Hydrogen Bond Switching among Flavin and Amino Acid Side Chains in the BLUF Photoreceptor Observed by Ultrafast Infrared Spectroscopy

• Published in: Biophysical journal 2008-11, Vol.95 (10), p.4790-4802
• Main 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.
• Format: Article
• Language: English
• Subjects: Amino acids; Amino Acids - chemistry; Amino Acids - radiation effects; Binding Sites; Biochemistry; Chemical compounds; Computer Simulation; Flavins - chemistry; Flavins - radiation effects; Hydrogen Bonding - radiation effects; Hydrogen bonds; Light; Light Signal Transduction; Models, Chemical; Photobiophysics; Photoreceptors, Microbial - chemistry; Photoreceptors, Microbial - radiation effects; Protein Binding; Protein Structure, Tertiary - radiation effects; Proteins; Spectrophotometry, Infrared - methods; Spectrum analysis
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1529/biophysj.108.139246

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 D 2O) 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.