Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine

Blue-light absorption by the flavin chromophore in light, oxygen, voltage (LOV) photoreceptors triggers photochemical reactions that lead to the formation of a flavin-cysteine adduct. While it has long been assumed that adduct formation is essential for signaling, it was recently shown that LOV photoreceptor variants devoid of the photoactive cysteine can elicit a functional response and that flavin photoreduction to the neutral semiquinone radical is sufficient for signal transduction. Currently, the mechanistic basis of the underlying electron- (eT) and proton-transfer (pT) reactions is not well understood. We here reengineered pT into the naturally not photoreducible iLOV protein, a fluorescent reporter protein derived from the Arabidopsis thaliana phototropin-2 LOV2 domain. A single amino-acid substitution (Q489D) enabled efficient photoreduction, suggesting that an eT pathway is naturally present in the protein. By using a combination of site-directed mutagenesis, steady-state UV/Vis, transient absorption and electron paramagnetic resonance spectroscopy, we investigate the underlying eT and pT reactions. Our study provides strong evidence that several Tyr and Trp residues, highly conserved in all LOV proteins, constitute the eT pathway for flavin photoreduction, suggesting that the propensity for photoreduction is evolutionary imprinted in all LOV domains, while efficient pT is needed to stabilize the neutral semiquinone radical.

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Supporting Methods Supporting Methods
Site directed mutagenesis Determination of fluorescence lifetimes using time correlated single photon counting (TCSPC) Analysis of TCSPC data Ultra-High-Performance Liquid Chromatography (UPLC) analyses. Singular value decomposition (SVD) and deconvolution of time sequences of the steady-state UV/Vis spectra of iLOV-Q489D at pH 7.2

Molecular dynamics (MD) simulations Supporting Data and Figures Figure S1
Close-up view of the FMN binding pocket of iLOV S9 Figure S2 Photoreduction of parental iLOV in the presence and absence of oxygen, Photoreduction of parental iLOV under anaerobic conditions in the presence of EDTA, Reoxidation kinetics of parental iLOV and iLOV-Q489D S10 Supporting Results: pH-dependence of iLOV-Q489D photoreduction S11-S13 Figure S3 Spectral changes associated with illumination of iLOV-Q489D at pH 2.6 to pH 10.6.

Figure S6
SVD analysis of the photoreduction data of iLOV-Q489D at pH 7.2

Figure S7
UPLC analysis of iLOV-Q489D at pH 7.2 and after photoreduction at pH 2.6 and 9.8.

S17
Supporting Results: UV/Vis spectra along with computational predictions suggest that the newly introduced Asp is protonated at neutral pH values S18-S19

Figure S8
UV/Vis spectra of parental iLOV, iLOV-Q489D at pH 7.2; Closeup view of the FMN binding pocket of iLOV-Q489D; H-bond analysis S19 Supporting Results: Generation of Trp • reference spectrum in solution S20 Figure S9 Comparison of the difference absorption spectra of FMN + Trp and FMN + Cys in the absence of apoprotein.

S20
Supporting Results: Electron paramagnetic resonance (EPR) spectroscopy reveals the presence of a stable radical pair in iLOV-Q489D

S21
Figure S11 X-band continuous wave (cw) EPR spectra of iLOV-Q489D S22 Supporting Results: Experimental and computational analysis of the eT pathway in iLOV-Q489D

Figure S12
Potential electron donating Tyr and Trp residues in parental iLOV S23 Table S1 Center of mass and edge-to-edge distances between the redox active amino acids Y416, Y459, Y484, W467 and the FMN chromophore in iLOV

Figure S14
DADS of iLOV-Q489D/W467F obtained via global analysis of a 2D TA data set measured on a 200 µs streak window

Figure S15
Close-up view of the crystal structure of A. thaliana Cry1 (PDB: 1U3D)

Table S2
Center of mass (black) and edge-to-edge (red) distances between the components of the Trp triad in Arabidopsis thaliana Cry1

