Electrochemically and catalytically active reconstituted horseradish peroxidase with ferrocene-modified hemin and an artificial binding site

A procedure for modification of hemin chloride by FcCH(2)NHw(2) (Fc = ferro cenyl) in the presence of 1-(dimethylaminopropyl)-3 -ethylcarbodiimide hydr ochloride and N-hydroxysuccinimide affords two main products 1 and 2 with m ono- and bis-amidated propionic acid residues. Monoamidated conjugate 1 was loaded into the apoenzyme of horseradish peroxidase (HRP) to afford an ele ctrochemically and catalytically active reconstituted enzyme Fc-HRP with re markably altered substrate specificity. With ABTS as substrate, the reactiv ity of Fc-HRP drops threefold compared with native HRP as a result of a low ering of the maximal rate V-m. Compared with HRP the reactivity of Fc-HRP t owards water-soluble ferrocenes is even higher at low concentrations of the latter, the rate increase being accompanied by a change in rate law: in co ntrast to first-order kinetics in ferrocenes for native HRP, there is a Mic haelis dependence for Fc-HRP. Molecular modeling suggests creation of an ar tificial hydrophobic binding site within a triangle confined by the ferroce nyl residue and the two phenyl rings of Phe 68 and 179. The site is believe d to be responsible for the kinetically meaningful binding between ferrocen e substrates and Fc-HRP which manifests in the saturation kinetics.