The radical that was never seen: one added hydrogen bond opens up the photoactivation mechanism of a blue-light receptor
A collaboration between researchers in the United States, the United Kingdom and Hungary has mutated the amino acid sequence of the AppA BLUF domain, a blue-light photoreceptor, and succeeded in capturing the proton-coupled electron transfer (PCET) radical intermediates that had never been observed in this protein. By introducing a double mutation (H44N, H78R) that adds a second hydrogen bond at the C2=O position of the flavin (FAD), the team steered AppA's photoactivation pathway toward the PixD model, in which radicals are observed. The work shows that a photoreceptor's "on" pathway (photoactivation) and its "off" pathway (dark state recovery) can be manipulated separately, while the authors also raise the possibility that these radicals were not newly created but were already present below an observability threshold and have simply been pushed above it. [Quantum Biology Society] Proton-coupled electron transfer (PCET), in which an electron and a proton move together and generate radicals, is a core mechanism of biological energy conversion and redox regulation. The BLUF (Blue Light Using FAD) domain, a photoreceptor that senses blue light, is understood to make use of it as well. Within the BLUF family, however, different proteins have given conflicting results, and the argument has persisted. In the cyanobacterial protein PixD, flavin radicals (FAD•⁻ and FADH•) are clearly observed during the photocycle, which established the PCET model. In the closely related protein AppA, from the purple bacterium Rhodobacter sphaeroides, no radical intermediates appeared in the ultrafast infrared spectra at all, and the same was true of BlsA. A study by a US, UK and Hungarian collaboration (corresponding authors Peter Tonge, Stephen Meech and Andras Lukacs), published in the Journal of the American Chemical Society in issue 1 of 2025, tackles that long-standing dilemma head-on. Instead of continuing the argument, the team chose experimental demonstration: deliberately reshaping the protein structure so that AppA switches over to the PixD mechanism. ■ Adding a hydrogen bond: in search of the missing intermediate The decisive difference the team focused on was the number of hydrogen bonds interacting with the C2=O position of the FAD molecule. In PixD two residues form hydrogen bonds there, while AppA has only one. The limits of a single mutation: when H44 in AppA was first replaced with other amino acids, the C2=O vibrational band shifted down from 1650 cm⁻¹ (to 1642 cm⁻¹ in H44N), but the radical marker near 1520 cm⁻¹ still did not appear. Capturing the radical with a double mutation: to mimic the role of R65 in PixD, the team replaced H78 in AppA with arginine, producing the double variant H44N,H78R. Time-resolved infrared (TRIR) spectra showed the C2=O band dropping to 1634 cm⁻¹, confirming that a second hydrogen bond had formed. Notably, the single H78R variant on its own showed no radical intermediate, so both substitutions were needed. Evidence for stepwise PCET: within 1 ps of illumination, new transient absorption bands appeared at 1507 cm⁻¹ and 1521 cm⁻¹, corresponding to the tyrosine cation radical (Tyr•⁺) and the flavin anion radical (FAD•⁻). At 8 ps, FAD* and Tyr•⁺ decayed as a band at 1532 cm⁻¹, assigned to the neutral semiquinone radical FADH•, appeared. Because decay of the 1521 cm⁻¹ band precedes that of the 1532 cm⁻¹ band, the authors take the electron and proton transfer to be stepwise rather than simultaneous. ■ Rewiring the activation pathway while the reverse reaction stays put To establish that the radicals observed were not incidental by-products but lie on the obligatory path to photoactivation, the team replaced the conserved tyrosine Y21 in the double variant with fluoro-tyrosines of higher acidity, which lower the phenol pKa from 9.9 in tyrosine to 8.4 for the monofluoro analogue and 6.4 for the trifluoro one. In the most acidic variant, 2,3,5F₃Y21, the transient at 1532 cm⁻¹ was absent, which the authors read as the increased acidity of Y21 preventing formation of the FADH• state and halting the photocycle at FAD•⁻. The protein vibrational mode at 1618 cm⁻¹, which appears when the final signalling state forms, also disappeared. Together this indicates that the radicals found in the double variant are key intermediates on the way to the light state. More striking still is the rate of recovery to the dark state, the reverse reaction. Despite the forward photoactivation pathway having been thoroughly rebuilt, the recovery rate of the double variant rose only about twofold relative to wild-type AppA. Its sensitivity to fluoro-tyrosine substitution also kept the wild-type AppA pattern rather than the PixD one: a 34-fold acceleration in 3FY21 H44N,H78R, close to the 51-fold seen in 3FY21 wild-type AppA, against only 5.8-fold in the corresponding PixD variant. This suggests that a photoreceptor's path into the light state and its path back out are chemically separable and can be manipulated independently. ■ Significance and open questions The study is a notable achievement in showing that manipulating specific amino acid residues alone can dramatically change how a PCET pathway inside a protein is activated. The authors do not gloss over how complicated the interpretation is. Recent quantum mechanics/molecular mechanics (QM/MM) calculations suggest that the PCET process is not a single clean step but a set of multiple relaxation pathways deeply entangled with the dynamics of the surrounding protein. Accordingly, the authors write that this work is probably not a matter of "creating PCET that wild-type AppA did not have", but rather of the C2=O interactions perturbing protein dynamics and charge-transfer state stabilisation so as to push the population and decay rate of metastable radical intermediates, previously hidden below an observability threshold, above that threshold. Work on another BLUF protein, OaPAC, has meanwhile reported that the forward PCET there proceeds by a concerted mechanism, so the detailed mechanism of the BLUF photocycle remains an open question requiring further study. #QuantumBiology #ProtonCoupledElectronTransfer #PCET #BLUF #Photoreceptor #Flavin #FAD #AppA #PixD #RadicalIntermediates #TimeResolvedInfrared #Fluorotyrosine #HydrogenBondNetwork #Optogenetics #Rhodobacter https://pmc.ncbi.nlm.nih.gov/articles/PMC11726546/