Magnetically Sensitive Radical Photochemistry of Non-natural

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Magnetically sensitive radical photochemistry of non-natural flavoproteins Tilo M. Zollitsch, Lauren E. Jarocha, Chris Bialas, Kevin B. Henbest, Goutham Kodali, P. Leslie Dutton, Christopher C. Moser, Christiane R. Timmel, P. J. Hore, and Stuart R. Mackenzie J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Journal of the American Chemical Society

Magnetically sensitive radical photochemistry of non-natural flavoproteins Tilo M. Zollitsch,1 Lauren E. Jarocha,1 Chris Bialas,2 Kevin B. Henbest,3 Goutham Kodali,2 P. Leslie Dutton,2 Christopher C. Moser,2 Christiane R. Timmel,*3 P. J. Hore,*1 and Stuart R. Mackenzie,*1 1

Department of Chemistry, University of Oxford, Physical and Theoretical Chemistry Laboratory, OX1 3QZ, United Kingdom 2

Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. 3

Department of Chemistry, University of Oxford, Centre for Advanced Electron Spin Resonance, Inorganic Chemistry Laboratory, OX1 3QR, United Kingdom * Corresponding Authors: [email protected] [email protected] [email protected]

1 Environment ACS Paragon Plus

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Abstract It is a remarkable fact that ~50 µT magnetic fields can alter the rates and yields of certain free radical reactions and that such effects might be the basis of the light-dependent ability of migratory birds to sense the direction of the Earth’s magnetic field. The most likely sensory molecule at the heart of this chemical compass is cryptochrome, a flavin-containing protein that undergoes intramolecular, blue-light-induced electron transfer to produce magnetically sensitive radical pairs. To learn more about the factors that control the magnetic sensitivity of cryptochromes, we have used a set of de novo designed protein maquettes that self-assemble as four-α-helical proteins incorporating a single tryptophan residue as an electron donor placed approximately 0.6, 1.1 or 1.7 nm away from a covalently-attached riboflavin as chromophore and electron acceptor. Using a specifically developed form of cavity ring-down spectroscopy, we have characterized the photochemistry of these designed flavoprotein maquettes to determine the identities and kinetics of the transient radicals responsible for the magnetic field effects. Given the gross structural and dynamic differences from the natural proteins, it is remarkable that the maquettes show magnetic field effects that are so similar to those observed for cryptochromes.


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Introduction Many animals sense the direction of the Earth’s magnetic field (25-65 µT) for the purposes of orientation and navigation. Migratory songbirds have a light-dependent magnetic compass the mechanism of which seems to be based on cryptochromes, blue-light photoreceptor proteins that contain a non-covalently bound flavin adenine dinucleotide (FAD) cofactor and a conserved chain of three or four tryptophan (Trp, W) residues. At least in vitro, photo-excitation of the fully oxidized FAD triggers a sequence of electron transfers along the Trp-triad or tetrad resulting in FAD and Trp radicals.1-4 To function as a compass sensor, the quantum yield of the signaling state of the protein must depend on the local direction of the Earth’s magnetic field. The origin of this sensitivity can be understood in terms of the radical pair mechanism, in which photo-induced electron transfer produces two radicals in a spin-correlated state. This radical pair interconverts coherently between its singlet (S) and triplet (T) states under the influence of hyperfine interactions with nearby nuclear spins and the Zeeman interaction with the external magnetic field. The latter modifies the energy levels of the pair, changing the extent and frequency of S ↔ T interconversion, and so leads to magnetic field effects (MFEs) on the lifetime of the radical pair and on the yields of the reaction products formed from it. MFEs have been measured in vitro for a plant cryptochrome,5 a (closely related) bacterial photolyase,5,6 and for Drosophila melanogaster cryptochrome.7 Most studies of MFEs on biomolecules have employed conventional optical techniques such as transient absorption.8-12 We have recently developed a range of optical cavity-enhanced absorption spectroscopies specifically to measure MFEs in proteins and in model systems.7,11-13 By increasing the optical path-length such methods can provide high signal-to-noise data from small volumes of samples at low concentration, using low photo-excitation power. Our recent study of Drosophila



