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.

Lower frequency region mid-infrared spectroscopy by chirped pulse upconversion

“…Figure 1(d) shows the decay dynamics at ~1550cm −1 after baseline subtraction. The trace was fitted with a single exponential decay with a time constant of 200ps, consistent with previous measurements at this frequency [6].…”

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

“…The absence of an obvious H/D exchange effect on the decay of FAD* [1][2][3][4] indicates that FAD* is deactivated through electron transfer, as for wild-type Slr1694 (19, 21, 22). The decay of FAD* is highly multiexponential and distributed between a few picoseconds and ∼250 ps in wild-type Slr1694 (19,21,22) and the W91F mutant, which is assigned to different conformational subpopulations of the tyrosine side chain having variations in the distance to FAD, with an ensuing distribution of electron transfer rates (9,20). According to the kinetic model of Figure 4 (upper) and that for wild-type Slr1694, a single proton transfer rate of about (5 ps) -1 governs protonation of FAD •-to FADH • .…”

“…H 2 O. A KIE is unexpected on the FAD* lifetime because in BLUF domains electron transfer usually is considered to constitute the primary photochemical reaction (19,20,22,23,48). In addition, if proton or hydrogen transfer underlies FAD* 1 in Figure 6A deactivation, one would under most circumstances expect a slowdown of the reaction upon H/D exchange, not a speeding up.…”

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

“…In various x-ray crystal and NMR solution structures (14)(15)(16)(17)(18)28), as well as spectroscopic (13,22,(39)(40)(41) and theoretical (42)(43)(44) studies, the dark-state orientation of Gln-63 has remained ambiguous. As illustrated in Fig.…”