Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illumination

Detailed analysis of the effects of 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 Tm values as well as

helical content loss. Prolonged UV illumination and Heme irradiation at 403nm has

pronounced effect on the fluorescence quantum yield, correlated with changes in the

prosthetic group pocket, leading to pronounced decrease in the heme's Soret absorbance

band. Analyzes of the picosecond resolved fluorescence emission of horseradish

peroxidase A2 with streak camera shows that UV illumination induces an exponential

change in the pre-exponential 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 photo-induced 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 ultra-fast

processes and how streak camera data can be correlated with protein structural changes.

Udgivelsesdato: MAR 15