Photoadduct Formation from the FMN Singlet Excited State in the

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Letter

Photoadduct Formation from the FMN Singlet Excited State in the LOV2 Domain of Chlamydomonas Reinhardtii Phototropin Jingyi Zhu, Tilo Mathes, Yusaku Hontani, Maxime T.A. Alexandre, Kee Chua Toh, Peter Hegemann, and John T.M. Kennis J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b02075 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 21, 2016

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The Journal of Physical Chemistry Letters

Photoadduct Formation from the FMN Singlet Excited State in the LOV2 Domain of Chlamydomonas reinhardtii Phototropin Jingyi Zhu†, Tilo Mathes†, Yusaku Hontani†, Maxime T.A. Alexandre†, K.C. Toh†, Peter Hegemann‡, and John T. M. Kennis†* †

Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, 1081 De Boelelaan, 1081HV Amsterdam, The Netherlands

‡

Department of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Invalidenstraße 42, 10115 Berlin, Germany

Corresponding Author *

[email protected]

Abstract The two Light, Oxygen, and Voltage domains of phototropin are blue-light photoreceptor domains which control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds. We demonstrate

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that in CrLOV2, a direct photoadduct formation channel originates from the initially excited singlet state, in addition to the canonical reaction through the triplet state. This direct photoadduct reaction is coupled by a proton or hydrogen transfer process, as indicated by a significant kinetic isotope effect of 1.4 on the fluorescence lifetime. Kinetic model analyses showed that 38% of the photoadducts are generated from the singlet excited state.

TOC GRAPHIC

KEYWORDS Phototropism, light oxygen voltage domain, photoreaction, ultrafast spectroscopy, optogenetic actuator, Signal transduction

LOV domains regulate a number of light responses in plants, bacteria and fungi1-3 and are activated through a light-dependent and reversible formation of a covalent thioether bond between a flavin chromophore and a conserved cysteine (Figure 1).4 This local event induces conformational changes at the LOV molecular surface, which communicate the photon absorption event to a signal output module.2-3, 5 LOV domains have a tremendous potential for biotechnology and bioengineering: because of the high degree of modularity with regard to input- and output modules in signaling proteins and LOV-domain based photoreceptors2-6 and the ubiquitous presence of flavins in cells and tissues. Indeed, LOV domains have emerged as an important platform for engineering optogenetic actuators,7-10 ACS Paragon Plus Environment

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fluorescent reporters11 and singlet oxygen generators.12 Hence, unraveling the molecular photoactivation mechanisms is crucial for a proper molecular understanding of the LOV domain signaling function. Upon illumination of plant phototropin LOV2, the flavin mononucleotide (FMN) singlet excited state undergoes intersystem crossing (ISC) to the FMN triplet state on a nanosecond time scale at yield of 60%.13-15 In turn, the FMN triplet converts to the covalent adduct state in microseconds.15-18 This process involves breaking of the S–H bond in the Cys, formation of the new bonds N(5)–H and C(4a)– S, and a change of the electronic spin multiplicity from triplet to singlet at the product state. Radical pair mechanisms were put forward with either a concerted formation of the adduct by simultaneous protonation at N(5) and bond formation between the thiol sulfur and C(4a), or formation of the sulfurcarbon bond without any prior proton transfer.16, 19-22 Although no FMN radicals were ever observed in the wild type LOV photoreaction, there is general agreement on their involvement given the spin-flip that has to occur; most likely their lifetime is too short to accumulate in a detectable transient concentration.19 The question arises if such reaction mechanism generally applies to LOV domains, as only a limited number of LOV domains have been investigated in any detail.13-25

Figure 1.

(a) Photoreaction in LOV domains. (b) Absorption spectra of AsLOV2 (dashed) and

CrLOV2 (solid) and the excitation wavelengths used for transient absorption. Here we used a combination of time resolved absorption and fluorescence spectroscopy, providing complete dynamics over the fs to µs time range for two LOV domains: AsLOV2, the well-studied photACS Paragon Plus Environment


