Dependence of heme accessibility in horseradish peroxidase on Ca2+

The rate constant for quenching, k(q), of the pulsed laser-induced phosphor escence of 6-bromo-2-naphthyl sulfate (B) was measured at room temperature to be 4.6 x 10(8) and 7.6 x 10(8) M-1 s(-1) for quenching by horseradish pe roxidase (HRP) with and without bound Ca2+ (CaD-HRP), respectively. Quenchi ng of B phosphorescence by apo-HRP was found to be biexponential and gave e vidence that quenching by HRP and CaD-HRP occurs predominantly without the formation of a bound complex of B-3 and protein. It was also concluded that quenching occurs predominantly after B migrates through the single substra te channel into the vicinity of the heme. Since the activation energy for q uenching was found to be insensitive to the presence of Ca2+, the conformat ion and dynamical motion within the substrate channel were concluded to cha nge little between CaD-HRP and HRP. It was concluded that if enzyme enhance ment induced by Ca2+ is due to structural stabilization of HRP, the stabili zing influence is not transmitted strongly into the substrate channel. (C) 1998 Elsevier Science Inc. All rights reserved.