Kinetics of cyanide binding to chloroperoxidase in the presence of nitrate: detection of the influence of a heme-linked acid group by shift in the appa

Abstract

The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant ( K CN) of the peroxidase-cyanide complex and both forward ( k +) and reverse ( k −) rate constants are independent of the H + concentration over the pH range 2.7 to 7.1. The values obtained are k cn = (9.5 ± 1.0) × 10 -5 M, k +. = (5.2 ± 0.5) × 10 4 M −1 sec −1 and k - = (5.0± 1.4) sec -1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher p K a values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the p K a of the heme-linked acid group.