cryptochrome employed a continuous-wave cavity enhanced absorption method which, while offering high sensitivity with broadband spectral coverage, does not provide time-resolved data.7 The experiments reported below employ a pump-probe technique with detection by cavity ringdown spectroscopy (CRDS),14 which permits a detailed analysis of the time-, wavelength-, and magnetic field-dependence of the transient radicals. In CRDS, a laser light pulse is injected into a high-finesse optical cavity.12 Intra-cavity absorption by transient species is detected via the time constant of the exponential decay of the light intensity within the cavity (the ring-down time). The detection sensitivity corresponds to absorbance changes of 10−6, representing an improvement over conventional transient absorption of two orders of magnitude as well as requiring considerably smaller sample volumes. Importantly, CRDS also allows lower light intensities to be used thereby reducing the need for the addition of an oxidant to restore the protein to its ground state.12 In this report, we present a CRDS study of MFEs on a class of de novo designed flavincontaining proteins,15 called flavomaquettes, that are intended to reproduce the key elements of the photochemistry of cryptochromes. In a previous study,16 using conventional single-pass transient absorption, we reported a room-temperature MFE of 17% for a flavomaquette thereby demonstrating the potential of these model systems for investigating the biophysical requirements for magnetoreception in proteins. Using more sensitive cavity ring-down absorption techniques that permit a detailed time- and wavelength-resolved analysis, we describe here the identification of the key transient species and measurement of the kinetic parameters that control the magnetic sensitivity of maquettes with different flavin-tryptophan separations.

Experimental


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A. Materials. The design principles, expression, and characterization of the flavomaquettes have been described previously.16 Briefly, flavomaquettes are de novo designed α-helical bundles. Each of the four 26-amino-acid helices is built around a hydrophobic-hydrophilic heptad repeat.17 The helices are linked together by flexible glycine-serine loops and self-assemble in solution (Figure 1). Riboflavin was covalently attached at carbon 8 in the isoalloxazine group to a core cysteine at position 9 on helix 2. Four flavomaquettes were used in this work: a control molecule that does not contain an amino acid capable of reducing the photo-excited flavin, and three designs (W13, W16, and W20) in which a core amino acid on helix 2 at position 13, 16 or 20 was replaced with a tryptophan. The distances between the β-carbons of these residues and that of the cysteine to which the flavin is attached are 5.6 Å, 11.2 Å, and 16.8 Å respectively. Flavomaquette samples (20-25 µM) in a 20 mM phosphate buffer (pH 6.5) with 0.1 M potassium chloride were centrifuged for 90 min to remove any aggregated protein or other particles that might scatter light and so interfere with the spectroscopic measurements.

B. Cavity Ring-Down Spectroscopy.

Figure 2 shows a schematic of the CRDS experiment used to make MFE measurements. The sample cell (Hellma, 165-QS, 0.2 mL sample volume, 1 mm path length) was placed in an optical cavity of length 0.6 m formed by two highly reflective mirrors M1 and M2 (Layertec, broadband coated, reflectivity > 99.7% in the wavelength range 450−690 nm). To help minimize the effects of photobleaching, the sample (total volume ca. 1 mL) was recirculated at 2.5 mL min−1 using a micropump (Bartels Mikrotechnik mp6). Pulsed photo-excitation at 450 nm (< 1 mJ pulse energy, 5 ns pulse length, 1 Hz repetition rate) was provided by a Nd:YAG-pumped dye laser (Continuum Surelite I, Sirah Cobra, coumarin 2 dye). The tunable probe pulse from an optical parametric



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oscillator (Opotek, Opolette) was introduced into the cavity via the front mirror. Light exiting the rear mirror was detected by a photomultiplier tube (Hamamatsu, H6780) and recorded on a digital oscilloscope (LeCroy, WaveSurfer 64MXs-A). Ring-down decays were measured alternately with and without photo-excitation for a range of pump-probe delay (PPD) times, t. The differential absorbance, ΔA(t), per pass of probe light through the cavity, was obtained using a least-squares curve-fitting routine. Several of the transient species detected had lifetimes comparable with the ring-down time (ca. 300 ns) meaning that the analysis had to take account of the changing absorber concentrations on the timescale of the measurement.18 This was done using a deconvolution method based on that described by Brown et al. (see Supporting Information for details).19 ΔA(t) spectra were recorded by scanning the probe wavelength in 10 nm steps between 460 nm and 690 nm with reference measurements taken at 520 nm after each wavelength step to enable a correction for sample photobleaching.

C. MFE measurements. External magnetic fields (strength B) in the range 0–30 mT were applied using home-built Helmholtz coils. At a given PPD, the change in ΔA arising from the presence of the field, ΔΔA(B), is defined as:

∆∆A ( B ) = ∆A ( B ) − ∆A ( 0 )

(1)

which can also be expressed as a percentage:

= % MFE

∆∆A ( B ) × 100 . ∆A ( 0 )

(2)

The dependence of ∆∆A ( B ) on B was determined by acquiring data at a set of randomly ordered field strengths, controlled by the current supplied to the Helmholtz coils. 6 Environment ACS Paragon Plus

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Results and Discussion A.

