Photochemistry of Wild‐Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy1. Issue 9 (24th February 2016)

• Record Type: Journal Article
• Authors: Müller, Pavel; Brettel, Klaus; Grama, Laszlo; Nyitrai, Miklos; Lukacs, Andras
• Is Part Of: Chemphyschem. Volume 17:Issue 9(2016)
• Page Start: 1329
• Page End: 1340
• Publication Date: 2016-02-24
• Subjects: CPD photolyase -- cryptochrome -- electron transfer -- proton transfer -- flavin radicals; Chemistry, Physical and theoretical -- Periodicals; 541.05
• DOI: 10.1002/cphc.201501077
• Languages: English
• ISSNs: 1439-4235

Abstract

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 ( Ec PL) has been studied for a long time and is often used as a reference system; however, Ec PL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of Ec PL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild‐type Ec PL 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 Ec PL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant Ec PL are discussed. Abstract : Keep your proton ! Photoreactions of oxidized Escherichia coli DNA photolyase and its N378D mutant (to mimic plant photoreceptor cryptochrome) are fully characterized on timescales from nanoseconds to tens of seconds. Unlike in plant cryptochrome, the photoinduced flavin adenine dinucleotide anion radical (FAD .− ) is protonated by D378 to only a very limited extent in the mutant photolyase (see scheme). Instead, it is merely stabilized by a hydrogen bond from D378, yielding a FAD radical with an unusual absorption spectrum.