Studies have shown that horseradish peroxidase works in 3 steps (taken from Maehly, 1960):
1) formation of green, active substrate complex I:
k1
HRP + H2O2 <========>
Complex I
k2
where HRP stands for Horseradish Peroxidase
2) transition of complex I to red, active complex II:
k7
Complex I + AH <====> Complex
II + A
where AH is the donor in reduced form, A is the donor in oxidized form
3) Recovery of enzyme by reduction of Complex II:
k4
Complex II + AH <====> HRP + A +
2H2O
4) Formation of product:
2A --> A2
The substrate specificity of HRP in forming Complex I is high. Hydrogen Peroxide (H2O2), Methyl Hydrogen Peroxide (MeOOH), and Ethyl Hydrogen Peroxide (EtOOH) are the only compounds that could combine with HRP to form the active substrate complex Complex I. However, The specificity of the enzyme substrate complexes for hydrogen donors in equations 2 and 3 is quite low. As such, AH2 in the equations represents a number of different compounds such as 4-aminoantipyrene, cytochrome c, ferrocyanide, phenols, amino phenols, diamines, indophenols, ascorbic acid, leucodyes, and some amino acids (Maehly, 1960).
The picture on the left (taken from http://metallo.scripps.edu/PROMISE/FPBPEROXIDASE.html) shows another representation of the general mechanism of the peroxidase oxidation-reduction reaction which shows how the heme iron is part of the process. In the mechanism shown, the first step involves the addition of H2O2 to the FeIII resting state of HRP, yielding Complex I, which is two oxidation equivalents above the FeIII resting state. The redox reaction involves two electrons, where H2O2 is reduced to H2O and the enzyme is oxidized. This Complex I oxidizes an organic substrate to give a substrate radical. It undergoes a second one electron oxidation to yield Complex II (Dawson, 1988). Lastly, Complex II reacts with another substrate producing another substrate radical. In this reaction, the iron in Complex II is reduced back to the native ferric state. (Dawson, 1988)
Formation of Complex I:
Click here for a picture of the proposed mechanism of complex I formation upon addition of Hydrogen Peroxide to Peroxidase(Fukuyama et al, 1995). The reaction occurs in three steps.
O-O Cleavage:
The O-O bond cleavage in the third step of the mechanism is interpreted as a "push-pull" concept (Dawson, 1988). Click here for a picture depicting this concept.