Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy

Pavel Müller, Klaus Brettel, L. Grama, M. Nyitrai, Andras Lukacs

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH- (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD.- and FADH. formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD.- (formed by ET from the tryptophan chain) in plant CRYs in about 1.5μs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD.- is converted in 3.3μs into a state with spectral features that are different from both FADH. and FAD.-. Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.

Original languageEnglish
JournalChemPhysChem
DOIs
Publication statusAccepted/In press - 2016

Fingerprint

Deoxyribodipyrimidine Photo-Lyase
Flavin-Adenine Dinucleotide
Photochemical reactions
adenines
Absorption spectroscopy
photochemical reactions
Escherichia coli
absorption spectroscopy
deoxyribonucleic acid
electron transfer
tryptophan
Tryptophan
Electrons
Cryptochromes
proteins
Flavoproteins
aspartic acid
reference systems
Asparagine
Protonation

Keywords

  • CPD photolyase
  • Cryptochrome
  • Electron transfer
  • Flavin radicals
  • Proton transfer

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Atomic and Molecular Physics, and Optics

Cite this

@article{34b6e21020034cce827fe7949169392d,
title = "Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy",
abstract = "DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH- (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD.- and FADH. formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD.- (formed by ET from the tryptophan chain) in plant CRYs in about 1.5μs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD.- is converted in 3.3μs into a state with spectral features that are different from both FADH. and FAD.-. Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.",
keywords = "CPD photolyase, Cryptochrome, Electron transfer, Flavin radicals, Proton transfer",
author = "Pavel M{\"u}ller and Klaus Brettel and L. Grama and M. Nyitrai and Andras Lukacs",
year = "2016",
doi = "10.1002/cphc.201501077",
language = "English",
journal = "ChemPhysChem",
issn = "1439-4235",
publisher = "Wiley-VCH Verlag",

}

TY - JOUR

T1 - Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy

AU - Müller, Pavel

AU - Brettel, Klaus

AU - Grama, L.

AU - Nyitrai, M.

AU - Lukacs, Andras

PY - 2016

Y1 - 2016

N2 - DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH- (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD.- and FADH. formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD.- (formed by ET from the tryptophan chain) in plant CRYs in about 1.5μs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD.- is converted in 3.3μs into a state with spectral features that are different from both FADH. and FAD.-. Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.

AB - DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH- (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD.- and FADH. formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD.- (formed by ET from the tryptophan chain) in plant CRYs in about 1.5μs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD.- is converted in 3.3μs into a state with spectral features that are different from both FADH. and FAD.-. Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.

KW - CPD photolyase

KW - Cryptochrome

KW - Electron transfer

KW - Flavin radicals

KW - Proton transfer

UR - http://www.scopus.com/inward/record.url?scp=84959432212&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84959432212&partnerID=8YFLogxK

U2 - 10.1002/cphc.201501077

DO - 10.1002/cphc.201501077

M3 - Article

C2 - 26852903

AN - SCOPUS:84959432212

JO - ChemPhysChem

JF - ChemPhysChem

SN - 1439-4235

ER -