Cavity ring-down spectroscopy. The photocycle for the Trp-containing flavomaquettes

is shown in Scheme 1. Photo-excitation of the fully oxidized FAD (1F) gives the excited singlet flavin (1F*) which undergoes intersystem crossing (ISC) to form the excited triplet state (3F*). Subsequent electron transfer (ET) from a Trp residue (WH) leads to the formation of a spincorrelated, intramolecular radical pair (RP1). Conservation of spin angular momentum dictates that RP1 is formed in a triplet state, 3[F●− WH●+], which interconverts coherently with the singlet state, 1[F●− WH●+]. Singlet RP1 can return to the ground state by back electron transfer while both singlet and triplet RP1 states can, via proton transfer reactions, form a secondary radical pair, RP2, consisting of the neutral radicals FH● and W●. Figure 3A shows the CRDS spectrum of W16 at PPD times up to 40 µs. The ΔA spectra show the characteristic absorption features expected for an excited flavin–Trp system, including ground state bleaching (< 500 nm), and a broad positive absorption band between 500 and 690 nm due to 3

F* and the flavin and tryptophan radicals.9 The same general features were observed for the other

flavomaquettes, but with different time-dependence (see Figures S1-S3). The triplet state of the flavin, 3F*, absorbs across this whole range of wavelengths, but is the dominant absorber between 670 and 690 nm.7,20-22 Figure 3B shows the time-dependence of ΔA in the range 670–690 nm for the four maquettes. The decay of ΔA in W13, W16 and W20 is significantly faster than in the control. The presence of a nearby Trp residue leads to quenching of 3F* by electron transfer (see Scheme 1).23 Since no other species absorb significantly at these wavelengths, fitting the data provides an estimate of the 3F* lifetime in each maquette: control, 7.1 ± 0.6 µs; W20, 5.4 ± 0.3 µs; W16, 3.7 ± 0.5 µs; W13, 1.3 ± 0.2 µs. The reduction in lifetime with decreasing flavin-Trp distance is consistent with the increased rate of electron transfer expected



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from the "Moser-Dutton ruler", which rationalizes electron transfer rate constants in natural and non-natural proteins considering solely the thermodynamic driving force, the reorganization energy, and the donor-acceptor distance.24 Figure 3C shows the corresponding time-dependence of the light–induced absorbance change observed at 600 nm. At this wavelength, both 3F* and the radical FH● (formed by protonation of the flavin anion radical on a microsecond timescale) contribute significantly to the overall signal.21 As a result, the decay of ΔA at 600 nm due to the loss of 3F* is partly compensated by an increase associated with the formation of FH● and consequently persists longer than in the 670–690 nm region (Figure 3B). The lifetime of FH● is expected to increase with increasing flavin-Trp distance as the rate of the back electron transfer decreases;24 a comparison of the ΔA decay rates for W13, W16, and W20 reflects this. The lifetimes of ΔA for the control maquette at 600 nm (8.7 ± 0.7 µs) and at 670–690 nm (7.1 ± 0.6 µs) are comparable because the absorbance is dominated by 3F* in the absence of an electron donor.

B.

Spectral deconvolution. To gain further insight into the photochemistry and to allow a

quantitative comparison of the three maquettes, the ΔA data at each PPD time were deconvolved. Experimental spectra, S(λ,t) were fitted to reference spectra (520−690 nm, Figure 4A) obtained from a combination of pulse radiolysis measurements (for W●) and transient absorption measurements on similarly substituted flavins.20,25 The observed ΔA spectra were deconvolved using a linear combination of the reference spectra: S (λ , t ) = ∑ ci (t ) Si ( λ )

(3)

i

where Si ( λ ) is the normalized reference spectrum for species i (i = 3F*, FH●, F●−, W●) and ci (t ) is the corresponding amplitude. A representative fit of the experimental data is shown in Figure


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4B for W16 at a PPD time of 1 µs (see Supporting Information for further details). The quantity ci (t ) represents the relative contribution of species i to the observed ΔA spectrum and is directly proportional to the concentration of species i. This type of spectral deconvolution permits the concentration of each species to be followed as a function of time without bias towards or restrictions introduced by using a particular kinetic model. This type of spectral deconvolution permits the concentration of each species to be followed as a function of time. Such a fitting of the 3F*, W●, F●− and FH● contributions to the overall ΔA signal is shown in Figure 4C for W16 and in Figure S5 for W13, W20, and the control. The timeprofile for each species agrees well with the mechanism shown in Scheme 1: photo-excited 3F* is rapidly reduced by electron transfer from the Trp, initially forming F●− which protonates to give FH●. As shown in Figure 4A, 3F* and FH● exhibit distinctive spectral features (around 660– 690 nm and 600 nm, respectively) and absorb approximately an order of magnitude more strongly than W● and F●−. Indeed, the weak, almost featureless absorbance spectra of W● and F●− in this region, means their contributions cannot be determined with the same certainty as those of 3F* and FH●. The remainder of the analysis thus focuses on quantitative comparison of the behaviour of 3

F* and FH● in the different flavomaquettes.

