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Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme

Nature Chemistry volume 14, pages 677–685 (2022)Cite this article

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Abstract

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•− isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine–aspartate salt bridge allows proton transfer from arginine to FAD•−. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

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Fig. 1: Reactions catalysed by DNA photolyases.
Fig. 2: Structure of oxidized MmCPDII photolyase.
Fig. 3: Structural changes during the photoreduction of MmCPDII.
Fig. 4: Mechanistic view of MmCPDII photoreduction.

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Data availability

Data supporting the findings of this study are available within the Article, the Supplementary Information or the source data. Structural models, along with the structure factors presented here, can be found under PDB accession codes 7F8T, 7VJ8, 7VIW, 7VIX, 7VIY, 7VIZ, 7VJ0, 7VJ1, 7VJ2, 7VJ3, 7VJ4, 7VJ5, 7VJ6, 7VJ9, 7VJA, 7VJB, 7VJC, 7VJE, 7VJG, 7VJH, 7VJI, 7VJJ, 7VJK and 7VJ7. Full details of MmCPDII TR-SFX structures are listed in Table 1, along with the detailed structural parameters summarized in Supplementary Tables 1–3. Source data are provided with this paper.

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Acknowledgements

This work was supported by Academia Sinica and the Taiwan Protein Project funded by the Ministry of Science and Technology (grants nos. AS-KPQ-105-TPP and AS-KPQ-109-TPP2; M.-D.T.), and in part by JSPS KAKENHI (16K01942) to Y.B., by the Air Force Office of Scientific Research (AFOSR; grant no. FA9550-14-1-0409) and the German Research Foundation (DFG, grant no. ES152/18) to L.-O.E. and by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) from the Japan Agency for Medical Research and Development (AMED) to S.I. The XFEL experiments were performed at BL2 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposals nos. 2017A8019, 2017B8052, 2018A8008, 2018B8031, 2019A8014 and 2019B8005). We thank T.-C. Hsiao and M.-l. Wu for their assistance with sample preparation, T. Tanaka, T. Arima, Y. Matsuura, H. Naitow, N. Kunishima, T. Kin and the members of the Engineering Support Team of SACLA for help during our X-ray experiments, T. Nakane for the introduction to CrystFEL, all staff members of the TPS05A beamline, NSRRC, a national user facility supported by MOST, R.O.C. (Taiwan) and in particular C.-C. Tseng and C.-K. Chou for help in setting up non-standard conditions for crystal testing, H.-L. Shr and S.-G. Shyu for assisting with crystallization under non-standard conditions, T. C. Terwilliger (Los Alamos National Laboratory) for his kind help in understanding the theoretical underpinnings of Bayesian difference refinement, G. Gotthard for assistance with using the icOS platform of the Grenoble Instruct-ERIC Center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003), and finally A. M. Reyna for help in setting up the necessary scripts and macros for calculating extrapolated structure factors.

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Authors and Affiliations

  1. Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan

    Manuel Maestre-Reyna, Cheng-Han Yang, Wei-Cheng Huang, Eka Putra Gusti Ngurah Putu, Wen-Jin Wu, Po-Hsun Wang, Hsiang-Yi Wu, Cheng-Chung Lee, Kai-Fa Huang, Yao-Kai Chang, Jiahn-Haur Liao, Jui-Hung Weng, Wael Gad, Chiung-Wen Chang, Allan H. Pang, Yoshitaka Bessho & Ming-Daw Tsai

  2. RIKEN SPring-8 Center, Sayo, Hyogo, Japan

    Eriko Nango, Michihiro Sugahara, Yasumasa Joti, Ayumi Yamashita, Rie Tanaka, Tomoyuki Tanaka, Fangjia Luo, So Iwata & Yoshitaka Bessho

  3. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, Japan

    Eriko Nango, Ayumi Yamashita, Rie Tanaka, Tomoyuki Tanaka, Fangjia Luo & So Iwata

  4. Department of Chemistry, Philipps University Marburg, Marburg, Germany

    Sophie Franz-Badur, Martin Saft, Hans-Joachim Emmerich, Stephan Kiontke & Lars-Oliver Essen

