Structure of a putative immature form of a Rieske-type iron-sulfur protein in complex with zinc chloride

1.

2.

3.

4.

5.

6.

7.

8.

9.

Beinert, H., Holm, R. H. & Münck, E. Iron-sulfur clusters: nature’s modular,

multipurpose structures. Science 277, 653–659 (1997).

Johnson, M. K. Iron-sulfur proteins: new roles for old clusters. Curr. Opin.

Chem. Biol. 2, 173–181 (1998).

Russell, M. J. & Martin, W. The rocky roots of the acetyl-CoA pathway. Trends

Biochem. Sci. 29, 358–363 (2004).

Heinen, W. & Lauwers, A. M. Organic sulfur compounds resulting from the

interaction of iron sulfide, hydrogen sulfide and carbon dioxide in an

anaerobic aqueous environment. Orig. Life Evol. Biosph. 26, 131–150 (1996).

Zanello, P. The competition between chemistry and biology in assembling

iron–sulfur derivatives. Molecular structures and electrochemistry. Part II.

{[Fe2S2](SγCys)4} proteins. Coord. Chem. Rev. 280, 54–83 (2014).

Zanello, P. The competition between chemistry and biology in assembling

iron–sulfur derivatives. Molecular structures and electrochemistry. Part III.

{[Fe2S2](Cys)3(X)} (X = Asp, Arg, His) and {[Fe2S2](Cys)2(His)2} proteins.

Coord. Chem. Rev. 306, 420–442 (2016).

Ciofi-Baffoni, S., Nasta, V. & Banci, L. Protein networks in the maturation of

human iron-sulfur proteins. Metallomics 10, 49–72 (2018).

Maio, N. & Rouault, T. A. Outlining the complex pathway of mammalian Fe-S

Cluster biogenesis. Trends Biochem. Sci. 45, 411–426 (2020).

Braymer, J. J., Freibert, S. A., Rakwalska-Bange, M. & Lill, R. Mechanistic

concepts of iron-sulfur protein biogenesis in Biology. Biochim. Biophys. Acta

Mol. Cell Res. 1868, 118863 (2021).

COMMUNICATIONS CHEMISTRY | (2023)6:190 | https://doi.org/10.1038/s42004-023-01000-6 | www.nature.com/commschem

COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-023-01000-6

10. Stehling, O. & Lill, R. The role of mitochondria in cellular iron-sulfur protein

biogenesis: mechanisms, connected processes, and diseases. Cold Spring Harb.

Perspect. Biol. 5, a011312 (2013).

11. Isaya, G. Mitochondrial iron-sulfur cluster dysfunction in neurodegenerative

disease. Front. Pharm. 5, 29 (2014).

12. Beilschmidt, L. K. & Puccio, H. M. Mammalian Fe-S cluster biogenesis and its

implication in disease. Biochimie 100, 48–60 (2014).

13. Wachnowsky, C., Fidai, I. & Cowan, J. A. Iron-sulfur cluster biosynthesis and

trafficking—impact on human disease conditions. Metallomics 10, 9–29

(2018).

14. Selvanathan, A. & Parayil Sankaran, B. Mitochondrial iron-sulfur cluster

biogenesis and neurological disorders. Mitochondrion 62, 41–49 (2022).

15. Roche, B. et al. Iron/sulfur proteins biogenesis in prokaryotes: formation,

regulation and diversity. Biochim. Biophys. Acta 1827, 455–469 (2013).

16. Blanc, B., Gerez, C. & Ollagnier de Choudens, S. Assembly of Fe/S proteins in

bacterial systems: Biochemistry of the bacterial ISC system. Biochim. Biophys.

Acta 1853, 1436–1447 (2015).

17. Gao, F. Iron-sulfur cluster biogenesis and iron homeostasis in cyanobacteria.

Front. Microbiol. 11, 165 (2020).

18. Baussier, C. et al. Making iron-sulfur cluster: structure, regulation and

evolution of the bacterial ISC system. Adv. Microb. Physiol. 76, 1–39 (2020).

19. Monfort, B., Want, K., Gervason, S. & D’Autréaux, B. Recent advances in the

elucidation of frataxin biochemical function open novel perspectives for the

treatment of Friedreich’s ataxia. Front. Neurosci. 16, 838335 (2022).

