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BLUF (blue light sensing using FAD) domains constitute a recently discovered class …

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

Photoactivation Mechanism in BLUF Domains. The observation of anionic and neutral semiquinones in the Slr1694 photocycle implies that light-driven ET is followed by PT between FAD and aromatic residues in the protein. The picosecond time scale of these events indicates that the residue(s) involved in the transfer must be in close proximity to the isoalloxazine ring of the flavin. 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 flavin/flavin•− is approximately −0.8 V and for Tyr/Tyr•+ is ≈0.93 V (25, 27), providing a driving force for ET of ΔG = −0.62 eV given that the energy of the 0–0 transition of S0S1 is ≈ 2.35 eV (1 eV = 1.602 × 10−19 J) (27).

In the various BLUF crystal structures, the dark-state orientation of the conserved Gln remained ambiguous: according to Anderson et al. (11) the amino group of the conserved Gln would donate hydrogen bonds to N5 and the conserved Tyr, whereas Jung et al. (13) and Kita et al. (12) favored an orientation where the Gln’s amino group donates a hydrogen bond to O4 and receives a hydrogen bond from the conserved Tyr. Fourier transform infrared results by Masuda et al. (17) indicated formation of hydrogen bonds to O4 and loss of a hydrogen bond to N5 of FAD upon illumination. Recent work by Unno et al. (28) showed that mutation of the conserved Gln in the AppA BLUF domain did not alter hydrogen bonding of O4. The latter observations are consistent with a dark orientation as shown in Fig. 1A. Thus, the accumulated evidence points favorably in the direction of a hydrogen-bond switching by means of a Gln flip, as proposed by Anderson et al. (11). 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*. This mechanism does not involve radical flavin intermediates and is clearly at odds with the present results.

We now are in a position to propose a photochemical reaction mechanism for the photoactivation of BLUF domains, which is schematically indicated in Fig. 5. In the dark state, the mutual orientation of Gln and FAD is arranged as indicated in Fig. 5A, with hydrogen bonds from the amino group of Gln-50 to Tyr-8 and the N5 atom of FAD. Blue-light excitation induces a change in the redox potential of FAD, which causes it to accept an electron, resulting in formation of the FAD anionic semiquinone FAD•−, as indicated in Fig. 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. The spectroscopic properties of FAD•− as determined from the target analysis indicate that it forms a CT complex, possibly with the oxidized Tyr-8 radical or perhaps with another positively charged residue in the vicinity of FAD.

Upon its reduction, the flavin becomes a strong base that attracts a proton. The closest proton source is the amino group of Gln-50, from which most likely a proton is abstracted. In turn, Gln-50 abstracts a proton from Tyr-8, which at the time is oxidized and highly reactive, resulting in the radical pair FADH–Tyr (Fig. 5C). The PT event takes 6 ps and slows down 3-fold upon deuteration. It is well established that protonation of the neutral flavin semiquinone radical FADH takes place at N5 (32), which inevitably breaks the hydrogen bond between N5 and Gln-50, leaving Gln-50 unhinged and free to rotate. The hydrogen-bond rupture constitutes the triggering event that allows an ≈180° flip of Gln-50, which results in a new hydrogen bond from the amino group of Gln-50 to the O4 of the flavin (Fig. 5D). Possibly, the carbonyl of Gln-50 transiently accepts a hydrogen bond from the newly protonated N5 (configuration not shown). After 65 ps, radical-pair recombination takes place between FADH and Tyr-8, whereby the hydrogen atom located on N5 of the flavin returns to Tyr-8, possibly using the carbonyl of Gln-50 as a stepping stone (Fig. 5E). The reprotonated Tyr then donates a hydrogen bond to the carbonyl group of Gln-50, locking it in place. This final step is subject to a KIE of 2.4 upon H/D exchange and is attributed to formation of the long-lived signaling state. In this conformation, the amino moiety of Gln-50 donates a hydrogen bond to O4 of the flavin, and the carbonyl moiety of Gln-50 accepts a hydrogen bond from Tyr-8. Possibly, the amino moiety of Gln-50 hydrogen bonds to N5 as well (configuration not shown). The protein remains in this conformation for a long time because of high energetic barriers.

The potential involvement of the conserved Trp (Trp-91 in Slr1694, Trp-104 in AppA) is not taken into account because earlier work has shown that Trp-104 is not required for photocycling in AppA (20). The driving force is larger for Tyr than for Trp (net ΔG = −0.62 eV for Tyr and −0.4 eV for Trp), and (from the AppA crystal structure) the distances from Tyr-21 and Trp-104 to N5 of FAD are 4.5 and 5.7 Å, respectively (11), indicating that Tyr has a better spatial overlap with the flavin than Trp. The crystal structures of the R. sphaeroides BlrB and Thermosynechococcus Tll0078 BLUF domains indicated a different orientation for the conserved Trp, with a significantly increased distance to FAD (12, 13).

With the present reaction model, predictions can be made with regard to the photochemistry of the nonphotocycling Gln-50 and Tyr-8 mutants. With Gln-50 deleted, ET and possibly PT may still occur from Tyr-8 upon excitation of the flavin. In the absence of the switchable Gln-50 residue, the resulting radical pair will very likely recombine to the original ground state without the production of long-lived photoproducts. Upon deletion of Tyr-8, the light-driven ET process that underlies the photoactivation reaction is shut off, which readily explains the inability of mutants to photocycle. However, the conserved Trp (Trp-91 in Slr1694, Trp-104 in AppA) may complement the ET process to FAD without initiating the photocycle. 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).

Comparison with Other BLUF Domains. Given the similarities in structure and sequence between all BLUF domains studied so far, a similar reaction mechanism very likely holds. In AppA, the FAD* lifetime is >1 order of magnitude longer than in Slr1694, with time constants of 90 and 590 ps. The red-shifted signaling state is apparently formed directly from FAD* (18). The reason that no transient reaction intermediates are observed probably stems from a rate-limiting initial ET rate from FAD*. Such differences in ET rates likely arise from different redox potentials of FAD, because these are sensitive to its hydrogen-bonding pattern and the vicinity of charged residues. It is interesting to note that in AppA, a positively charged His-44 hydrogen bonds to the O2 in lieu of a neutral Asn-31 in Slr1694. In the context of the present results, the formation of the FAD•− anionic radical in AppA would occur with time constants of 90 and 590 ps, which rapidly evolves into the FADH intermediate and long-lived product AppARED on much faster timescales. In this way, the FAD•− and FADH intermediates do not transiently accumulate during the reaction and therefore escape detection. A rate-limiting light-driven ET rather than a hydrogen transfer in the AppA BLUF domain is consistent with the absence of a KIE on the FAD excited-state lifetime upon H/D exchange (20).

Light-Induced ET: A General Theme in Flavin-Binding Photoreceptors? Three families of flavin-binding photoreceptors have thus far been identified: the phototropins [with light, oxygen, or voltage (LOV) domains as flavin-binding domains], the cryptochromes, and the BLUF domains. Although their modes of action appear very distinct, there are interesting parallels. The present results show that the hydrogen-bond switch in BLUF domains is driven by initial ET from aromatic residues to the oxidized flavin. In the cryptochromes, similar ET processes appear to underlie their activity, with Trps as likely electron donors (10, 33). The question remains whether in LOV domains, ET or PT from a conserved Cys to the FMN chromophore constitutes the primary photochemical event (9, 3436). If the former situation applies, we may indeed conclude that it is the redox potential of the flavin chromophore that defines the light sensitivity of flavin-binding photoreceptors, very much so as it defines the catalytic activity in most flavoenzymes (37).


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