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1 LOV2 domain of Avena sativa, and CrLOV2, the LOV2 domain of Chlamydomonas reinhardtii phototropin. In CrLOV2, fluorescence experiments previously showed that the FMN fluorescence lifetime was shorter than in other LOV domains, and it was suggested that electron transfer from the singlet excited would take place.26 Here, we demonstrate that in CrLOV2, covalent adduct formation takes place directly from the initially excited singlet state, revealing a previously unidentified reaction mechanism in this important class of photoreceptors. Transient absorption changes from near-UV to visible spectral region were recorded after excitation at 404 nm, with an extended time window from femtoseconds to 25 microseconds. Selected timeresolved difference spectra and global-fit extracted evolution-associated difference spectra (EADS) are shown in the Supporting Information (Figure S1, S2). For AsLOV2, we find lifetimes of 3.1 ps, 2.1 ns, 1.9 µs and a nondecaying component which are assigned to the hot singlet,27-28 the vibrationally relaxed singlet, the triplet and the covalent adduct states of FMN, respectively, in agreement with earlier studies.13, 15, 18, 29 In CrLOV2, the lifetimes are 1.7 ps, 1.0 ns, 0.2 µs and a nondecaying component. Both the FMN singlet and triplet states lifetime in CrLOV2 are significantly shorter than those in AsLOV2. To gauge the CrLOV2 data against the well-known photoreaction of AsLOV2, we compare their raw transient absorption spectra at specific delay times. Figure 2a,b shows the spectra that represent the FMN hot singlet (0.5 ps) and vibrationally relaxed singlet excited states (20 ps) respectively, for AsLOV2 (blue) and CrLOV2 (red). The 0.5 ps and 20 ps spectra of CrLOV2 can be scaled by a factor of 1.7 to overlap with those of AsLOV2 throughout the spectral window. The resulting CrLOV2-minusAsLOV2 difference spectra (Figure 2a, b, black symbols) show some residuals only at the blue and red edges. The clear offset at ~380 nm is likely caused by the red shifted S0S2 absorption in AsLOV2 as compared to CrLOV2 (Figure 1b) that accordingly causes a difference in the corresponding bleach, while the minor oscillatory residuals at the red edges are caused by white light fluctuations of the probe. Similar differences are observed for the covalent adduct state at 20 µs (Figure 2d), except that the scaling factor is different. ACS Paragon Plus Environment

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Figure 2c shows the spectra at 12 ns that, for AsLOV2, represent the triplet state.13, 16, 18, 25 In contrast to the other time delays, the 12 ns spectra cannot be scaled throughout the spectral window. A scale factor of 3.4 is required to overlap the 500-800 nm spectral range, which is dominated by triplet absorption. However, this results in a strong difference for the ground state bleach near 450 nm (cf. the blue and red curves for AsLOV and CrLOV2, respectively). Strikingly, the resulting CrLOV2-minusAsLOV2 difference spectrum is similar to that of the photoadduct difference spectrum. Thus, the larger scale factor of the triplet state compared to that of the singlet states indicates that in CrLOV2, less triplet states are generated after singlet excited state relaxation, which agrees with the shorter singlet lifetime (1.2 ns vs. 2.1 ns). In addition, the difference in the bleach region indicates that in parallel with the triplet state, another state is formed which likely corresponds to an early thioadduct.



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Figure 2. Comparison of raw transient absorption spectra of AsLOV2 and CrLOV2 by scaling and subsequent subtraction of CrLOV2-minus-AsLOV2. (a) Hot singlet excited states at 0.5 ps. (b) Relaxed singlet excited states at 20 ps. (c) Triplet state (for AsLOV2) at 12 ns. (d) Adduct states at 20 µs. The spectra of CrLOV2 were red shifted by 2 nm to match the S1 bleach difference of the samples. To further examine the CrLOV2 photodynamics, we carried out a H2O/D2O dependent timeresolved fluorescence experiment. Figure 3 shows the fluorescence decay of AsLOV2 and CrLOV2. Fitted time constants are included in SI (Table S1). For AsLOV2, the fluorescence lifetime hardly changes upon H2O/D2O exchange (2.1 ns vs. 2.3 ns). In contrast, for CrLOV2, the fluorescence lifetime ACS Paragon Plus Environment

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is 1.15 ns in H2O and 1.6 ns in D2O. The kinetic isotope effect (KIE), defined here as the fluorescence lifetime in D2O divided by that in H2O, thus amounts to 1.4 in CrLOV2. Because photoadduct formation requires breaking of the Cys S–H bond and formation of the new bonds N(5)–H and C(4a)–S, a proton or hydrogen transfer to N(5) should be involved. Thus, the KIE observed in CrLOV2 is consistent with a direct photoadduct formation pathway from the FMN singlet state.

