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

Abstract

Proton relays through H-bond networks are essential in realizing the functionality of protein machines such as in photosynthesis and photoreceptors. It has been challenging to dissect the rates and energetics of individual proton-transfer steps during the proton relay. Here, we have designed a proton rocking blue light using a flavin (BLUF) domain with the flavin mononucleotide (FMN)–glutamic acid (E)–tryptophan (W) triad and have resolved the four individual proton-transfer steps kinetically using ultrafast spectroscopy. We have found that after the photo-induced charge separation forming FMN· –/E-COOH/WH· +, the proton first rapidly jumps from the bridging E-COOH to FMN– (τfPT2 = 3.8 ps; KIE = 1.0), followed by a second proton transfer from WH· + to E-COO– (τfPT1 = 336 ps; KIE = 2.6) which immediately rocks back to W· (τrPT1 = 85 ps; KIE = 6.7), followed by a proton return from FMNH· to E-COO– (τrPT2 = 34 ps; KIE = 3.3) with the final charge recombination between FMN· – and WH· + to close the reaction cycle. Our results revisited the Grotthuss mechanism on the ultrafast timescale using the BLUF domain as a paradigm protein.

Elementary Reactions in the Functional Triads of the Blue-Light Photoreceptor BLUF Domain

“…We tackle this problem by designing mutants to modulate the driving force of the proton relay, thereby capturing the PR intermediates and understanding their nature before we move to functional motifs. Hence, we designed Y6W/Q48E/W90F (referred to as FMN-Glu-Trp) in which Tyr6 is mutated into Trp to decouple the ET from the proton relay and Gln48 is substituted with Glu to reverse the sequence of the two PT steps . The PT from Glu to FMN happens before the PT from Trp to Glu due to the side chain of Glu (p K a = 4.15 in water) having a much lower p K a value than that of the native Gln (p K a (−NH 2 ) ∼ 15.1). , In this way, the purported PR intermediate has increased its accumulation from ∼3.3% relative to the FMN* population in the functional FMN-Gln-Tyr (from the kinetics simulation) to a significant 64% in the designed FMN-Glu-Trp, leading to the capture and quantification of the PR intermediate in FMN-Glu-Trp (Figure B).…”

“…Hence, we designed Y6W/Q48E/W90F (referred to as FMN-Glu-Trp) in which Tyr6 is mutated into Trp to decouple the ET from the proton relay and Gln48 is substituted with Glu to reverse the sequence of the two PT steps . The PT from Glu to FMN happens before the PT from Trp to Glu due to the side chain of Glu (p K a = 4.15 in water) having a much lower p K a value than that of the native Gln (p K a (−NH 2 ) ∼ 15.1). , In this way, the purported PR intermediate has increased its accumulation from ∼3.3% relative to the FMN* population in the functional FMN-Gln-Tyr (from the kinetics simulation) to a significant 64% in the designed FMN-Glu-Trp, leading to the capture and quantification of the PR intermediate in FMN-Glu-Trp (Figure B). By elucidating all of the elementary steps, we found that after the photoinduced forward ET (1.0 ps, KIE = 1.0) forming the CS intermediate (FMN •– /Glu-COOH/TrpH •+ ), it will proceed with the forward PT2 (3.8 ps, KIE = 1.0) between the bridge and FMN •– forming the PR intermediate (FMNH • /Glu-COO – /TrpH •+ ) followed with forward PT1 (336 ps, KIE = 2.6) between Trp and the bridge forming the DR intermediate (FMNH • /Glu-COOH/Trp • ) (Figure ).…”

“…30 From the "proton relay" section, we deduce that the absence of the PR intermediate in the dark-state photocycle is likely due to kinetics reasons of a slow rise (fPCET1) and fast decay (fPT2) reaction, as shown in Figure 5C,D. 63 Taken as a whole, our work points out that the key to resolve the dark-state photocycle is to search for trace evidence of the PR intermediate since it is one important missing piece in the puzzle. Besides the PR intermediate (FMNH • /Glu-COO − / TrpH •+ ) in FMN-Glu-Trp when the forward PT2 proceeds before the forward PT1, 63 we recently have captured the PR intermediates in YnF mutants from the AppA and SyPixD BLUF domain where the central Tyr is mutated into Phe, retaining the nearby Trp.…”

“…65,66 In this way, the purported PR intermediate has increased its accumulation from ∼3.3% relative to the FMN* population in the functional FMN-Gln-Tyr (from the kinetics simulation) to a significant 64% in the designed FMN-Glu-Trp, leading to the capture and quantification of the PR intermediate in FMN-Glu-Trp (Figure 5B). By elucidating all of the elementary steps, 63 we found that after the photoinduced forward ET (1.0 ps, KIE = 1.0) forming the CS intermediate (FMN •− /Glu-COOH/ TrpH •+ ), it will proceed with the forward PT2 (3.8 ps, KIE = 1.0) between the bridge and FMN •− forming the PR intermediate (FMNH • /Glu-COO − /TrpH •+ ) followed with forward PT1 (336 ps, KIE = 2.6) between Trp and the bridge forming the DR intermediate (FMNH • /Glu-COOH/Trp • ) (Figure 5). In the reverse processes, the DR intermediate undergoes reverse PT1 (85 ps, KIE = 6.7) reforming the PR intermediate and subsequently proceeds with reverse PT2 (34 ps, KIE = 3.3) to reform the CS intermediate and finally charge recombine (25 ps, KIE = 1.3) to regenerate the ground state (FMN/Glu-COOH/TrpH) (Figure 5), demonstrating a bidirectional proton relay or a double proton rocking process.…”

Photodegradable by Yellow-Orange Light degFusionRed Optogenetic Module with Autocatalytically Formed Chromophore

“…One type incorporates FMN as a chromophore: light-oxygen-voltage domain 2 (LOV2) [ 7 ], GIGANTEA [ 8 ], Vivid domain (VVD) [ 9 ] and its derivatives, including Magnets [ 10 ] and TULIPS [ 11 ] (VVD and its derivatives incorporate both FMN and FAD). The second type incorporates only FAD as a chromophore: cryptochrome 2 (CRY2) [ 12 ] and BLUF domain [ 13 ]. The third type incorporates p-coumaric acid as a chromophore: photoactivatable yellow protein (PYP) [ 7 ].…”