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 1.5 μs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD(.-) is converted in 3.3 μs 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.
authors: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France. pavel.muller@cea.fr.
authors: Pavel
authors: 0000-0002-3245-0810
authors: P
authors: Müller
authors: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France. klaus.brettel@cea.fr.
authors: Klaus
authors: 0000-0001-6764-7042
authors: K
authors: Brettel
authors: Department of Biophysics, Medical School, University of Pecs, 12 str. Szigeti, 7624, Pecs, Hungary.
authors: Laszlo
authors: 0000-0003-2848-576X
authors: L
authors: Grama
authors: Department of Biophysics, Medical School, University of Pecs, 12 str. Szigeti, 7624, Pecs, Hungary.
authors: Miklos
authors: 0000-0002-6229-4337
authors: M
authors: Nyitrai
authors: Department of Biophysics, Medical School, University of Pecs, 12 str. Szigeti, 7624, Pecs, Hungary. andras.lukacs@aok.pte.hu.
authors: Andras
authors: 0000-0001-8841-9823
authors: A
authors: Lukacs
mesh_terms: D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction
chemical_list: D005182:Flavin-Adenine Dinucleotide; D004255:Deoxyribodipyrimidine Photo-Lyase
keywords: CPD photolyase; cryptochrome; electron transfer; flavin radicals; proton transfer