I. Triplet 3F*. Figure 5A shows the concentration of 3F* as a function of PPD time for each maquette studied. The primary processes involved in the loss of 3F* are: 3

3

k

1 3F F∗ → F,

k

ET F∗  → F•− .

(4) (5)

In the control molecule, the loss of 3F* is well-described by a single exponential decay (solid line in Figure 5A) with a rate constant k3F = 0.133 ± 0.008 µs−1. This value, obtained by the 9 Environment ACS Paragon Plus


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deconvolution method, agrees well with the independently measured lifetime of 3F* reported above: (7.1 µs)−1 = 0.14 µs−1. In W20, W16, and W13, 3F* is also removed by electron transfer (Equation (5)), leading to an overall decay rate constant, k2 = k3F + kET. In principle, this should also lead to single-exponential decays, but such fits were unsatisfactory. In the maquettes, unlike in cryptochromes, the flavin cofactor does not have a well-defined binding pocket to restrict its position and orientation. As a result, there is likely to be a range of flavin-Trp distances and hence a distribution of electron transfer rates. In all cases, however, k3F represents a concentration-weighted average over the subset of maquettes that have a triplet lifetime in excess of the ca. 250 ns time-resolution of the measurement. In an attempt to account for this dynamic heterogeneity, Figure 5A shows fits of the relative 3F* concentrations to the functional form,

[ 3 F∗ ](= t)

A2 exp ( −k2t ) + A3F exp ( −k3Ft ) ,

(6)

in which k2 = k3F + kET (as above) and A3F represents the fraction of maquettes with kET W16 > W20 consistent with the respective flavin-Trp separations.24 For W20, kET is in good agreement with the value (1.2 × 105 s−1) expected from the Moser–Dutton ruler for a distance of around 16.8 Å.16 For W16 and W13, the values of kET in Table 1 are smaller than expected (2.75 × 108 s−1 and 6.31 × 1011 s−1, respectively16). This discrepancy arises from a combination of a distribution of electron transfer rates and the lack of data during the first 250 ns after the pump pulse when the electron transfer primarily takes place.


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Table 1. Rate constants and pre-exponential factors obtained from fitting the decay of 3F* and FH●.

A3F

k3F /µs−1

A2

k2 /µs−1

kET /µs−1

Control

1.00 ± 0.02

0.133 ± 0.008

–

–

–

W20

0.29 ± 0.14

0.133*

0.71 ± 0.14

0.304 ± 0.054

0.171 ± 0.054

W16

0.62 ± 0.03

0.133*

0.38 ± 0.04

1.22 ± 0.29

1.09 ± 0.29

W13

0.13 ± 0.01

0.133*

0.87 ± 0.04

2.23 ± 0.18

2.10 ± 0.18

#

#

*

k3F was fixed at 0.133 µs−1 (the value obtained from the control maquette).

#

The amplitudes have been scaled so that A3F + A2 = 1.

II. Neutral FH• radical In Figures 4A and S5, the decay rate of the radical anion ΔA signal appears similar to the growth of the neutral radical for both W13 and W16. This correlation between the increase in FH● concentration and the decrease in F•− concentration is consistent with the proposed photochemistry in Scheme 1. However, the relative contribution of FH● to the total ΔA is smaller than might be expected, given that the relative absorption of FH● is higher than that of F•− in this region of the spectrum. This is explained if the quantum yield of formation of FH● from F•− is not unity, and some of the F•− radicals undergo back electron transfer or reduction back to the ground state instead of protonation. Figure 5B shows the time-dependence of [FH●] for the four maquettes obtained from the spectral deconvolution. In the case of W13, W16 and W20, a significant FH● concentration was already present before the first measurements at 250 ns. The dotted lines in Figure 5B show fits of the FH● time-profiles to a simplified version of Scheme 1 including 1F, 3F*, F•−, and FH● with irreversible



first-order reaction kinetics. Although the agreement is satisfactory for PPD times less than 10 µs, the model is unable to account for the W13 and W16 data at the later times, where it underestimates the concentration of FH● that remains. Closer inspection suggests a multiphasic decay with more than half of the FH● reacting on a 1–5 μs timescale and the rest having a lifetime longer than 10 μs. In W20, by contrast, the FH● concentration plateaus at around 1 μs and then remains constant for 40 μs following photo-excitation. Comparing the three Trp-maquettes, it is clear that the yield of FH● decreases markedly with increasing F-W distance. Also visible in Figure 5B, for the control maquette, is a slowly formed signal from a small amount of a neutral flavin radical arising from a photo-reaction of the isoalloxazine ring and the ribityl chain of the riboflavin.26-28 This may explain the presence of a long lived FH● radical in the ΔA of W13 and W16, and the inability of the simple kinetic model to fit the concentration of these species at long times, since any radical formed from photodegradation of the riboflavin in these maquettes will not return to the ground state by the same chemical processes with the same rate as radicals that are part of a magnetically sensitive RP formed via ET.

