Spectroscopic and Mutational Analysis of the Blue-Light Photoreceptor AppA:  A Novel Photocycle Involving Flavin Stacking with an Aromatic Amino Acid

Abstract

The flavoprotein AppA is a blue-light photoreceptor that functions as an antirepressor of photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides. Heterologous expression studies show that FAD binds to a 156 amino acid N-terminal domain of AppA and that this domain is itself photoactive. A pulse of white light causes FAD absorption to be red shifted in a biphasic process with a fast phase occurring in <1 micros and a slow phase occurring at approximately 5 ms. The absorbance shift was spontaneously restored over a 30 min period, also in a biphasic process as assayed by fluorescence quenching and electronic absorption analyses. Site-directed replacement of Tyr21 with Leu or Phe abolished the photochemical reaction implicating involvement of Tyr21 in the photocycle. Nuclear magnetic resonance analysis of wild-type and mutant proteins also indicates that Tyr21 forms pi-pi stacking interactions with the isoalloxazine ring of FAD. We propose that photochemical excitation of the flavin results in strengthening of a hydrogen bond between the flavin and Tyr 21 leading to a stable local conformational change in AppA.

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

“…This mechanism also can explain why the photoactivated state is so stable and lives from seconds in Slr1694 to a half-hour in AppA. In support of this hypothesis, mutation of the conserved Tyr or the conserved Gln results in a complete loss of photocycling activity (12,15,16,28,29). With the R. sphaeroides BlrB structure at hand, Jung et al (13) presented an alternative that would imply a light-induced PT from Arg-32 to the O2 of the flavin from FAD*.…”

“…Previous studies on flavo-enzymes have revealed that rapid light-driven ET may occur from aromatic residues to flavin (25,26). Tyr-8 (Tyr-21 in AppA) is a good candidate because it is the closest aromatic residue to the flavin and has been shown to be critical for photocycling activity in BLUF domains (12,15,16). The redox properties of flavin and Tyr gives a favorable driving force for the ET reaction: the midpoint potential for flavin͞flavin •Ϫ is approximately Ϫ0.8 V and for Tyr͞Tyr •ϩ is Ϸ0.93 V (25,27), providing a driving force for ET of ⌬G ϭ Ϫ0.62 eV given that the energy of the 0-0 transition of S 0 3 S 1 is Ϸ 2.35 eV (1 eV ϭ 1.602 ϫ 10 Ϫ19 J) (27).…”

“…Early studies suggested that an increased -stacking between Tyr and FAD, or deprotonation of FAD, would induce signaling-state formation (15,16). Jung et al (13) proposed that in the BlrB BLUF domain, proton transfer (PT) takes place from an arginine (Arg) residue to the O2 atom of the isoalloxazine ring upon blue-light absorption, triggered by the increased basicity of O2 upon promotion of FAD to the singlet excited state.…”

PixE promotes dark oligomerization of the BLUF photoreceptor PixD

“…The main interfacing residues occur along a loop (Ile-83-Val-90) located between the fourth and fifth ␤-strands, the start of the fifth ␤-strand (Trp-91-Met-93), and two C-terminal helices. At the joint between this loop and the fifth ␤-strand are highly conserved residues Trp-91 and Met-93 that undergo major conformational changes in response to light as suggested by crystallographic analysis of several BLUF proteins, FTIR, and static fluorescence studies (10,11,13,24) (Fig. 5B).…”

A eukaryotic BLUF domain mediates light-dependent gene expression in the purple bacterium Rhodobacter sphaeroides 2.4.1

“…Recently, details of the AppA photo-excitation process emerged (16,17). Whereas the fully oxidized AppA at high oxygen tension and the fully reduced AppA at low oxygen tension mediate the redox signal independently of light, at intermediate oxygen concentrations light determines whether AppA releases the repressing effect of PpsR (7).…”