Supplementary Methods
Site directed mutagenesis. The double variants iLOV-Q489D/Y416F, iLOV-Q489D/Y459F, iLOV-Q489D/Y484F and iLOV-Q489D/W467F were generated by Quikchange PCR according to the instructions given by the manufacturer (Stratagene, La Jolla, CA). The plasmid pET28a_iLOV-Q489D was used as template together with the primers QC_iLOV-Y416F_f (5'-GACAGAGTTTTCGCG CGAGGAAATATTGGG-3') and QC_iLOV-Y416F_r Reoxidation under aerobic conditions. To evaluate the efficacy of FMN reoxidation after complete photoreduction, photoreduction measurements were carried out as described in the main manuscript. Subsequently the light was switched off and flavin reoxidation was monitored for 2 hours by recording a UV/Vis spectrum every 2 minutes.
The efficacy of the process was quantified by determining the reoxidition lifetime τ FMNox by plotting the rise in absorbance at 450 nm after photoreduction due to reoxidation (Abs 450nm ) against the time in the dark and fitting the experimental data to a single-exponential decay function: (1)

Determination of fluorescence lifetimes using time correlated single photon counting
(TCSPC). The fluorescence lifetimes of the excited S 1 states were determined by TCSPC with a time resolution in the nsrange at room temperature. The samples were excited at 443 nm with a pulsed LED (NanoLED-450, Horiba Jobin Yvon, λ max = 443 nm, FDHM = 1.1 ns, S5 FWHM = 27 nm, ω = 100 KHz) and the emission was observed in a 90° geometry relative to excitation at 495 nm with a photomultiplier tube (PMT). The electronic signal was processed digitally resulting in histograms representing the fluorescence decay curves of the samples.
The response function of the LED was detected separately at 450 nm close to λ max .
Analysis of TCSPC data. The duration of the excitation pulse of the LED (FDHM = 1.1 ns) cannot be neglected when determining fluorescence lifetimes in the ns time range via TCSPC.
Therefore, the measured data correspond to the fluorescence decay function, f(t), convoluted with the response function of the LED, g(t). (2) Analysis of these data was performed using home-written software, which uses a sum of N exponential functions as model function f model (t). The square deviation between f(t) and the data is minimized, taking the experimental instrument response for g(t  Subsequently, a sequence of approximations A (K) to the data matrix is formed and compared to the original data matrix A plot of () K  vs K indicates how many components K make a significant contribution to the data. In all our experiments we found K = 2, with the third components contributing less than 1 % of the first two.
The reduced data matrix A (2) was then decomposed into the product of two spectra and two concentration time profiles, Here, S is a matrix with two columns containing the species spectra, C is a matrix with two columns containing the concentration-time profiles of the two species, W (2) contains only the first two diagonal elements of the matrix W, and X and Y are 2x2 matrices with XY = 1.
Since only one species (i.e. the neutral radical) absorbs at 615 nm, we take the corresponding row of A (2) as the concentration profile of this species, i.e.

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(2) Were α is a scaling factor that determines the yield of the reaction at the final time. The concentration of the other species (i.e. the oxidized flavin) is defined by c (1) + c (2) Due to the orthogonality of the matrix V. Inversion of Y gives the matrix X and the species spectra according to (2) The only adjustable parameter in this procedure is the scaling factor α. If α is too small, the calculated spectrum of species 2 will have negative intensity in the region of strong absorption of species 1, if α is too large, the peaks of species 1 will also appear in the spectrum of species 2.