  5. Japan Synchrotron Radiation Research Institute, Sayo, Hyogo, Japan

    Shigeki Owada, Yasumasa Joti & Kensuke Tono

  6. Division of Chemistry, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka, Japan

    Yuhei Hosokawa & Junpei Yamamoto

  7. Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan

    Kai-Cheng Hsu

  8. Fritz Haber Center for Molecular Dynamics, Institute of Chemistry, The Hebrew University of Jerusalem,, Jerusalem, Israel

    Igor Schapiro

  9. Dipartamento di Scienze e tecnologie, Universita degli studi del Sannio, Benevento, Italy

    Roberta Spadaccini

  10. Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), Grenoble, France

    Antoine Royant

  11. European Synchrotron Radiation Facility, Grenoble, France

    Antoine Royant

  12. Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan

    Ming-Daw Tsai

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  1. Manuel Maestre-Reyna
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Contributions

M.M.-R., S.I., L.-O.E., Y.B. and M.-D.T. conceived the research and designed experiments. M.M.-R., C.-H.Y., E.N., W.-C.H., E.P.G.N.P., W.-J.W., P.-H.W., S.F.-B., M.S., H.-J.E., H.-Y.W., C.-C.L., K.-F.H., Y.-K.C., J.-H.L., J.-H.W., W.G., C.-W.C., A.H.P., M.S., S.O., Y.H., A.Y., R.T., T.T., F.L., K.T., R.S., A.R., J.Y., L.-O.E. and Y.B. performed experiments. M.M.-R., C.-H.Y., E.N., Y.J., S.K., I.S., L.-O.E., Y.B. and M.-D.T. analysed the data. M.M.-R., E.P.G.N.P., L.-O.E. and A.R. performed in crystallo spectroscopy. M.M.-R. and C.-H.Y. established and analysed the refinement protocol. K.-C.H. performed the structure-based PDB search. M.M.-R., W.-J.W., C.-H.Y., A.R., L.-O.E., Y.B. and M.-D.T. wrote the manuscript.

Corresponding authors

Correspondence to Lars-Oliver Essen, Yoshitaka Bessho or Ming-Daw Tsai.

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The authors declare no competing interests.

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Nature Chemistry thanks Dongping Zhong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Structural changes from TR-SFX experiments for the FADox→FAD•- transition.

(a) Structures of the isoalloxazine ring and the nearby Arg378-Asp409 salt bridge, at time t as indicated. Eox/t-Eox/dark difference maps [|Fobs| (Eox/t)-|Fcalc| (Eox/dark) with phase information from Eox/dark] are shown for each panel at a 3σ level (cyan for positive and magenta for negative). The Eox/ss omit maps were least-squares superimposed via coot. Eox/10µs is also shown in Fig. 3e. Distances shown are in Å. (b) Structures of the isoalloxazine ring and the nearby Asn403 sidechain at time t are shown. Eox/t-Eox/dark difference maps are shown as in (a). Additionally, 2σ contoured composite omit maps of the corresponding structure and Eox/ss are shown in gold and grey, respectively. Eox/10µs is also shown in Fig. 3h. Distances shown are in Å.

Extended Data Fig. 2 Structural changes from TR-SFX experiments for the FADH•→FADH− transition.

(a) Structures of the isoalloxazine ring and the nearby Arg378-Asp409 salt bridge, from Str12 to Str23. Each structure is superposed over the EX/Y-Ered/ss difference maps. Difference density is shown in cyan (positive) and magenta (negative) at a 3.5σ contour level. The isoalloxazine moiety of Esemi/ss and Esemi/300ns are also shown in Fig. 3, f and g, respectively. (b) Same as (a) except with the EX/Y-Eox/ss difference maps (at a 4σ contour level) instead of EX/Y-Ered/ss. The Arg378-Asp409 pair (lower part) of Esemi/ss and Esemi/300ns are also shown in Fig. 3, f and g, respectively. (c) Structures of the isoalloxazine ring and the nearby Asn403 sidechain at time t are shown, with EX/Y-Eox/ss difference maps as in (b). Additionally, 2σ contoured composite omit maps of the corresponding structure and Esemi/ss are shown in gold and grey, respectively. Esemi/ss and Esemi/300ns are also shown in Fig. 3, i and j, respectively. All distances shown are in Å.

