Abstract

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

Proc Natl Acad Sci U S A. 2006 July 18; 103(29): 10895–10900. Open Access Article.

 

Abstract

 

BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae, where they control a range of physiological responses including photosynthesis gene expression, photophobia, and negative phototaxis. Other than in well known photoreceptors such as the rhodopsins and phytochromes, BLUF domains are sensitive to light through an oxidized flavin rather than an isomerizable cofactor. To understand the physicochemical basis of BLUF domain photoactivation, we have applied femtosecond transient absorption spectroscopy to the Slr1694 BLUF domain of Synechocystis PCC6803. We show that photoactivation of BLUF domains proceeds by means of a radical-pair mechanism, driven by electron and proton transfer from the protein to the flavin, resulting in the transient formation of anionic and neutral flavin radical species that finally result in the long-lived signaling state on a 100-ps timescale. A pronounced deuteration effect is observed on the lifetimes of the intermediate radical species, indicating that proton movements underlie their molecular transformations. We propose a photoactivation mechanism that involves a successive rupture of hydrogen bonds between a conserved tyrosine and glutamine by light-induced electron transfer from tyrosine to flavin and between the glutamine and flavin by subsequent protonation at flavin N5. These events allow a reorientation of the conserved glutamine, resulting in a switching of the hydrogen-bond network connecting the chromophore to the protein, followed by radical-pair recombination, which locks the glutamine in place. It is suggested that the redox potential of flavin generally defines the light sensitivity of flavin-binding photoreceptors.

 

 

 

 

 

Organisms have evolved extensive sensory mechanisms to perceive information carried by light. Their responses are mediated by photoreceptor proteins, which are sensitive to light through prosthetic chromophore molecules. The past decade has witnessed the discovery of a large number of novel flavin-binding photoreceptors, notably the phototropins, the cryptochromes, and BLUF (blue-light sensing using FAD) domains (1). Phototropins are primarily found in plants and control several physiological responses such as phototropism, chloroplast movement, and stomatal opening (2), whereas cryptochromes are known to regulate growth and development in plants and circadian rhythms in plants and insects (3, 4). BLUF domains are a distinct family of flavin-binding photoreceptors that show no significant relationship to other sensor proteins in sequence or structure. The BLUF domain was first discovered as the N-terminal part of the flavoprotein AppA from the purple photosynthetic bacterium Rhodobacter sphaeroides and was shown to control photosynthesis gene expression in response to high-intensity blue-light irradiation and variation of the oxygen tension (5, 6). In later work, the BLUF-containing photoactivated adenylyl cyclase (PAC) photoreceptor was demonstrated to mediate photophobic responses in the green alga Euglena gracilis (7).

The generation of a light signal by photoreceptor proteins relies on the formation of a long-lived signaling state, where initial local structural changes resulting from light absorption by the chromophore are relayed to the protein surface, which undergoes specific alterations that may be sensed by signaling partner proteins. In “traditional” photoreceptors such as the rhodopsins, phytochromes and the xanthopsins, the initial local change is achieved by rapid E/Z isomerization photochemistry of their chromophore, which in turn initiates larger conformational changes by creating steric conflicts, or by charge movements among chromophore and apoprotein (1). In the absence of isomerizable groups, flavin-binding photoreceptors rely on different modes of light activation and thus provide an alternative window on how photon absorption may be coupled to biological sensory function. In the phototropins, for instance, signaling occurs through the light-induced formation of a covalent adduct between the flavin chromophore and a conserved cysteine in light, oxygen, or voltage (LOV) domains, which eventually leads to autophosphorylation of a C-terminal Ser/Thr kinase (8, 9). In analogy with the related photolyases, cryptochromes are thought to function by means of light-induced electron transfer (ET) reactions (10), but at present their mode of action remains largely obscure.

Recently, crystal and solution structures of several BLUF domains were obtained (11–14). The BLUF domain shows a ferredoxin-like fold consisting of a five-stranded β-sheet with two α-helices packed on one side of the sheet, with the noncovalently bound isoalloxazine ring of flavin adenine dinucleotide (FAD) positioned between the two α-helices. Fig. 1 shows a close-up of the FAD-binding pocket of the AppA BLUF domain (11). FAD is involved in an extensive hydrogen-bond network with residues lining the FAD binding pocket, including a highly conserved tyrosine (Tyr) and glutamine (Gln). Upon illumination with blue light, BLUF domains show a characteristic spectral red-shift of their absorption spectrum by ≈10 nm, which is a unique feature of this photoreceptor family and is thought to correspond to the signaling state (5).

The photochemical mechanism by which BLUF domains are activated by blue light is under considerable debate. Early studies suggested that an increased π–π 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. Masuda et al. (17) interpreted their results from Fourier transform infrared (FTIR) spectroscopy by a hydrogen-bond rearrangement around the FAD chromophore upon signaling-state formation. Anderson et al. (11) have proposed a photoactivation model that involves such a hydrogen-bond rearrangement accompanying a 180° rotation of the conserved Gln, as shown in Fig. 1. In this view, photon absorption leads to the breaking of hydrogen bonds from the Gln amino group to the N5 of flavin and to the Tyr and formation of a hydrogen bond to the O4 of the flavin. Ultrafast spectroscopy on the AppA BLUF domain showed that the red-shifted product state is formed in <1 ns, directly from the FAD singlet excited state and without any apparent reaction intermediate (18), yielding no clues on the validity of any of these scenarios.

 

Here, we use femtosecond transient absorption spectroscopy to investigate the photochemistry in the Slr1694 BLUF domain of the cyanobacterium Synechocystis PCC6803. We show that formation of the red-shifted signaling state proceeds by means of a radical-pair mechanism, driven by ET and PT processes to FAD from its protein environment and resulting in the transient formation of anionic and neutral FAD radical species. We propose a photoactivation mechanism that involves a successive rupture of hydrogen bonds between the conserved Tyr and Gln by ET and between the Gln and FAD by light-induced protonation at N5, which allows a reorientation of the conserved Gln and subsequent switch of hydrogen-bond network connecting the chromophore to the protein.