Molecular dynamics (MD) simulations.
All MD simulations were performed as described previously for parental iLOV and iLOV-Q489K (2). Starting from the X-ray crystal structure of parental iLOV (PDB-code: 4EES, 1.8 Å (3) ), a structural model of the iLOV-Q489D variant was constructed in YASARA Structure version 13.9.8 (4) using the YASARA-FoldX plugin (5) and employing the FoldX method (6). In order to explore alternative conformations of Q489D and the surrounding side chains below 6 Å distance from residue Q489, 10 independent runs including rotamer search employing a probability-based rotamer library were performed during the FoldX energy minimization. From the analysis of stabilization energy and interaction energies the most stable conformer was chosen as starting structures for MD simulations of iLOV-Q489D variant. The details of the FoldX prediction can be found elsewhere (2). Molecular dynamics (MD) simulations were performed using the AMBER14 program.(7) The AMBER99SB force field (8,9) parameters for the protein were augmented by the general AMBER force field (GAFF) (10) parameters for flavin mononucleotide (FMN) with the AMBER ff99 compatible RESP partial charges suggested in our previous work (2). The protonation states of titratable residues were assigned on the basis of pKa prediction carried out by using the PROPKA 3.1 program (11) and visual inspection. His471 and His495 were treated as HIE (NE2 protonated) and HIP (both ND1 and NE2 protonated), respectively. Side S8 chains of Asn and Gln residues were checked for possible flipping. Hydrogen atoms were added employing the tleap module of AmberTools14.(7) Crystal water molecules were kept.
We performed the MD simulations with two possible different protonation states of the D489, either the protonated or deprotonated state. The phosphate group of flavin was deprotonated, carrying a charge of -2, which leads to a total charge of -4e/-5e for protonated or deprotonated D489 variant. The protein was solvated in an octahedral TIP3P (12) water box centered at the center of mass to ensure a water layer of 12 Å around the protein and neutralized by replacing solvent water molecules that were at least 5.5 Å away from any protein atoms by the sodium ions. The systems contained ~ 81,000 atoms in total, including ~26,500 TIP3P (12) water molecules.
In all MD simulations, long range electrostatic interactions were computed by using the Particle Mesh Ewald (PME) (13) method. To calculate the electrostatic interactions a cutoff of 10 Å with periodic boundary conditions was used. Initially, the solvent and the ions followed by the whole system were subjected to minimization using 10,000 steps of steepest descent followed by 3,000 steps of conjugate-gradient minimization. Following the energy minimization, the system was then slowly heated from 0 to 300 K for 50 ps. After heating, the systems were equilibrated for 1000 ps at 300 K. Finally, three independent MD simulations for the NPT ensemble at 300 K (for 50 ns) were carried out for iLOV-Q489D in each of the protonation states of D489 with a 2 fs integration step and a total length of 50 ns. No restraints were imposed on the coordinates in the simulations. VMD (14), Pymol (15), and AmberTools 14 (7)   Photoreduction of parental iLOV is only possible in the absence of oxygen and the addition of excess amounts of the sacrificial electron donor EDTA. To enforce photoreduction of parental iLOV, the protein sample (6.5 µM) was mixed with approx. 150-fold excess EDTA (1 mM) and the solution was degassed by bubbling Argon for 15 minutes through the solution in a quartz cuvette closed with a rubber septum. The resulting solution was illuminated for up to 45 minutes. All measurements were carried out in in 200 mM sodium phosphate buffer pH 7.2 supplemented with 10 mM NaCl. (D) Reoxidation kinetics of iLOV-Q489D (red circles) and parental iLOV (blue rectangles) after photoreduction. iLOV-Q489D was photoreduced in the presence of oxygen as shown in Figure 2, A of the main manuscript. Subsequently, reoxidation of the flavin chromophore was monitored for 2 hours in the dark and recording sequential spectra. Parental iLOV was photoreduced as described in panel C and subsequently flavin reoxidation was monitored for 2 hours in the dark. Reoxidation efficacy was quantified by plotting the normalized rise in absorbance at 450 nm against the incubation time in the dark. Experimental data was fit using a single exponential decay function (blue and red solid line) to obtain the reoxidation lifetime τ FMNox = 1502 ± 27 s (iLOV-Q489D) and τ FMNox = 1376 ± 520 s (parental iLOV). Please note that the error associated with the reoxidation measurement of parental iLOV is quite large probably due to the overlay of oxygen diffusion and reoxidation. Additionally, the degassing of the protein solution within the cuvette results in variable volume loss further complicating the measurement. Error bars correspond to the standard deviation of the mean derived from three independent measurements. S11