Extended Data Fig. 3 Evolution of FAD redox states in MmCPDII.

(a) In crystallo spectra of the oxidized (black line), neutral semiquinone (grey line), and fully reduced (dotted black line) states adopted by MmCPDII crystals. The occupancies of 100% for the oxidized and reduced states, as well as 65% for the neutral semiquinone state, were estimated by use of equations 1-3. (b) Projected occupancy of the FAD•- species over time. The projection was calculated by integrating a differential equation system resulting from previously published time constants and a quantum yield of 48%10. Numerical integration was performed using the Tinkercell software68. Integration was performed from 100 ps to 5 ms.

Source data

Extended Data Fig. 4 The intramolecular electron-transfer chain of MmCPDII.

(a, b) The electron-transfer chain of Eox/dark (Str2, grey) is compared to that of Eox/10ns and Eox/1µs (orange, a and b, respectively), showing no apparent conformational changes after electron transfer during RS1. Similar results were obtained for RS2. (c) The Trp388 environment are shown here for the Eox/dark, Eox/10ns, Eox/50ns, Eox/400μs, Eox/1ms and Eox/5ms (all orange) versus Eox/ss (magenta) during RS1. Eox/t-Eox/dark difference maps [|Fobs| (Eox/t)-|Fcalc| (Eox/dark) with phase information from Eox/dark] are shown for each panel at a 3σ level (cyan for positive and magenta for negative). Additionally, 2σ contoured composite omit maps of the corresponding structure and Eox/ss are shown in gold and grey, respectively. Both omit maps and structures were least-squares superimposed via coot. 10 ns after illumination, conformational changes have occurred in Asn361 and two histidines (343 and 356) near Trp388, the last electron donor in MmCPDII’s intramolecular electron-transfer chain. These correlate well with previous spectroscopic studies, which showed very rapid, subnanosecond deprotonation of the cationic Trp388•+ to Trp388•10. These conformational changes were observed for RS1 because reduction/protonation of W388• to W388 is a slow step (Fig. 1b). Interestingly, these changes, which cannot be seen in the dark, are maintained up to 0.4 to 1 ms, and only relax back between 1 and 5 ms after excitation, correlating well with the timing of reoxidation in the RS1 series (Fig. 3a,c). Further, and confirming the lack of light contamination of the dark state, no substantial conformational changes could be observed between Eox/dark and Eox/ss. Since RS2 was performed in the presence of reducing agent DTT, W388• can be regenerated to W388 continuously and the ultrafast conformational change could not be observed. (d) Non-isomorphous crystallographic changes upon light excitation. On the left side, the distances along the c-axis between the dark1 and dark2 images (Dark) and the corresponding light and dark images for each time point are shown. Although these changes are small, it is quite evident that they are relevant due to their time dependence. On the right panel, one of the main MmCPDII crystal contacts for the structures in (c) is shown, with molecules belonging to difference asymmetric units separated by a dotted line representing the symmetry axis. Note the conformational change occurring in Asp18 and Asn109 as a result of the changes in H345 and H356.

Source data

Extended Data Fig. 5 Functional analysis of the R378K mutant.

(a) FPLC profiles showing the low (ca. 20%) stoichiometry of FAD uptake by R378K (dashed lines) relative to WT (solid lines). The 280 nm traces in blue are characteristic of protein absorption, while the 450 nm traces (red) for oxidized FAD. (b) Effect of heat denaturation on the spectroscopic properties of wild type MmCPDII (black lines) and the R378K mutant (red lines). The left panel shows the raw spectra, with the characteristic FAD absorption pattern between 300 and 500 nm, while the right panel shows difference spectra. The fine structures of the WT spectrum, which are caused by tight protein-chromophore interactions69, disappear upon FAD being released into the solvent due to protein denaturation, as evidenced by the spectrum becoming smooth after denaturation, and by the maxima and minima between 350 and 500 nm in the difference spectrum. On the other hand, R378K does not showcase fine structures either in the bound, or in the denatured state, resulting in a featureless difference spectrum. The native MmCPDII spectra were obtained by measuring oxidized 10 mg/mL MmCPDII samples, which had been equilibrated in the dark for at least 24 h, with a Nanodrop 1000 (Thermo Scientific) spectrophotometer. For heat denaturation to achieve FAD release from the protein moiety, the protein sample was boiled for 10 min at 99 °C, then centrifuged at 18,000 g for 3 min to remove the protein precipitate and leave a free FAD solution.

