The mechanism of Compound I formation revisited.

The most recently proposed mechanisms for the formation of the Compound I intermediates of the peroxidases and catalases have been based on the crystallographic elucidation of the enzyme structures. It has been assumed that these mechanisms are compatible with an earlier proposal of the formation of a reversible enzyme-substrate intermediate called Compound 0, which was based on data that pre-dated the availability of the enzyme structures. However, it is argued here that this is not the case and some modifications of the existing mechanism are proposed which reconcile the structural, kinetic and energetic data for the reactions. This paper focuses attention on horseradish peroxidase isoenzyme C and particularly on the acid-base properties of the imidazole side chain of distal histidine 42. This imidazole group has an exceptionally low pK(a) value in the resting enzyme, which is higher in Compound I and higher still in Compound II. The pK(a) value must also be greatly increased following Compound 0 formation so that the imidazole can become an effective proton acceptor. An explanation is offered in a dielectric insertion (DI) model, in which the peroxide substrate, or fragments thereof, screens the influence of the positively charged heme iron on the pK(a) value of the imidazole group. It is proposed that Compound 0 is converted to a second intermediate, Compound 0*, by intramolecular proton transfer along a pre-existing hydrogen bond, a process which reduces the energy requirements of charge separation in the deprotonation of hydrogen peroxide.