pH-dependence of iLOV-Q489D photoreduction
One possible explanation for low photoreduction yields at low acidic and high basic pH values could be a pH-dependent unfolding of iLOV-Q489D during the photoreduction measurement, which would result in the release of the flavin chromophore from the protein, hence abolishing the possibility of photoreduction by the protein. At a pH < 4, the corresponding UV/Vis spectra ( Figure S3, A, B and Figure S4, A, B) show evidence for unfolding of iLOV-Q489D during the photoreduction measurement, i.e. the vibronic fine structure with a maximum at around 470 nm is lost during the photoreduction measurement and the whole spectra are broadened which results in a specific rise in absorbance at 495 nm (see Figure S4, A and B). At all other pH values no evidence for unfolding of iLOV-Q489D during the measurement is observed (see Figure S3, C-J). To quantify the pH-dependent unfolding of iLOV-Q489D at the two lowest pH values (pH 2.6 and pH 3.0), we measured sequential UV/Vis spectra under identical conditions as used in the photoreduction experiment but did not illuminate the sample ( Figure S4, A and B). As control, the pH stability of iLOV-Q489D at the two highest pH values (pH 9.0 and 10.6) was measured (see Figure S4, C and D). At pH 2.6 ( Figure S4, A) and pH 3.0 ( Figure S4 Figure S3: Spectral changes associated with illumination of iLOV-Q489D at pH 2.6 to pH 10.6. For clarity only one exemplary measurement out of three independent experiments per pH value is shown. S13 Figure S4: pH stability of iLOV-Q489D at pH 2.6 (A), pH 3.0 (B), pH 9.0 (C) and pH 10.6 (D). iLOV-Q489D was diluted to an OD 450nm of approx. 0.1 in either 200 mM phosphate/citrate buffer (pH 2.6 and pH 3.0) supplemented with 10 mM NaCl or in 200 mM Glycine/NaOH buffer (pH 9.0 and pH 10.6) supplemented with 10 mM NaCl. The samples were placed in a 1 cm quartz cuvette and incubated at 25 °C in the spectrophotometer for up to 15 minutes. Sequential UV/Vis spectra were recorded at a time interval of 30 sec (shown in rainbow coloring). At pH 2.6 (A) and pH 3.0 (B) the UV/Vis spectra are indicative of unfolding and the release of the flavin chromophore of iLOV-Q489D, which was followed over time by plotting the rise in absorbance at 495 nm (red arrow) against the incubation time (inset). The resulting data could be fitted using a single-exponential decay function. The unfolding proceeds with a lifetime of τ pH = 148 ± 9 seconds at pH 2.6 and τ pH = 191 ± 11 seconds at pH 3.0. This corresponds to an illumination time of about 25 and 32 seconds at pH 2.6 and pH 3.0, respectively. At pH 9.0 (C) and pH 10.6 (D) no spectral changes are observed, which suggests that at those pH values iLOV-Q489D is stable during the incubation time. Error bars correspond to the standard deviation of the mean derived from three independent measurements. S14 Spectral changes associated with photoreduction of iLOV-Q489D at pH 7.2, pH 2.8 and pH 9.8 At pH 7.2 the respective spectra ( Figure S3, F and Figure S5, A) provide clear evidence for the formation of FMNH • , with the process being fully reversible in the dark ( Figure S5, B).
We performed singular value decomposition (SVD) of this data set ( Figure S6). The plot of σ (K) vs K ( Figure S6, A, inset) indicates, that only two components contribute significantly to the spectra at pH 7.2. We considered the absorption in the range 250 -700 nm, and up to 290 s. Applying a two component model to the data yields the reconstructed spectra in the range 0.8 < α < 0.9 and the concentration time profiles for α = 0.85 of FMN ox and the product FMNH • (Figure S6, A; B). Note that this reconstruction conserves the isosbestic points in the two spectra.   2 (A, B), pH 2.8 (C, D) and pH 9.8 (E, F). The dark-adapted sample (solid black line) was illuminated and sequential spectra were recorded (rainbow coloring, illumination time increment: 10 s) until no further spectral changes did occur (dashed blue line). The inset shows the absorbance trace at 615 nm indicative of FMNH • formation and conversion. After complete photoreduction, the samples were incubated in the dark and sequential spectra were recorded (data not shown). As marker for FMN reoxidation the absorbance at 450 nm was plotted against incubation time (B, D, F). Only at pH 7.2 complete FMN reoxidation is observed which proceeds with a lifetime of τ FMNox = 1502 ± 27 s. The respective data was fit to a single exponential decay function (B; red line). Error bars correspond to the standard deviation of the mean derived from three independent measurements. S16 Figure S6: SVD analysis of the photoreduction data of iLOV-Q489D at pH 7.2 (data from Figure S5  The native chromophore content of iLOV-Q489D was determined by extracting the flavins from a dark adapted sample adjusted to pH 7.2 (chromatogram: C, elution peak spectra: D). At pH 2.6 and pH 9.8 the samples were illuminated until no further spectral changes occurred (60 min) and protein bound flavins were extracted as described in the Supporting Methods section. Subsequently, the extracted flavins were analysed by UPLC. The corresponding chromatograms and elution peak spectra are shown in panel E, F (pH 9.8) and G, H (pH 2.6), respectively. Please note, that at pH 2.6 the overall signal intensity was 3-fold lower than for the pH 9.8 sample, although similar amounts of protein were denatured to release the protein bound flavins.