Source data

Extended Data Fig. 6 Representative active site structures of six families of flavin-dependent oxidoreductases.

FAD is highlighted in gold, residues in grey, and substrate or inhibitor in blue. The FAD orientation is identical to Fig. 1b. PDB codes for all structures can be found on the bottom right of each panel. Source organism, enzyme name, and protein family ID are indicated over each structure.

Extended Data Fig. 7 Difference maps showing significant peaks at the active site mainly.

(a) Chemical structures of the tricyclic isoalloxazine moiety of oxidized (FADox, left), semiquinone radical (FADH•, middle), and fully reduced hydroquinone (FADH−, right). (b) Overall structures of the three states of MmCPDII shown in grey, blue and dark green, respectively. The bound FAD is shown in gold sticks. The Esemi/ss-Eox/ss map, corresponding to the FADox → FADH• transition is superposed to the semiquinone FADH• structure (middle), and the Ered/ss-Eox/ss map, corresponding to the FADox → FADH− transition, to the FADH− structure (right). Positive and negative difference electron densities (cyan and magenta, respectively; contouring level: 4 σ) are almost exclusively located near the coenzyme site. (c) Since DTT is present in Esemi/ss and Ered/ss but not Eox/ss, maps of bound DTT from Esemi/ss-Eox/ss (left) and Ered/ss-Eox/ss (right) are clearly observed, serving as a good quality control for sample preparation. The corresponding maps are 4σ contoured.

Extended Data Fig. 8 Control analysis of the steady-state datasets from SFX.

Each of the datasets was randomly divided in half, then processed and solved independently. Next, |Fobs,1/2| -|Fobs,2/2| maps were generated for each of them. Here, the corresponding difference maps are shown with a 4 σ-cut-off for (a) the full proteins for all three datasets, (b) the bound DTT for Esemi/ss and Ered/ss, and (c) the active sites of all three proteins. As expected, only very few peaks can be observed overall, and none at the active site.

Extended Data Fig. 9 Examples of the FAD geometry correlation analysis.

(a) Superposition of all three steady-state real space correlation analysis (Eox/ss, Str1, in shades of grey, Esemi/ss, Str12, in shades of green, and Ered/ss, Str23, in shades of blue). The overall restraint space derived from our computational analysis is shown as a dotted square surrounding the data extracted from Phenix refinement. Individual data points represent the actual ρC and ρN values obtained from the refined structures. Values corresponding to good fits (top 95 to 100% CC values) are highlighted by a darker shade. (b) Real space correlation analysis examples of the first time-resolved series. The dark control structure (Eox/dark, Str2) results are shown in black, while the ones for the 10 µs time-point (Eox/10µs, Str7) in shades of orange, with good fits in a dark shade, while bad fits in a light one. As the conformational space for FAD•- was unknown, the restraint space for Eox/10µs varied from -90 to 90 degrees. (c) Real space correlation analysis results for the control dark structure (Esemi/dark, Str13) of the second time-resolved series. For real space correlation analysis, the FADH− restraint space was used, and accordingly, data is shown in shades of blue (dark blue for good fits, light blue for bad fits). (d) Real space correlation analysis results for the 300 ns time-point (Esemi/300ns, Str17) of the second time-resolved series. For real space correlation analysis, the FADH− restraint space was used, and accordingly, data is shown in shades of blue (dark blue for good fits, light blue for bad fits). (e - g) 2D ρC and ρN heat maps for the initial computational analysis. Occupancy of each bin is shown in a blue (low occupancy) to red (high occupancy) scale. (e) Oxidized state computational analysis. (f) Semiquinone state computational analysis. (g) Reduced state computational analysis assuming a neutral Arg378-Asp409 pair.

Source data

Supplementary information

Supplementary Information

Supplementary Methods, Tables 1–6 and references.

