Preparation and Initial Characterization of the Compound I, II, and III States of Iron Methylchlorin-Reconstituted Horseradish Peroxidase and Myoglobin: Models for Key Intermediates in Iron Chlorin Enzymes
“…Panel A shows the conversion of resting HRP:sol−gel to HRP−I:sol−gel. The isosbestic behavior and growth of the bands at 580 and 650 nm are consistent with a clean conversion between these two species . Panel B shows the spectra recorded between 40 and 100 s after addition of H 2 O 2 .…”
This study addresses the viability of sol-gel encapsulated HRP (HRP:sol-gel) as a recyclable solid-state catalytic material. Ferric, ferric-CN, ferrous, and ferrous-CO forms of HRP:sol-gel were investigated by resonance Raman and UV-visible methods. Electronic and vibrational spectroscopic changes associated with changes in spin state, oxidation state, and ligation of the heme in HRP:sol-gel were shown to correlate with those of HRP in solution, showing that the heme remains a viable ligand-binding complex. Furthermore, the high-valent HRP:sol-gel intermediates, compound I and compound II, were generated and identified by time-resolved UV-visible spectroscopy. Catalytic activity of the HRP:sol-gel material was demonstrated by enzymatic assays by using I(-), guaiacol, and ABTS as substrates. Encapsulated HRP was shown to be homogeneously distributed throughout the sol-gel host. Differences in turnover rates between guaiacol and I(-) implicate mass transport of substrate through the silicate matrix as a defining parameter in the peroxidase activity of HRP:sol-gel. HRP:sol-gel was reused as a peroxidation catalyst for multiple reaction cycles without loss of activity, indicating that such materials show promise as reusable catalytic materials.
“…Panel A shows the conversion of resting HRP:sol−gel to HRP−I:sol−gel. The isosbestic behavior and growth of the bands at 580 and 650 nm are consistent with a clean conversion between these two species . Panel B shows the spectra recorded between 40 and 100 s after addition of H 2 O 2 .…”
This study addresses the viability of sol-gel encapsulated HRP (HRP:sol-gel) as a recyclable solid-state catalytic material. Ferric, ferric-CN, ferrous, and ferrous-CO forms of HRP:sol-gel were investigated by resonance Raman and UV-visible methods. Electronic and vibrational spectroscopic changes associated with changes in spin state, oxidation state, and ligation of the heme in HRP:sol-gel were shown to correlate with those of HRP in solution, showing that the heme remains a viable ligand-binding complex. Furthermore, the high-valent HRP:sol-gel intermediates, compound I and compound II, were generated and identified by time-resolved UV-visible spectroscopy. Catalytic activity of the HRP:sol-gel material was demonstrated by enzymatic assays by using I(-), guaiacol, and ABTS as substrates. Encapsulated HRP was shown to be homogeneously distributed throughout the sol-gel host. Differences in turnover rates between guaiacol and I(-) implicate mass transport of substrate through the silicate matrix as a defining parameter in the peroxidase activity of HRP:sol-gel. HRP:sol-gel was reused as a peroxidation catalyst for multiple reaction cycles without loss of activity, indicating that such materials show promise as reusable catalytic materials.
“…Reaction of heme with peroxide/peracid can lead to a ferric peroxide which can either cleave the O–O bond homolytically to form compound II or heterolytically to form compound I. Heme peroxides are characterized by absorption at 526–540 nm and 556–575 nm while compound II is characterized by absorption at 525–551 and 556–586 nm 33–38. Alternatively, compound I is characterized by absorption at 645–690 nm 37–46. Absorption in this region results in a green colour and is characteristic of a ferryl porphyrin cation radical (Fe IV O Por˙ + ) or compound I (Table S1†).…”
Compound I is an active oxidant responsible for the peroxidase activity of heme–Aβ and can cause oxidative degradation of neurotransmitters like serotonin, a marker of Alzheimer's disease.
“…The major change is associated with a shift in the Soret band. Regardless of the starting state (ferric or compound II) or the presence of the substrate, the final state of the protein appears to be the oxyferrous form, which is also known as compound III in the peroxidase literature ( − ). 7 Time-dependent spectra for the turnover of TCP by DHP and three mutants Y38F, H55R, and H89G obtained using a photodiode array spectrophotometer.…”
Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H2O2. This observation suggests that the mechanism of DHP relies on H2O2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products.
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