UV/Vis spectra of iLOV-Q489D along with computational predictions suggest that the newly introduced Asp is protonated at neutral pH values. Alteration of the H-bonding
network around the FMN-N5 atom in iLOV, e.g. by substitution of Q489 for Asp or by the presence of either a protonated or unprotonated (and hence charged) Asp side chain in iLOV-Q489D at different pH values, is expected to influence the flavin absorbance band in the violet/blue region of the spectrum (2). To address this issue we compared UV/Vis spectra of parental iLOV and iLOV-Q489D at pH 7.2, pH 2.8 and pH 9.8 ( Figure S8).
Parental iLOV ( Figure S8, A, black dashed line) shows an absorbance maximum at around 447 nm (corresponding to the S0-S1 transition). At pH 7.2 (red solid line) and pH 2.8 (green solid line) the absorbance maximum of iLOV-Q489D is slightly blue shifted by 4 or 5 nm, respectively. In contrast, at pH 9.8 the absorbance maximum of iLOV-Q489D is clearly blue shifted by 7 nm to about 440 nm. To study how the Q489D substitution influences the structure of iLOV, the iLOV-Q489D variant was constructed in silico, starting from the parental iLOV crystal structure, and a rotamer search was conducted with FoldX exploring alternative conformations of the surrounding side chains. To investigate possible conformational changes associated to the introduced Q489D mutation, we performed three independent 50 ns MD simulations of the iLOV-Q489D variant starting from the most stable FoldX predicted structure in both protonated and deprotonated states of D489. Analysis of the D489 side chain highlights the structural changes surrounding residues D489, near to FMN chromophore during MD simulations ( Figure S8, B and C). Figure S8, C shows the analysis of the interatomic distances for the D489-FMN interaction along MD trajectories, indicating that protonated D489 preserves an H-bond with the FMN-O4 atom. Independent H-bond analysis of the trajectories shows that in ~70% of the trajectories protonated D489 forms an H-bond with FMN-O4. In contrast, in the simulation containing deprotonated D489 the D489 side chain samples multiple conformations along the trajectories but does at no time form an H-bond to the FMN chromophore. Inspection of the simulation reveals that D489 flips away from the FMN chromophore followed by the entrance of some water molecules to the FMN binding pocket ( Figure S8, B and C). Detailed information about the tree independent MD simulations can be found in Figure S16-S21. The 7 nm blue-shift observed for iLOV-Q489D at pH 9.8 can readily be explained based on our MD simulation of iLOV-Q489D with deprotonated D489, where the side chain of D489 flips away from the chromophore ( Figure   S8, B and C), with the loss of H-bonding between D489 and FMN resulting in a blue-shifted absorption maximum similar to the recently characterized iLOV-Q489K variant (2). Likewise the small 4-5 nm blue-shift observed at pH 7.2 and pH 2.8, respectively, can be explained by S19 altered protein-FMN interactions compared to parental iLOV. In parental iLOV the NE2 atom of Q489 is in close proximity to the FMN-N5 atom (<3.5 Å) (2) and forms an H-bond with the FMN-O4 atom. While the H-bond to FMN-O4 is retained in our MD simulations of iLOV-Q489D with protonated D489, the average distance between the OD2 atom of D489 and FMN-N5 is increased (>4.5 Å) ( Figure S8, C), which may result in a blue-shifted absorbance maximum. Figure S8: UV/Vis spectra of parental iLOV (black dashed line), iLOV-Q489D at pH 7.2 (red solid line), pH 2.8 (green solid line) and pH 9.8 (blue solid line) (A). Close-up view of the FMN binding pocket of iLOV-Q489D in cartoon representation (B). Representative structure of the FMN chromophore binding pocket of iLOV in protonated D489 and deprotonated D489 taken from the MD trajectories. The snapshot was selected based on cluster analysis of the MD trajectories. Average distance distribution curve of the interatomic distances for the indicated residues (in Å) calculated over three independent MD trajectories of iLOV-Q489D with protonated and deprotonated D489 (C). H-bond analysis (with geometric cutoff for hydrogen-bond distance and angle values of 3.2 Å and 150°, respectively) shows that D489-OD2 preserves a hydrogen bond with FMN-O4 ~70% of the time in the simulations with protonated D489. The depicted distance distribution curves were derived from the data shown in Figure S16, S18 (protonated D489) and Figure S17, S19 (deprotonated D489).

