The nature of proton-coupled electron transfer in a blue light using flavin domain

Abstract

Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be ∼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNH˙. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.

Dissecting the Ultrafast Stepwise Bidirectional Proton Relay in a Blue-Light Photoreceptor

“…The KIE values (k H /k D ) for the photochemistry in FMN-E-W are calculated from the acquired rates (Table 1). Note that the KIE for the entire proton relay process has been reported previously, 16,17,29 and our work here allows the dissecting of each elementary step. The pure ET processes fET (1.0) and reverse ET (rET) (1.3) have very small KIE values.…”

“…16,17 However, individual PT steps in the forward and reverse relay pathways have not been dissected for the past 20 years, and it is unknown whether the proton relay in the BLUF domain is concerted or step-wise due to the absence of relay intermediates. 13,14,16,17 In this work, we have designed a BLUF domain-based proton rocking machine featuring a central FMN-E-W motif (Figure 1b). Using ultrafast spectroscopy and kinetics analysis, we captured relay intermediates and unambiguously determined the rates and kinetic isotope effects (KIE) of all four fundamental PT steps.…”

Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction

“…The hydrogen-bond patterns that connect the FAD to its neighboring residues and their relation to the photodynamics in BLUF photoreceptors have remained controversial, despite many studies with X-ray crystallography, ,,− NMR, − Fourier-transform infrared spectroscopy (FTIR), − computational studies, ,− and time-resolved spectroscopies. ,,,,,,,− This issue largely finds its origin in the difficulty in X-ray crystallography to distinguish between O and N nuclei, and the general elusiveness of specific Gln signals in spectroscopy. Figure d,e shows four models that may account for hydrogen bond realizations in DA and LA states: model 1 and 2 derive from X-ray crystallographic structures, ,,− while model 3 and 4 were put forward on the basis of computational studies. , Note that the positions of a conserved Met and a semiconserved Trp , have not been considered here.…”

“…24 In the Oscillatoria acuminata BLUF domain, a concerted PCET reaction on the hundreds of ps to result in FADH • was found. 25 Remarkably, in the AppA and BlrB BLUF domains from Rhodobacter sphaeroides, the hydrogen bond rearrangement proceeds without the apparent involvement of radical species, 26−30 species have escaped detection due to kinetic limitations. As a result of the hydrogen-bond switch reaction, conformational changes occur in the BLUF fold on longer timescales 31,32 that may involve a slide or extension of the β5 strand, 33 as demonstrated for Slr1694.…”

“…First, FAD •– is formed in a multiphasic fashion in 7, 40, and 180 ps , (Figure S1) upon Tyr–FAD electron transfer. , Subsequently, proton transfer proceeds in 6 ps, resulting in the formation of FADH • . , Finally, reoxidation of FAD and a hydrogen bond rearrangement are completed in 65 ps. , Similar dynamics involving FAD radicals were observed in the PapB BLUF domain of Rhodopseudomonas palustris . In the Oscillatoria acuminata BLUF domain, a concerted PCET reaction on the hundreds of ps to result in FADH • was found . Remarkably, in the AppA and BlrB BLUF domains from Rhodobacter sphaeroides, the hydrogen bond rearrangement proceeds without the apparent involvement of radical species, − but it remains unclear whether such species have escaped detection due to kinetic limitations.…”