Mechanism of the AppA_BLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 pK_a on the Forward and Reverse Ground-State Reactions

Abstract

The transcriptional antirepressor AppA is a blue light using flavin (BLUF) photoreceptor that releases the transcriptional repressor PpsR upon photoexcitation. Light activation of AppA involves changes in a hydrogen bonding network that surrounds the flavin chromophore on the nanosecond timescale, while the dark state of AppA is then recovered in a light independent reaction with a dramatically longer half-life of ~18 min. Residue Y21, a component of the hydrogen bonding network, is known to be essential for photoactivity. Here we directly explore the effect of the Y21 pKa on dark state recovery by replacing Y21 with fluorotyrosine analogs that increase the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements confirm that the structure of AppA is unperturbed by fluorotyrosine substitution, and that there is a small (3-fold) change in the photokinetics of the forward reaction over the fluorotyrosine series. However, reduction of 3.5 pH units in the pKa of Y21 increases the rate of dark state recovery by 4,000-fold with a Brønsted coefficient of ~ 1, indicating that the Y21 proton is completely transferred in the transition state leading from light to dark adapted AppA. A large solvent isotope effect of ~6-8 is also observed on the rate of dark state recovery. These data establish that the acidity of Y21 is a crucial factor for stabilizing the light activated form of the protein, and have been used to propose a model for dark state recovery that will ultimately prove useful for tuning the properties of BLUF photosensors for optogenetic applications.

Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

“…Although little impact was observed on the forward light driven reaction in AppA BLUF , 21 the F-Tyr analogues are shown here to have a profound impact on the photoactivation mechanism of PixD. In wild-type PixD there is evidence from ultrafast infrared spectroscopy for the sequential formation of FAD •− followed by FADH • on the reaction coordinate for light state formation.…”

“…Further, as was observed for AppA BLUF , reduction in the Y8 p K a monotonically accelerates the rate of dark state recovery (Table 2). However, the relative increase in the rate of recovery is much less marked in PixD: in H 2 O, the rate increases 970-fold between wild-type AppA BLUF and 3,5-F 2 Y21 AppA BLUF , 21 compared to only 15-fold for PixD, while in D 2 O the change in rate is 850-fold for AppA BLUF and 6-fold for PixD. The corresponding Brønsted plots for PixD have shallower slopes of 0.43 in H 2 O and 0.28 in D 2 O (Figure 8), compared to slopes of ~1 for AppA BLUF .…”

“…21 3-FY8 PixD exhibits the highest similarity to the wild-type protein although the rate of spectral evolution is described by significantly longer time constants compared to wild-type PixD (Table 1). The light state, characterized by the transient at 1690 cm −1 forms within 230 ps, although a considerable amount of the excited state is still present at this stage, again consistent with the presence of multiple conformations and an associated distribution of rates.…”

“…In some BLUF protein structures a conserved Trp is within hydrogen bonding distance of the network, and indeed the variable location of the Trp side chain, between this Trp in conformation and a Trp out conformation, supports proposed mechanisms in which a light-induced change in Trp conformation is on the reaction coordinate for photoactivation. 7, 20–21 …”

“…34 We further confirmed that electron transfer did not occur during the photoactivation of AppA BLUF using the site-specific incorporation of fluorotyrosine (F-Tyr) analogs into position Y21. 21 The observation of radical formation in PixD, 34, 38 and in the BLUF protein PapB, 39 thus suggests that significant differences exist in the mechanism of photoactivation within the BLUF protein family.…”