Generation of Trp • reference spectrum in solution.
We excited FMN at 447 nm in the presence of Trp and measured TA on a 20 µs time window. The resulting difference spectrum is shown in Figure S9. The spectrum remained unchanged from 9 -20µs and was averaged.
The spectral signature exhibits similarity to the DADS2 of iLOV-Q489D (Figure 3

Electron paramagnetic resonance (EPR) spectroscopy reveals the presence of a stable radical pair in iLOV-Q489D
EPR spectra of the photoexcited FMN triplet state. Figure S10 shows echo-detected EPR spectra recorded 500 ns after laser excitation of parental iLOV and iLOV-Q489D at 30 K. Besides the narrow central signal due to the photoaccumulated radical and radical pair products, the typical spectrum of the flavin excited triplet state is detected (18). The small changes of the fine structure parameters indicate the sensitivity of triplet electronic structure to the Q489D mutation in the vicinity of the flavin.
Figure S10: X-band echo detected EPR spectra of parental iLOV (black trace) and iLOV-Q489D (blue trace) detected 500 ns after 450 nm laser flash at 30 K using t p --2t p Hahn echo sequence with t p = 20ns and  = 200ns.

Simulation of radical pair EPR spectrum.
The radical pair spectrum was analysed using the EasySpin toolbox for the Matlab program package (19,20). Because the counter radical in the pair is unknown, the spectrum was calculated using inhomogeneous Gaussian linewidth of 3.0 mT. The best agreement between experimental and calculated spectra was found for |D| = 4.0 ± 1.0 mT and E=0 (see Figure   S11). Figure S11: X-band continuous wave (cw) EPR spectra of iLOV-Q489D (blue trace) recorded after 20 min illumination of the dark adapted sample. The spectrum was corrected for FMNH  contribution using the EPR spectrum recorded after sample annealing (see Figure 5 of the main manuscript). The dashed line shows the best fit using a numerical solution of the spin Hamiltonian accounting for fine (zero field) structure with |D| = 4.0 mT, E=0. The residual is shown by red line.

Experimental and computational analysis of the eT pathway in iLOV-Q489D
As potential electron donating redox active amino acids iLOV contains three tyrosines (Y416, Y459 and Y484) as well as one tryptophan (W467) ( Figure S12). Figure S12: Potential electron donating Tyr and Trp residues in parental iLOV (A). Parental iLOV (PDB: 4EES) is shown in transparent cartoon representation with the FMN chromophore depicted as sticks with yellow carbon atoms. All tyrosine and tryptophan residues of parental iLOV are shown as sticks with carbon atoms in light blue. Nitrogen, oxygen and phosphorous atoms are shown in dark blue, red and orange, respectively. Dashed lines highlight the distance between the center of mass of the respective aromatic ring system and the center of mass of the FMN isoalloxazine ring. For clarity only center of mass distances between the amino acids and FMN are shown. All other measured distances can be found in Table S1 below. The center of mass of each group (the tricyclic isoalloxazine of the FMN, the indole part of Trp or the benzene ring of Tyr) was determined as the mean of the crystallographic coordinates of all the ring carbon and nitrogen atoms. The center of mass distance was then determined between the corresponding center coordinates. $ : The edge-to-edge distance was determined as the smallest separation between C or N ring atoms of the respective groups.   1U3D). ArCry1 is shown in transparent cartoon representation with the FAD chromophore depicted as sticks with yellow carbon atoms. The tryptophan triad (W400, W377, W324) is shown as sticks with carbon atoms in light blue. Nitrogen, oxygen and phosphorous atoms are shown in dark blue, red and orange, respectively. Dashed yellow lines highlight the distance between the center of mass of the respective aromatic ring system and the FAD C4a atom. For clarity only center of mass distances between the amino acids and FMN are shown. The corresponding distances (in Å) are given in red.   Figure S18: Distance distribution curve of the interatomic distances for the indicated residue (atom) pairs (in Å) calculated over the MD trajectories of iLOV-Q489D variant with protonated D489; (A) FMN-O4 … D489-OD2 distance distribution, (B) FMN-N5 … D489-OD2 distance distribution for three independent 50 ns MD runs (run_1, red; run_2, green; run_3, blue). The data was used to generate the distance distribution curves shown in Figure  S8, C (orange and blue lines).  Figure S19: Distance distribution curve of the interatomic distances for the indicated residue (atom) pairs (in Å) calculated over the MD trajectories of iLOV-Q489D variant with deprotonated D489; (A) FMN-N5 … D489-OD2 distance distribution, (B) FMN-O4 … D489-OD2 distance distribution for three independent 50 ns MD runs (run_1, red; run_2, green; run_3, blue). The data was used to generate the distance distribution curves shown in Figure S8, C (light grey and dark grey lines).