Figure 3. Fluorescence decay of (a) AsLOV2 and (b) CrLOV2 in H2O and D2O.

To quantitatively estimate the efficiency of photoadduct formation from the FMN singlet excited state, we performed target analysis with a branched kinetic model (Figure 4a). The model involves initial population of the vibrationally hot FMN singlet excited state (1FMN*hot), which vibrationally relaxes to 1FMN* on a picosecond timescale. From 1FMN*, branching takes place: 1FMN* can evolve to the FMN triplet state (3FMN) at rate kISC, and to the FMN ground state at the summed internal conversion rate kIC and radiative rate kRAD. An additional reaction path with rate kSA was added from 1FMN* directly to the adduct A. For AsLOV2, a zero rate for kSA was set, thus closing ACS Paragon Plus Environment


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this channel and reflecting the classical triplet-driven photocycle (Figure S3). The AsLOV2 analysis shows that adduct formation occurs from 3FMN with around 60% efficiency and an overall adduct quantum yield of 30%, as previously reported.13,

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Notably, the rate constants that describe the

branching from the 1FMN* state were essentially the same as estimated earlier,13 with the ISC rate kISC at (3.3 ns)-1 and internal conversion (IC) rate kIC and radiative rate kRAD summing up to (5 ns)-1. For CrLOV2, the kSA channel from 1FMN* to A was allowed to assume a nonzero value. We required that spectral shape and ratio of triplet and product state species spectra were approximately the same as for AsLOV2 (Figure S4). The CrLOV2 target analysis shows that about 10% of the 1

FMN* population directly forms the adduct with a rate constant kSA of (12 ns)-1. Furthermore, 53%

and 37% of the initially excited states evolve to the ground state and to 3FMN at kIC + kRAD = (2.0 ns)-1 and kISC = (2.8 ns)-1, respectively. The overall quantum yield for production of the covalent adduct A was estimated at 26%, of which 62% is formed via 3FMN and 38% through 1FMN*. This overall quantum yield of 26% is much lower than that estimated earlier by Holzer et al, 90%.26 We consider the latter value unreliable as it was estimated indirectly from time-resolved fluorescence and steady-state fluence curves. The KIE of 1.4 on the CrLOV2 fluorescence lifetime (Figure 3) primarily results from the singlet-state photoadduct reaction (kSA in Figure 4a). However, in the model of Figure 4a, this KIE could at most be 1.1: if kSA becomes infinitely slow in D2O, the 1.1 ns lifetime would then become 1.2 ns (by considering (kIC + kRAD + kISC)-1 in Figure 4a). To provide an explanation for the occurrence of the KIE, the kinetic model may be modified according to Figure 4b. Here, an intermediate state X is tentatively introduced between 1FMN* and A, and is formed with rate constant kSA. Crucially, state X is required to be so short-lived that it does not accumulate in an


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appreciable transient concentration, i.e., k1 >> kSA, and hence is not observed in the time-resolved experiments.

Figure 4. Target analysis of CrLOV2. (a) Kinetic model and estimated rate constants. (b) Modified kinetic model that incorporates the experimentally determined KIE on the fluorescence lifetime. Black compartments denote experimentally observed states, red compartments spectrally unobserved compartments. See text for details.

Although the transient absorption and time-resolved fluorescence data are too sparse to explicitly apply this kinetic scheme, we may estimate some of the rate constants. First, we note that in the