Reporting Summary

Supplementary Video 1

Summary of the first TR-SFX series, showing the FADox to FAD•− transition. Here, first the overall topology of the photolyase is shown for Eox/dark (Str2, N-terminal domain in blue, C-terminal domain in orange). Then, the active site is zoomed into, and the changes in FAD (gold) buckling versus movement of Arg378 and Asp409 (orange) are shown from two different viewpoints (front and top). For the composition of this video, Eox/t structures were used (Str2–11), and intermediate frames were generated as described in the ‘Data visualization’ section of the Methods.

Supplementary Video 2

Summary of the second TR-SFX series, showing the FADH• to FADH− transition. Here, first the overall topology of the photolyase is shown for Esemi/dark (Str13, N-terminal domain in blue, C-terminal domain in cyan). Then, the active site is zoomed into, and the changes in FAD (gold) buckling versus movement of Arg378 and Asp409 (cyan) are shown from two different viewpoints (front and top). For the composition of this video, Esemi/t structures were used (Str13–22), and intermediate frames were generated as described in the ‘Data visualization’ paragraph of the Methods.

Supplementary Video 3

A structural view of photoactivation: composite of both photoreduction steps. The initial frame corresponds to panels a to c of Fig. 4. From that point on, panel a divides, showing both the ‘front’ orientation (left) of the FAD isoalloxazine moiety, Arg378 and Asp409, and the ‘top’ orientation (right), which is more adequate for following the Asn403-isoalloxazine interactions. The video follows the forward reactions between the FAD and the redox sensor triad via colour-coded dashed lines indicating their interatomic distances. These lines become solid when two atoms are close enough to each other for hydrogen bonding. These are also highlighted by the graph below the structural representations, which follows the hydrogen-bonding score, and the ρC and ρN angles over time. The video finishes with a superposition of all four FAD redox states investigated here.

Supplementary Data 1

Raw data from hydrogen-bonding analysis. Spreadsheet containing all raw data for the hydrogen-bonding analysis between the FAD isoalloxazine N5 nitrogen, Arg378, Asn403 and Asp409 (distances in Ångstrom, angles in degrees). The spreadsheet contains a total of 24 tables, divided into three subgroups, that is eight for RS1, eight for RS2 and eight for the steady-state structures. Within each subgroup, each table describes a single hydrogen bond in any given structure by the five criteria described in the corresponding Supplementary Methods section.

Supplementary Data 2

Computational data for real-space correlation analysis of FAD geometry at each time point. Panels a to j correspond to the RS1 structures, and panels k to t to the RS2 ones. Individual data points represent the actual ρC and ρN values obtained from the refined structures. Values corresponding to good fits (top 95–100% CC values) are highlighted by a darker shade. a–j, RS1 analysis. The RS1 dark control structure (Eox/dark, Str2) results are shown in black (a), as it was analysed against the oxidized conformational space. All other RS1 structures are in shades of orange, with good fits in a dark shade and bad fits in a light one. As the conformational space for FAD•− was unknown, the restraint space for all RS1 data varied from −90 to 90°. k–t, RS2 analysis. For real-space correlation analysis, the FADH− restraint space was used and, accordingly, data are shown in shades of blue (dark blue for good fits, light blue for bad fits), while the overall restraint space derived from our computational analysis is shown as a dotted square surrounding the data.

Supplementary Data 3

PDB structure-based search results. Two searches were performed as described in the Supplementary Methods. The detailed results are listed in this file. Specific results are shown in Supplementary Table 6 and Extended Data Fig. 6.

Source data

Source Data Fig. 3

Numerical data for plots in panels a to d.

Source Data Fig. 4

Numerical data for plots in panels c and e.

Source Data Extended Data Fig. 3

Numerical data for absorption spectra (panel a) and simulation (panel b).

Source Data Extended Data Fig. 4

Numerical data for plot on panel b.

Source Data Extended Data Fig. 5

Numerical data for all plots.

Source Data Extended Data Fig. 9

Source numerical data for all plots in ED Fig. 9.

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Maestre-Reyna, M., Yang, CH., Nango, E. et al. Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme. Nat. Chem. 14, 677–685 (2022). https://doi.org/10.1038/s41557-022-00922-3

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