Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from Rhodobacter sphaeroides

Abstract

AppA is a blue-light and redox-responding regulator of photosynthesis gene expression in Rhodobacter sphaeroides. Detailed time-resolved fluorescence spectroscopy and subpicosecond transient absorption spectroscopy study of the BLUF domain is presented for wild-type AppA (AppAwt) and a photoinactive Y21F mutant of AppA. The main findings discussed here are that (1) time-resolved laser excitation studies on dark-adapted protein show that AppAwt and Y21F mutant protein exhibits a fluorescence decay with a lifetime of 0.6 ns. Dark-adapted AppAwt but not Y21F also exhibits slower fluorescence decay with a lifetime of 1.7 ns. Analysis of AppAwt that was light-excited to a stable light-adapted form prior to data collection shows monoexponential fluorescence decay with a lifetime of 1.0 ns. This component disappeared after 1 min of data collection after which the original "dark-adapted" values were recovered, demonstrating the presence of a approximately 1 min lifetime intermediate during the return of AppA from light- to dark-adapted form. (2) Transient absorption spectral analysis reveals a very fast rising of transient absorption (<1 ps) for AppAwt. This fast component is missing in the Y21F mutant, which lacks Tyr21, giving rise to a slower transient absorption at 4-6 ps. In the AppAwt transient spectra, most ground states recover within approximately 30 ps, compared to approximately 90-130 ps in the mutant Y21F. We propose that a temporary electron transfer occurs from Tyr21 to the N5 of flavin in AppAwt and is a triggering event for subsequent hydrogen-bond rearrangements. Dynamics of the AppA photocycle is discussed in view of the currently solved crystallographic structure of AppA.

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

“…5B. In view of the close proximity between FAD and the conserved Tyr, we propose that Tyr-8 acts as the electron donor as suggested for AppA (30,31). This event breaks the hydrogen bond between Tyr-8 and Gln-50 as a result of increased electrostatic repulsion by Tyr-8.…”

“…Indeed, recent experiments on the Tyr-21 mutant of AppA showed light-driven ET from Trp-104 on a picosecond timescale, followed by radical-pair recombination on the (sub) nanosecond timescale to the original ground state (ref. 31 and M.G., W. Laan, I.H.M.v.S., R.v.G., K. J. Hellingwerf, and J.T.M.K., unpublished data).…”

Role of active site conformational changes in photocycle activation of the AppA BLUF photoreceptor

“…Experimentally, the photocycle of the AppA BLUF domain has been found to have essentially the same features as that of the full AppA protein (34). It shows multiexponential decay kinetics following photoexcitation that could arise from conformational heterogeneity in the ground-state ensemble and/or multiple decay pathways (35). Our free-energy calculations indicate that the active site region of the AppA BLUF domain does indeed have significant conformational flexibility in the ground state, characterized by conformational changes of residues Tyr21, Gln63, Trp104, and Met106.…”

Why BLUF photoreceptors with roseoflavincofactors lose their biological functionality

“…These include AppA, [11][12][13][14][15][16] BlrB, 17,18 Slr1694 [19][20][21][22][23] and Tll0078. [24][25][26] Most of them have different physiological functions but share a common feature, namely the change in their UV/Vis-, as well as in their IR-spectra upon signaling state formation.…”