Dependence of heme accessibility in horseradish peroxidase on Ca 2+

Abstract

The rate constant for quenching, k q, of the pulsed laser-induced phosphorescence of 6-bromo-2-naphthyl sulfate (B) was measured at room temperature to be 4.6 × 10 8 and 7.6 × 10 8 M −1s −1 for quenching by horseradish peroxidase (HRP) with and without bound Ca 2+ (CaD-HRP), respectively. Quenching of B phosphorescence by apo-HRP was found to be biexponential and gave evidence that quenching by HRP and CaD-HRP occurs predominantly without the formation of a bound complex of 3B and protein. It was also concluded that quenching occurs predominantly after B migrates through the single substrate channel into the vicinity of the heme. Since the activation energy for quenching was found to be insensitive to the presence of Ca 2+, the conformation and dynamical motion within the substrate channel were concluded to change little between CaD-HRP and HRP. It was concluded that if enzyme enhancement induced by Ca 2+ is due to structural stabilization of HRP, the stabilizing influence is not transmitted strongly into the substrate channel.