Advanced

Flash Photolysis of Cutinase: Identification and Decay Kinetics of Transient Intermediates Formed upon UV Excitation of Aromatic Residues

Neves-Petersen, Maria Teresa; Klitgaard, Soren; Pascher, Torbjörn LU ; Skovsen, Esben; Polivka, Tomas; Yartsev, Arkady LU ; Sundström, Villy LU and Petersen, Steffen B. (2009) In Biophysical Journal 97(1). p.211-226
Abstract
Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under... (More)
Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after similar to 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR center dot-), with concomitant faster decay kinetics and near disappearance of the Trp(center dot) radical peak at 330 nm, indicating possible additional formation of TyrO(center dot) formed upon reaction of Trp(center dot) with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR center dot-. Additional mechanisms that may lead to the near disappearance of Trp(center dot) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biophysical Journal
volume
97
issue
1
pages
211 - 226
publisher
Cell Press
external identifiers
  • wos:000267871000021
  • scopus:68949118397
ISSN
1542-0086
DOI
10.1016/j.bpj.2009.01.065
language
English
LU publication?
yes
id
b5d64bcf-404a-4fea-a341-993c7436605d (old id 1462833)
date added to LUP
2009-08-19 08:25:47
date last changed
2017-10-01 03:35:40
@article{b5d64bcf-404a-4fea-a341-993c7436605d,
  abstract     = {Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after similar to 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR center dot-), with concomitant faster decay kinetics and near disappearance of the Trp(center dot) radical peak at 330 nm, indicating possible additional formation of TyrO(center dot) formed upon reaction of Trp(center dot) with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR center dot-. Additional mechanisms that may lead to the near disappearance of Trp(center dot) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation.},
  author       = {Neves-Petersen, Maria Teresa and Klitgaard, Soren and Pascher, Torbjörn and Skovsen, Esben and Polivka, Tomas and Yartsev, Arkady and Sundström, Villy and Petersen, Steffen B.},
  issn         = {1542-0086},
  language     = {eng},
  number       = {1},
  pages        = {211--226},
  publisher    = {Cell Press},
  series       = {Biophysical Journal},
  title        = {Flash Photolysis of Cutinase: Identification and Decay Kinetics of Transient Intermediates Formed upon UV Excitation of Aromatic Residues},
  url          = {http://dx.doi.org/10.1016/j.bpj.2009.01.065},
  volume       = {97},
  year         = {2009},
}