Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illumination

Neves-Petersen, Maria Teresa; Klitgaard, Soren; Leitao Carvalho, Ana Sofia; Petersen, Steffen B.; de Barros, Maria Raquel Aires; Melo, Eduardo

http://hdl.handle.net/10400.1/11670

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Authors

Neves-Petersen, Maria Teresa

Klitgaard, Soren

Leitao Carvalho, Ana Sofia

Petersen, Steffen B.

de Barros, Maria Raquel Aires

Melo, Eduardo

Abstract(s)

Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H2O2 to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value,. fluorescence emission,. fluorescence lifetime distribution,. fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T-m values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the. fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved. fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's. fluorescence lifetimes, leading to an exponential increase of the mean. fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.

Keywords

Aromatic-Amino-Acids Cytochrome-C Peroxidase Excited-State Chemistry Unusual Fluorescence Behavior Dilute Aqueous-Solution Tryptophan Fluorescence Circular-Dichroism Hydrated Electron Global Analysis Inactivation