Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illumination.
Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H(2)O(2) 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.
Neves-Petersen
Maria Teresa
MT
Department of Physics and Nanotechnology, NanoBiotechnology Section, UltrafastBioSpectroscopy Group, Aalborg University, Aalborg, Denmark. tnp@nanobio.aau.dk
Klitgaard
Søren
S
Carvalho
Ana Sofia Leitão
AS
Petersen
Steffen B
SB
Aires de Barros
Maria Raquel
MR
Pinho e Melo
Eduardo
E
D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000528:radiation effects; D006418:Heme / Q000737:chemistry* / Q000528:radiation effects; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000528:radiation effects* / Q000648:ultrastructure; D010777:Photochemistry / Q000379:methods; D010825:Physics / Q000379:methods; D011487:Protein Conformation / Q000187:drug effects; D011829:Radiation Dosage; D013696:Temperature; D014466:Ultraviolet Rays
D006418:Heme; D006735:Horseradish Peroxidase