C.

Magnetic field effects on flavomaquettes. As shown in Scheme 1, an externally applied

magnetic field can affect the mixing between the singlet and triplet states of the [F●− WH●+] radical pair (RP1). The electron Zeeman interaction with the external magnetic field separates the T±1 triplet energy levels from the S and T0 levels and leads to a decrease in the efficiency of S↔T interconversion driven by the hyperfine interactions. Charge recombination in a spin-correlated radical pair is only allowed from the singlet state. In triplet-born pairs, the application of a magnetic field stronger than the hyperfine interactions reduces T → S conversion, decreases the likelihood of singlet recombination, and increases the concentration of radical pairs and of the products


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formed from them. This is reflected in an increase in ΔA upon application of a magnetic field, i.e. a positive MFE at wavelengths where the radical pair absorbs, and a negative MFE in the ground state-absorption region. The opposite is expected for singlet-born radical pairs. The magnetic field effect develops during the lifetime of the spin-correlated radical pair (RP1) and is inherited by downstream products. As such, the time- and wavelength-dependence of the MFE can provide information on F●−, even when its signature in the spectral deconvolution of the ΔA data is difficult to observe. I. Spectral analysis of the ΔΔA measurements. The species observed in the ΔΔA spectra of the flavomaquettes are those whose concentrations depend on the applied magnetic field. As shown in Figure 6, the ΔΔA spectra of W16 show two distinct MFEs: a positive effect between 500 and 650 nm matching the absorption spectra of the radicals (Figure 4A), and a negative effect in the flavin ground state bleach region below 500 nm. The respective signs of these MFEs reflect the triplet-born nature of RP1 (Scheme 1). Where the transient absorption is dominated by the nonmagnetically sensitive 3F* (e.g., 670 < λ < 690 nm), the ΔΔA is negligible. The shape of the ΔΔA spectrum of W13 is similar to W16, while the signal-to-noise ratio in W20 is too small for spectral analysis (see Figure S3), and no significant MFE was observed for the control maquette (see Figure 7). In the positive MFE region in Figure 6, the wavelength-dependence provides clues to the photochemical pathways. The ΔΔA spectrum in the region 510–540 nm arises from RP1: following a rapid initial rise (< 1 µs), reflecting the primary charge separation, this signal decays as the F●– and W● radicals disappear (Figure 6, inset). However, between 560 and 630 nm, where the FH● radical absorbs predominantly, the ΔΔA signal first grows to a maximum at 1–3 µs as a result of



the protonation of F●– to form FH● (Figure 6, inset). The FH● radical in RP2 thus inherits its MFE from F●– in RP1. While the signs and magnitudes of the MFEs in the ground-state bleach and excited-state absorption regions provide strong evidence for the predominantly triplet-born nature of the radical pairs observed in the CRDS experiment, due to the limited time-resolution of the experiment, fast formation and decay of a singlet-born radical pair cannot be ruled out entirely. However, singletborn radical pairs would only be expected if electron transfer out-competes intersystem crossing, which is only likely in maquettes with short donor-acceptor distances, e.g. W13. The possibility of singlet photochemistry occurring in W13 is supported, indirectly, by the data in Figure S4. For solutions of the same concentration, W13 shows ΔA and ΔΔA signals that are about half those observed for W16. This is consistent with a lower concentration of triplet-born radical pairs in W13 on the microsecond timescale as a result of competition with singlet photochemistry. Back electron transfer of singlet-born radical pairs is likely to be efficient at the short donor-acceptor distances in W13, especially because the recombination is spin-allowed. Such radical pairs could be formed and recombine before undergoing significant spin-mixing and generating a MFE. Given the time-resolution of the CRDS experiment (with the first data available at 250 ns), it is unsurprising that singlet-born chemistry is not observed. II. Time-dependence of ΔΔA. Figure 7 shows a comparison of the time-dependence of the positive MFEs for all species, measured at B = 30 mT. For clarity, the full 510–620 nm spectral region is averaged and the data are presented both as absolute ΔΔA(t) (Figure 7A, B) and as a percentage MFEs (Figure 7C, D). In the Trp–containing flavomaquettes, the formation of RP2 from RP1 is fast enough to compete with the loss of RP1 due to back electron transfer (see Figure


