Hydrogen bond switching among flavin and amino acid side chains in the BLUF photoreceptor observed by ultrafast infrared spectroscopy

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 D(2)O) 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 approximately 180 degrees through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.

FIGURE 1

X-ray structure of the Synechocystis Slr1694 BLUF domain (35) with the hydrogen bond patterns lining the FAD binding pocket in proposed dark state (A) and light state (B) configurations. The inset of A shows structure and numbering of the FAD isoalloxazine ring.

FIGURE 2

DAS that follow from a global analysis of time-resolved fluorescence experiments on the Synechocystis Slr1694 BLUF domain in H₂O (A) and D₂O (B). The lifetime of each DAS in given in picoseconds with the relative contribution to the total fluorescence in parenthesis. The fifth DAS (bold line) is due to a minor fraction of unbound flavin and has a lifetime of 4.5 ns. The contribution of the free flavin was subtracted in the estimation of the relative contribution of the single DAS to the total fluorescence.

FIGURE 3

Kinetic traces at indicated mid-IR vibrational frequencies of Synechocystis Slr1694 BLUF domain in D₂O (A–F) and H₂O (G–L) (circles). The excitation wavelength was 475 nm. The result of the target analysis is shown as a solid line. Note that the time axis is linear from −10 to 10 ps, and logarithmic thereafter.

FIGURE 4

Kinetic scheme used for target analysis of the time-resolved data on the Slr1694 BLUF domain from Synechocystis. See text for details.

FIGURE 5

(A) SADS that follow from a target analysis of the time-resolved mid-IR data of the Synechocystis Slr1694 BLUF domain in D₂O on excitation at 475 nm; (B and C): same as A for the Slr1694 BLUF domain in H₂O. See text for details.

FIGURE 6

Reaction mechanism for the photo-induced hydrogen-bond network switch reaction in the Synechocystis Slr1694 BLUF domain. Hydrogen bonds are represented by dashed lines. See text for details.