Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues

Teresa Neves Petersen, Søren Klitgaard, Esben Skovsen, Torbjorn Pascher, Tomas Polivka, Steffen B. Petersen, Arkady Yartsev, Villy Sundström

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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 approximately 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 *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* 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 *-. Additional mechanisms that may lead to the near disappearance of Trp(*) 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.
Original languageEnglish
JournalBiophysical Journal
Volume97
Issue number1
Pages (from-to)211
ISSN0006-3495
DOIs
Publication statusPublished - 2009

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Photolysis
Tryptophan
Electrons
Disulfides
Ultraviolet Rays
Muramidase
Lighting
Proteins
Light
Aromatic Amino Acids
Fusarium
Skin Neoplasms
Enzymes
cutinase
Sulfhydryl Compounds
Cataract
Amino Acids

Cite this

Neves Petersen, Teresa ; Klitgaard, Søren ; Skovsen, Esben ; Pascher, Torbjorn ; Polivka, Tomas ; Petersen, Steffen B. ; Yartsev, Arkady ; Sundström, Villy. / Flash photolysis of cutinase : identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues. In: Biophysical Journal. 2009 ; Vol. 97, No. 1. pp. 211.
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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 approximately 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 *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* 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 *-. Additional mechanisms that may lead to the near disappearance of Trp(*) 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.",
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Flash photolysis of cutinase : identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues. / Neves Petersen, Teresa; Klitgaard, Søren; Skovsen, Esben; Pascher, Torbjorn; Polivka, Tomas; Petersen, Steffen B.; Yartsev, Arkady; Sundström, Villy.

In: Biophysical Journal, Vol. 97, No. 1, 2009, p. 211.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Flash photolysis of cutinase

T2 - identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues

AU - Neves Petersen, Teresa

AU - Klitgaard, Søren

AU - Skovsen, Esben

AU - Pascher, Torbjorn

AU - Polivka, Tomas

AU - Petersen, Steffen B.

AU - Yartsev, Arkady

AU - Sundström, Villy

N1 - Paper id:: doi:10.1016/j.bpj.2009.01.065

PY - 2009

Y1 - 2009

N2 - 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 approximately 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 *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* 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 *-. Additional mechanisms that may lead to the near disappearance of Trp(*) 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.

AB - 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 approximately 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 *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* 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 *-. Additional mechanisms that may lead to the near disappearance of Trp(*) 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.

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DO - 10.1016/j.bpj.2009.01.065

M3 - Journal article

VL - 97

SP - 211

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 1

ER -