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5 and Figure S5). In this way the magnetic field effect on RP1 is passed to the radicals that comprise RP2 which are long-enough lived to be detected. Both W16 and W13 exhibit a rapid rise in ΔΔA within the first 1 µs reflecting rapid production of F●– followed by protonation to give FH●. In both cases, the signal then decays with a lifetime on the order of 10 µs. The larger absolute ΔΔA of W16 simply reflects the higher concentrations of RP1 and RP2 in W16 (see Figure 5 and Figure S5B) and is not reflected in the percentage MFE. Since we would not expect the quantum yield of the electron transfer process to be higher in W16 that W13, this finding is also consistent with radical pair recombination being faster for shorter flavin-Trp distances, and more radical pairs in W13 than in W16 having recombined during the first 250 ns. The percentage magnetic field effect (%MFE) is determined by both ΔΔA and ΔA (Equation 2). Because the latter has contributions from species such as 3F* whose concentrations are timedependent but not magnetically sensitive, the time-dependence of %MFE is not straightforward to interpret. For example, this effect can be seen in both the observed rise of the %MFE of W13 and W16 within the first 3 μs and the subsequent drop in the %MFE of W13 and the plateau observed for W16. This difference between the two maquettes reflects, at least in part, the distancedependence of the quenching of 3F* reported above and the relative efficiency of radical pair formation. From Figure S5, it is clear that the radical contribution to the overall ΔA is greater in W13 than it is in W16, and that the contribution from 3F* decays more rapidly. This leads to both the larger %MFE and the more pronounced time-dependence of the %MFE in W13. A significantly smaller MFE is observed for W20 (see Figure 7A), which shows qualitatively different behavior to W13 and W16 in that it grows in over a 5 µs timeframe and then plateaus without any appreciable decay in the next 40 µs. This is, in part, because there is a substantially 15 Environment ACS Paragon Plus


larger contribution to the ΔA from the non-magnetically sensitive 3F*. These observations are consistent with the idea that radical pair recombination depends strongly on the radical separation. They further agree with the spectral deconvolutions of the ΔA data (Figure 5) where no significant decay in FH● was detected in W20 within the observed time frame of 40 µs. As such, the steady increase in the % MFE beyond the first 5 μs can be rationalized by the decrease in the concentration of 3F* and, hence, ∆A. III. Magnetically altered reaction yield profiles. Figure 8 shows the magnetically altered reaction yield (MARY) profiles for the maquettes studied here measured at 600 nm and a 0.5 μs PPD time. As can be seen from Figure 7B, at this delay time, the %MFEs observed in W13 and W16 are of comparable magnitude and significantly larger than that observed in W20. The magnetic field strength, B1/2, at which the %MFE reaches half of its maximum magnitude, is characteristic of the magnetically sensitive RP1, depending on intramolecular properties such as hyperfine couplings and the environment of the radical pair.29 Lorentzian fits to the MARY profiles shown in Figure 8 gave B1/2 values of 14.4 ± 0.7 mT (W13), 13.6 ± 0.4 mT (W16), and 16.3 ± 5.4 mT (W20). The similarity of these B1/2 values is consistent with the fact that the same magnetically sensitive RP1 is formed in all three maquettes. This value is also substantially larger than expected for a flavin-Trp radical pair. According to the Weller formula, B1/2 for the [F●− WH●+] radical pair is approximately 3.1 mT.30 However the values reported here for the flavomaquettes are consistent with values measured for this radical pair in protein environments, where broadening of the MARY curves has been attributed to the effects of spin relaxation and spin dephasing.5,30 31 The similarity in the B1/2 values for W13, W16, and W20 is somewhat surprising. Although the photo-induced radicals and their hyperfine interactions are identical in the three maquettes, and


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the molecular tumbling and local internal motions are likely to be similar, the electron-electron exchange and dipolar interactions and the global molecular dynamics could be very different. Given the different attachment points of the tryptophan residues in the maquettes and the distancedependence of these interactions, one might predict very different magnetic responses. Strong exchange interactions can lead to "2J-resonances" as a result of the avoided crossing of S and T+1 or T−1 energy levels, particularly for biradicals.32,33 We see no evidence for such features here and discuss them no further. In the following we suggest three hypothetical explanations for the similar magnetic response of the three molecules: further insight will have to wait for additional experiments. First, it cannot completely be excluded that the similarity is merely coincidental. Given the structural heterogeneity of the maquettes, the exchange interactions may have a complex dependence on the separation of the radicals, with (potentially) through-space and through-bond contributions. Both exchange and dipolar interactions will be modulated by correlated internal motions that are unlikely to be identical in the three molecules, giving rise to both S-T and T-T dephasing and hence broadening of the MARY profiles and increased values of the B1/2 parameter.31,34 It is difficult to predict which factors are most significant in each maquette and to what extent they contribute to spin dephasing, and thus, difficult to be confident that dissimilar B1/2 values are inevitable, despite the manifest differences between the three proteins. Second, it is possible that modulation of exchange and dipolar interactions is not the dominant spin relaxation mechanism. If the molecular conformations that are principally responsible for the observed field-dependence have large radical-radical separations, then it could be that the relaxation arises mainly from the dipolar components of the hyperfine interactions modulated by



