Polarized transient absorption to resolve electron transfer between tryptophans in DNA photolyase.
Transient absorption spectroscopy is a powerful tool for studying biological electron-transfer chains, provided that their members give rise to distinct changes of their absorption spectra. There are, however, chains that contain identical molecules, so that electron transfer between them does not change net absorption. An example is the chain flavin adenine dinucleotide (FAD)-W382-W359-W306 in DNA photolyase from E. coli. Upon absorption of a photon, the excited state of FADH* (neutral FAD radical) abstracts an electron from the tryptophan residue W382 in approximately 30 ps (monitored by transient absorption). The cation radical W382*+ is presumably reduced by W359 and W359*+ by W306. The latter two reactions could not be monitored directly so far because the absorption changes of the partners compensate in each step. To overcome this difficulty, we used linearly polarized flashes for excitation of FADH*, thus inducing a preferential axis in the a priori unoriented sample (photoselection). Because W359 and W306 are very differently oriented within the protein, detection with polarized light should allow us to distinguish them. To demonstrate this, W306 was mutated to redox-inert phenylalanine. We show that the resulting anisotropy spectrum of the initial absorption changes (measured at 10 ns time resolution) is in line with W359 being oxidized. The corresponding spectrum in wildtype photolyase is clearly different and identifies W306 as the oxidized species. These findings set an upper limit of 10 ns for electron transfer from W306 to W359*+ in wildtype DNA photolyase, consistent with previous, more indirect evidence [Aubert, C.; Vos, M. H.; Mathis, P.; Eker, A. P. M.; Brettel, K. Nature 2000, 405, 586-590].
Byrdin
Martin
M
Laboratoire de Photocatalyse et Biohydrogène, iBiTecS, CEA, 91191 Gif sur Yvette, France. Martin.Byrdin@cea.fr
Villette
Sandrine
S
Espagne
Agathe
A
Eker
Andre P M
AP
Brettel
Klaus
K
D016880:Anisotropy; D018360:Crystallography, X-Ray; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry*; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals / Q000737:chemistry; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry*
D005609:Free Radicals; D014364:Tryptophan; D004255:Deoxyribodipyrimidine Photo-Lyase