Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy** Pavel Müller,* [a] Klaus Brettel,* [a] Laszlo Grama, [b] Miklos Nyitrai, [b] and Andras Lukacs* [b] 1. Introduction DNA repair enzymes photolyases (PLs) use blue or UVA light to repair damage caused by genotoxic UVB light. These flavopro- teins developed in the very early stages of the evolution of life on earth and they [and/or their descendants—photoreceptors cryptochromes (CRYs)] are, even now, still present in organisms belonging to all kingdoms of life (bacteria, archaea, and eukar- yotes), including higher animals. [1–3] A PL repairing major UVB-induced lesions—the cyclobutane pyrimidine dimers (CPDs)—was discovered in Escherichia coli almost 60 years ago. [4] Since then, this protein, EcPL, and the mechanism by which it repairs this lesion, have been thor- oughly studied. [1–3, 5, 6] EcPL has become a benchmark for other proteins from the PL/CRY flavoprotein superfamily discovered later. PLs and CRYs share a noncovalently bound flavin adenine di- nucleotide (FAD) cofactor (Figure 1). Varying between different proteins and depending on their functional state, the FAD co- factor adopts the fully reduced (FADH  ), fully oxidized (FAD ox ), semireduced neutral radical (FADHC), or semireduced anion rad- ical states (FADC  ). DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling func- tions. 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 sur- face. Four redox states of FAD are relevant for the various func- tions of PLs and CRYs: fully reduced FADH  (required for DNA photorepair), fully oxidized FAD ox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FADC  and FADHC 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 FAD ox has so far not been investigated on all relevant timescales. Herein, a de- tailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FAD ox 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 FADC  (formed by ET from the trypto- phan chain) in plant CRYs in about 1.5 ms, are characterized. Surprisingly, the mutant protein does not show this protona- tion. Instead, FADC  is converted in 3.3 ms into a state with spectral features that are different from both FADHC and FADC  . Such a conversion does not occur in wild-type EcPL. The chem- ical nature and formation mechanism of the atypical FAD radi- cal in N378D mutant EcPL are discussed. Figure 1. The photoactive center of a) wild-type (WT) E. coli PL (X-ray struc- ture 1DNP [14] in the RSCB Protein Data Bank) and b) the photolyase homolo- gy region (PHR) of a plant CRY (Arabidopsis thaliana CRY1, X-ray structure 1U3D [15] ). The FAD cofactors (FADHC in EcPL and FAD ox in AtCRY1) are shown in yellow and the amino acids facing the N5 atoms of their isoalloxazine rings (asparagine N378 in EcPL and aspartic acid D396 in AtCRY1) are shown in green. Shown in gray are the first members of the electron-transferring tryptophan chains (W382 in EcPL and W400 in AtCRY1), the amino acids, to the backbone carbonyl of which D396 in AtCRY1 and possibly also D378 in N378D mutant EcPL make a hydrogen bond prior to FAD excitation (E363 in EcPL and M381 in AtCRY1), and the remaining two amino acids in the imme- diate vicinity of FAD, which are different in the two proteins: methionine 345 and tryptophan 384 in EcPL and valine 363 and tyrosine 402 in AtCRY1. [a] Dr. P. Müller, Dr. K. Brettel Institute for Integrative Biology of the Cell (I2BC), CEA CNRS, Univ Paris-Sud, UniversitØ Paris-Saclay 91198 Gif-sur-Yvette cedex (France) E-mail : pavel.muller@cea.fr klaus.brettel@cea.fr [b] Dr. L. Grama, Dr. M. Nyitrai, Dr. A. Lukacs Department of Biophysics, Medical School University of Pecs, 12 str. Szigeti, 7624 Pecs (Hungary) E-mail : andras.lukacs@aok.pte.hu [**] FAD = flavin adenine dinucleotide Supporting Information and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.doi.org/10.1002/ cphc.201501077. An invited contribution to a Special Issue on Fast Spectroscopy: Biosys- tems ChemPhysChem 2016, 17, 1 – 13  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 & These are not the final page numbers! ÞÞ These are not the final page numbers! ÞÞ Articles DOI: 10.1002/cphc.201501077