molecular tumbling and uncorrelated motions of the two radicals.35 In this case, one could expect similar broadening of the MARY profiles of the three maquettes. The third possibility, which we marginally prefer over the other two, is that the larger than expected B1/2 values arise from similar exchange and dipolar interactions in the three molecules because the radical pairs that dominate the magnetic sensitivity have similar distributions of separations. Although much smaller radical-radical distances are possible in W13 than in W16 or W20, it may be that these conformations contribute little to the MARY profiles. There are two reasons why this could be. (a) Conformations in which the exchange interaction is much stronger than the hyperfine and Zeeman interactions are unlikely to contribute much to the observed fielddependence because spin mixing will be suppressed by the large S-T energy gap. (b) The ground state conformations with small flavin-tryptophan distances may undergo rapid electron transfer to give singlet radical pairs which recombine rapidly, leaving the conformations with the larger donor-acceptor separations to undergo intersystem crossing and form the triplet radical pairs that are responsible for the magnetic field effects. This interpretation is consistent with the observations here, which suggest that a substantial portion of the radicals formed in W13 may undergo back electron transfer during the 250 ns before the first CRDS measurement, and with our previous observation of a lower quantum yield of fluorescence at shorter flavin-Trp distances.16

Conclusions We have designed, expressed and characterized three flavomaquettes as models of the first of the sequential photo-induced electron transfer steps in cryptochromes. Although they contain the same electron donor (tryptophan) and photoactivated acceptor (flavin), the maquette sequences and secondary and tertiary structures bear no relationship to natural cryptochromes. Nevertheless,


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the maquettes share the same basic photochemistry (Scheme 1). Magnetically sensitive [F•− TrpH•+] radical pairs (RP1) are formed in all three maquettes and the magnetic sensitivity arises, as it does in cryptochromes, from a competition between singlet recombination of RP1 and (de)protonation reactions that produce the stabilized RP2 state. We observe clear trends in the behaviour of the three maquettes that correlate with the expected distance between the Trp residue and the flavin. However, comparing the maquettes with cryptochromes, there are also differences in behaviour. The most obvious difference is the presence in the maquette of just a single tryptophan residue thereby removing the possibility of sequential electron transfer. The maquettes have much greater conformational heterogeneity leading to a distribution of timescales for radical pair formation not seen for the natural proteins. Maquettes are also considerably more dynamic: radical pairs are probably formed most efficiently for conformations which bring the tryptophan close to the flavin. Nevertheless, charge separation is unable to compete effectively with intersystem crossing with the result that RP1 in the maquettes is created in a triplet state rather than a singlet state and exhibits magnetic field effects of opposite phase to those found for natural cryptochromes. Remarkably, given these structural and dynamic differences, the B1/2 values of the maquettes are i) similar to one another, ii) similar to those reported for AtCry and EcPL, and iii) considerably larger than expected on the basis of the hyperfine interactions in FAD•− and TrpH•+.5 A range of potential explanations for the similarity of B1/2 values for different maquettes are discussed. The discrepancy in measured B1/2 values measured from those predicted almost certainly results from electron spin relaxation and spin-dephasing in RP1 which, in the maquettes, could be dominated by modulation of the exchange interaction by fluctuations in the radical separation. Such



fluctuations have a similar effect in cryptochromes, which have much less conformational freedom but can undergo sequential electron transfer along the tryptophan chain which can also modulate the exchange interaction in RP1.5 Although efficient charge separation is most likely when the flavin and tryptophan are close to one another, the magnetic field effect on the spin dynamics due to S-T0 mixing cannot develop until the radicals separate to a point where the exchange interaction no longer dominates the hyperfine and Zeeman interactions. Arguably, the conformational dynamics in maquettes plays a similar role to the subsequent TrpH → TrpH•+ electron transfer steps in cryptochromes.5 Be that as it may, it seems clear that the multi-step charge separation down a Trp chain in the natural proteins has advantages in terms of magnetic sensitivity: it allows efficient and rapid forward electron transfer, to form singlet radical pairs, and slow reverse electron transfer that gives enough time for weak Zeeman interactions to affect the spin dynamics. Experiments are currently in progress on flavomaquettes containing a second and a third electron donor at progressively larger distances from the flavin that extend these functional de novo maquette designs to reflect more closely the natural cryptochromes.


