Hydrogen bond switching among flavin and amino acid side chains in the BLUF photoreceptor observed by ultrafast infrared spectroscopy |
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Authors: | Bonetti Cosimo Mathes Tilo van Stokkum Ivo H M Mullen Katharine M Groot Marie-Louise van Grondelle Rienk Hegemann Peter Kennis John T M |
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Institution: | * Biophysics Group, Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands † Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, D-10115 Berlin, Germany |
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Abstract: | BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD•− and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD•− to result in FADH• on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH• C=N stretch marker mode, with tyrosine as the likely proton donor. FADH• is reoxidized in 67 ps (180 ps in D2O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by ∼180° through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch. |
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Keywords: | BLUF blue-light sensing using FAD DAS decay-associated spectrum EADS evolution-associated difference spectrum FAD flavin adenine dinucleotide FAD singlet excited-state FMN flavin mononucleotide IA induced absorption KIE kinetic isotope effect LOV light oxygen or voltage RBTA riboflavin tetraacetate SADS species-associated difference spectrum IR infrared |
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