
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The reactivity of potassium fluoride with wild-type cytochrome c peroxidase (CcP) and a mutant of cytochrome c peroxidase in which the distal histidine (His-52) is replaced with leucine [CcP(H52L)] has been studied from pH 4 to 8 in phosphate buffer at 0.1 M ionic strength. The spectrum of the fluoride complex of CcP(H52L) has been characterized. The kinetics of fluoride binding and the equilibrium constant for complex formation for both wild-type CcP and CcP(H52L) have been determined as a function of pH. The results of this study indicate that HF binds to wild-type CcP when the distal histidine is unprotonated. However, the association rate constant for fluoride binding to CcP(H52L) is only a factor of ten slower than binding to CcP at pH 5.5 and about the same at pH 8.0. A possible explanation is that another group, such as Arg-48 or the heme-bound hydroxide group in CcP(H52L), provides a function similar to that of His-52 in wild-type CcP. A major effect of the H52L mutation is to increase the rate of fluoride dissociation by a thousand-fold.