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Acknowledgements We are grateful to the following for financial support: the European Research Council (under the European Union’s 7th Framework Programme, FP7/2007-20013/ERC Grant Agreement No. 340451 to P.J.H.), and the Air Force Office of Scientific Research (Air Force Materiel Command, USAF Award No. FA9550-14-1-0095 to P.J.H., C.R.T., and S.R.M.). C.B. is grateful to the NIH for a graduate fellowship (T32 GM008275 - Structural Biology & Molecular Biophysics Training Program).

Notes The authors declare no competing financial interest.

Supporting Information Temporal deconvolution of ring-down traces, spectral analysis.



Scheme and Figures

Scheme 1. Photochemical reaction scheme appropriate for Trp-containing flavomaquettes. See text for more details.


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Figure 1. Flavomaquette designs based on α-helical bundles with a riboflavin chromophore (orange) covalently attached to a core cysteine residue: a control, which does not contain an electron donor, and three maquettes with a single tryptophan at position 13, 16 or 20 on helix 2 at increasing distances from the flavin. W13, W16 and W20 can undergo photo-induced electron transfer akin to that observed in cryptochromes.



Figure 2. Schematic of the CRDS apparatus used to measure MFEs as a function of PPD time (t), probe wavelength (λ) and magnetic field strength (B). The differential absorbance (ΔA) is determined as a function of the ring-down times measured with (τ) and without (τ0) photoexcitation.


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Figure 3. A) Differential absorbance (ΔA) spectra of ca. 20 µM W16 flavomaquette in pH 6.5 phosphate buffer at various PPD times. 3F* absorbance dominates the spectrum between 670 and 690 nm (marked with *). The peak of the FH● absorption band at 600 nm is indicated by **. B) and C) Time-dependence of ΔA in wavelength ranges 670−690 nm and 600 nm, respectively.



Figure 4. A) Reference spectra of the flavin and Trp species used in the spectral deconvolution (520−690 nm). Solid lines represent the spectra for F●− (blue) and W● (red), taken from the literature (left hand y-axis), while the dashed lines represent ΔA spectra (right hand y-axis) of FH● (orange) and 3F* (purple) determined in this work (Figures S2 and S3).20,25 The relative scale of the left and right hand axes was chosen to reflect the ratios of the extinctions typically found in comparable flavin species.9,21 B) The fit (dashed line) to the experimental ΔA spectrum (black squares) of W16 recorded at 1 µs PPD time is shown as a linear combination of the normalized reference spectra. C) The contribution coefficients for the individual species observed for W16 as a function of PPD time. Representative uncertainties are shown at PPD = 250 ns.


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Figure 5. Normalized time-profiles of A) 3F* and B) FH● from W20 (blue), W16 (green), W13 (red), and the control (black) estimated by spectral deconvolution. All signals are scaled to the amplitude of 3F* measured at 0.25 µs. The error bars shown at 0.5 µs show the estimated standard deviations and are representative of the uncertainty at all PPD times. The solid lines in A) represent bi-exponential fits of the concentration time profiles from which the phenomenological rate constants in Table 1 were derived.



Figure 6. ΔΔA(30 mT) spectra of ca. 20 µM flavomaquette W16 in pH 6.5 phosphate buffer taken at various PPD times. The inset shows the time dependence of the ΔΔA averaged over the wavelength regions around 510–540 nm and 580–630 nm, marked with * and ** (see text), respectively. In all cases, solid lines are spline fits to guide the eye.


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Figure 7. Time evolution of the magnetic field effect in each of the four flavomaquettes as a function of PPD expressed as ΔΔA(30mT) A), B) and as %MFE, C), D). The data are averages of twelve ΔΔA time traces recorded between 510 and 620 nm for W20 (blue), W16 (green), W13 (red), and the control (black open circles). Representative uncertainties (one standard error of the mean) are shown at early PPD times. Dotted lines are shown to guide the eye.



Figure 8. Magnetically Altered Reaction Yield (MARY) plots of the percentage MFE measured at 600 nm and 0.5 μs PPD time (close to the peak of the ΔΔA signal) as a function of magnetic field strength: W20 (blue), W16 (green), W13 (red), and the control (black, no field effect). The MFE curves shown are the average of 5 accumulations. The error bars shown at 30 mT show one standard error of the mean and are representative for each curve.


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Magnetically Sensitive Radical Photochemistry of Non-natural

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