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* yield. In the presence of HRP compound II, the lifetime of the dye at 520 nm decreased approximately 75% relative to the presence of native HRP that suggests MB* 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 (KMB) observed for the binding of dye to HRP was 5.4+/-0.6 microM and 0.57+/-0.05 microM in argon and air-saturated media, respectively. In argon-saturated medium, the rate of the conversion of HRP compound II to native HRP was significantly higher, k2argon=(2.1+/-0.1)x10(-2) s(-1), than that obtained in air-equilibrated medium, k2air=(0.73+/-0.02)x10(-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: k2/KMB=3.9x10(3) and 12.8x10(3) M(-1) s(-1).
Soares
Vanessa A
VA
Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil.
Severino
Divinomar
D
Junqueira
Helena C
HC
Tersariol
Ivarne L S
IL
Shida
Cláudio S
CS
Baptista
Maurício S
MS
Nascimento
Otaciro R
OR
Nantes
Iseli L
IL
D004578:Electron Spin Resonance Spectroscopy; D006735:Horseradish Peroxidase / Q000378:metabolism*; D008027:Light; D008751:Methylene Blue / Q000737:chemistry*; D010084:Oxidation-Reduction; D010777:Photochemistry; D019163:Reducing Agents / Q000737:chemistry*
D019163:Reducing Agents; D006735:Horseradish Peroxidase; D008751:Methylene Blue