Light-driven horseradish peroxidase cycle by using photo-activated methylene blue as the reducing agent

In this work, the regeneration of native horseradish peroxidase (HRP), following the consecutive reduction of oxo-ferryl pi-cation (compound I) and oxo-ferryl (compound II) forms, was observed by UV-visible spectrometry and electron paramagnetic resonance (EPR) in the presence of methylene (MB+), in the dark and under irradiation. In the dark, MB+ did not affect the rate of HRP compound I and II reduction, compatible with hydrogen peroxide as the solely reducing species. Under irradiation, the dye promoted a significant increase in the native HRP regeneration rate in a pH-dependent manner. Flash photolysis measurements revealed significant redshift of the MB+ triplet absorbance spectrum in the presence of native HRP. This result is compatible with the dye binding on the enzyme structure leading to the increase in the photogenerated MB center dot yield. In the presence of HRP compound II, the lifetime of the dye at 520 nm decreased similar to 75% relative to the presence of native HRP that suggests MB center dot as the heme iron photochemical reducing agent. In argon and in air-saturated media, photoactivated MB+ led to native HRP regeneration in a time- and concentration-dependent manner. The apparent rate constant for photoactivated MB+-promoted native HRP regeneration, in argon and in air-saturated medium and measured as a function of MB+ concentration, exhibited saturation that is suggestive of dye binding on the HRP structure. The dissociation constant (K-MB) observed for the binding of dye to HRP was 5.4 +/- 0.6 mu m and 0.57 +/- 0.05 mu m in argon and air-saturated media, respectively. In argon-saturated medium, the rate of the conversion of HRP compound 11 to native HRP was significantly higher k(2argon) = (2.1 +/- 0.1) x 10(-2)S(-1), than that obtained in air-equilibrated medium, k(2air) = (0.73 +/- 0.02) x 10(-2)S(-1). Under these conditions the efficiency of photoactivated MB+-promoted native HRP regeneration was determined in argon and air-equilibrated media as being, respectively: k(2)/K-MB = 3.9 x 10(3) and 12.8 x 10(3) M-1 S-1.