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CrLOV2 target analysis of Figure 4a, kIC and kRAD sum up to (2.0 ns)-1, while they sum up to (5.5 ns)-1 in AsLOV2. There is no a priori reason to assume that internal conversion and radiative decay would occur at any different rates in CrLOV2 and AsLOV2, so we assume that kIC and kRAD sum up to (5.5 ns)-1 in CrLOV2. With this assumption and the intersystem crossing rate kISC of (2.8 ns)-1 as determined from the target analysis of Fig. 4a, and the experimentally determined fluorescence lifetime of 1.1 ns, we may now estimate the rate kSA from 1FMN* to X at (2.7 ns)-1 in H2O. In D2O, the experimentally given fluorescence lifetime of 1.6 ns results in a kSA of (12 ns)-1. Thus, this rate is H2O/D2O dependent and underlies the observed KIE of 1.4 of the fluorescence lifetime in CrLOV2. State X then forms the adduct A at rate k1, which is undetermined but much faster than kSA. In the context of the scheme of Fig. 4b, State X is formed with a quantum yield of 40% (i.e., (2.7 ns)-1 / (1.1 ns)-1 ). To satisfy the condition that the overall 1FMN* to A quantum yield amounts to 10% (as determined from the target analysis of Figure 4a), state X needs to decay to the FMN ground state at a rate 3 x k1 in competition with formation of adduct A, resulting in a 0.25 fraction evolving to the adduct A and a 0.75 fraction going to the FMN ground state. We consider two possibilities for the molecular identity of intermediate state X. Here, we need to take into account that the rate kSA depends on whether CrLOV2 is dissolved in H2O (kSA = (2.7 ns)-1 ) or D2O (kSA = (12 ns)-1 ). Thus, kSA must involve a proton transfer process. (i) A radical pair mechanism where concerted electron-proton transfer occurs from Cys to 1

FMN* at a rate kSA, forming the FMNH• – S• radical pair X, which recombines to form the covalent

(C4a) thioadduct A at rate k1. Such a model would be similar to that of the canonical triplet thioadduct reaction such as in AsLOV2,16, 19, 21 where a KIE on the adduct formation reaction was observed as well.25 The flavin singlet excited state features a lower redox potential than the triplet state and hence is a less favorable electron acceptor accordingly,31 yet the initial formal hydrogen


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transfer from the FMN singlet state would be more than 100 times faster than from the triplet state in CrLOV2. This apparent discrepancy may be explained by the notion that no spin flip is required in the singlet-borne reaction. (ii) In 1FMN*, the basicity of FMN N5 significantly increases,32 which could result in proton abstraction from Cys to form a Cys thiolate - protonated 1FMN* pair X at rate kSA, followed by nucleophilic attack to form the (C4a) thioadduct at rate k1. Such a model is similar to early proposals for the AsLOV2 triplet-state borne covalent adduct reaction,13, 18 that were later discarded given the energetics and spin forbidden nature leading to unfavorable proton transfer kinetics.19-24, 33 However, the available free energy from the singlet excited state (~20,000 cm-1) is higher than that of the triplet (16,600 cm-1)29,

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and here the spin multiplicity remains identical in reactant and product,

which could render this reaction feasible. Both reaction models require that the distance between Cys and FMN is significantly shorter in CrLOV2 as compared to AsLOV2. This is consistent with the significantly shorter triplet lifetime of CrLOV2 (216 ns) as compared to AsLOV2 (1.9 µs) (Fig. S1, S2). Both models are incompatible with an earlier proposal for adduct formation from the FMN singlet through electron transfer,26 where no KIE would be expected on the fluorescence lifetime. FTIR experiments on CrLOV2 indicated that no hydrogen bonding takes place between FMN and Cys in the dark,35 which seemingly contradicts our assertion that FMN and Cys must be in close vicinity. However, those FTIR experiments were performed on CrLOV2 fused to a His tag, which shows canonical LOV dynamics36 in contrast to CrLOV2 fused to a maltose binding protein studied here. In summary, we have established a direct photoadduct formation channel from the FMN singlet excited state in CrLOV2. By comparative model analysis of the transient absorption data, it was found that the FMN singlet reaction channel accounts for 38% the total quantum yield of the adduct


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formation, providing a more comprehensive understanding of the photochemical reaction dynamics in LOV domains.

ASSOCIATED CONTENT Supporting Information. Additional experimental details, global fitting analysis and transient absorption spectra are provided in Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author [email protected]

ACKNOWLEDGMENT Funding Sources No competing financial interests have been declared. This work was supported by NWO. We thank Melanie Meiworm for excellent technical assistance.

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35. Bednarz, T.; Losi, A.; Gartner, W.; Hegemann, P.; Heberle, J. Functional Variations among LOV Domains as Revealed by FT-IR Difference Spectroscopy. Photochemical & Photobiological Sciences 2004, 3, 575-579. 36. Holzer, W.; Penzkofer, A.; Hegemann, P. Absorption and Emission Spectroscopic Characterisation of the LOV2-His Domain of Phot from Chlamydomonas reinhardtii. Chem Phys 2005, 308, 79-91.


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Photoadduct Formation from the FMN Singlet Excited State in the

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