20. Bilder, P. W., Ding, H. & Newcomer, M. E. Crystal structure of the ancient,

Fe-S scaffold IscA reveals a novel protein fold. Biochemistry 43, 133–139

(2004).

21. Ramelot, T. A. et al. Solution NMR structure of the iron-sulfur cluster

assembly protein U (IscU) with zinc bound at the active site. J. Mol. Biol. 344,

567–583 (2004).

22. Shimomura, Y., Wada, K., Fukuyama, K. & Takahashi, Y. The asymmetric

trimeric architecture of [2Fe-2S] IscU: implications for its scaffolding during

iron-sulfur cluster biosynthesis. J. Mol. Biol. 383, 133–143 (2008).

23. Kim, J. H. et al. Structure and dynamics of the iron-sulfur cluster assembly

scaffold protein IscU and its interaction with the cochaperone HscB.

Biochemistry 48, 6062–6071 (2009).

24. Iwema, T. et al. Structural basis for delivery of the intact [Fe2S2] cluster by

monothiol glutaredoxin. Biochemistry 48, 6041–6043 (2009).

25. Marinoni, E. N. et al. (IscS-IscU)2 complex structures provide insights into

Fe2S2 biogenesis and transfer. Angew. Chem. Int. Ed. Engl. 29, 5439–5442

(2012).

26. Boniecki, M. T., Freibert, S. A., Mühlenhoff, U., Lill, R. & Cygler, M. Structure

and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.

Nat. Commun. 8, 1287 (2017).

27. Cory, S. A. et al. Structure of human Fe-S assembly subcomplex reveals

unexpected cysteine desulfurase architecture and acyl-ACP-ISD11

interactions. Proc. Natl. Acad. Sci. USA 114, E5325–E5334 (2017).

28. Fox, N. G. et al. Structure of the human frataxin-bound iron-sulfur cluster

assembly complex provides insight into its activation mechanism. Nat.

Commun. 10, 2210 (2019).

29. Freibert, S. A. et al. N-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster

synthesis by ISCU2 dimerization. Nat. Commun. 12, 6902 (2021).

30. Gervason, S. et al. Physiologically relevant reconstitution of iron-sulfur cluster

biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and

frataxin. Nat. Commun. 10, 3566 (2019).

31. Lin, C. W., McCabe, J. W., Russell, D. H. & Barondeau, D. P. Molecular

mechanism of ISC iron-sulfur cluster biogenesis revealed by high-resolution

native mass spectrometry. J. Am. Chem. Soc. 142, 6018–6029 (2020).

32. Lin, C. W., Oney-Hawthorne, S. D., Kuo, S. T., Barondeau, D. P. & Russell, D.

H. Mechanistic insights into IscU conformation regulation for Fe-S cluster

biogenesis revealed by variable temperature electrospray ionization native ion

mobility mass spectrometry. Biochemistry 61, 2733–2741 (2022).

33. Srour, B. et al. Iron Insertion at the assembly site of the ISCU scaffold protein

is a conserved process initiating Fe-S cluster biosynthesis. J. Am. Chem. Soc.

144, 17496–17515 (2022).

34. Zhang, Z. et al. Electron transfer by domain movement in cytochrome bc1.

Nature 392, 677–684 (1998).

35. Solmaz, S. R. & Hunte, C. Structure of complex III with bound cytochrome c

in reduced state and definition of a minimal core interface for electron

transfer. J. Biol. Chem. 283, 17542–17549 (2008).

36. Kleinschroth, T. et al. X-ray structure of the dimeric cytochrome bc1 complex

from the soil bacterium Paracoccus denitrificans at 2.7-Å resolution. Biochim.

Biophys. Acta 1807, 1606–1615 (2011).

37. Esser, L. et al. Inhibitor-complexed structures of the cytochrome bc1 from the

photosynthetic bacterium Rhodobacter sphaeroides. J. Biol. Chem. 283,

2846–2857 (2008).

38. Gabellini, N., Harnisch, U., McCarthy, J. E., Hauska, G. & Sebald, W. Cloning

and expression of the fbc operon encoding the FeS protein, cytochrome b and

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

ARTICLE

cytochrome c1 from the Rhodopseudomonas sphaeroides b/c1 complex. EMBO

J. 4, 549–553 (1985).

Sattley, W. M. et al. Complete genome of the thermophilic purple sulfur

Bacterium Thermochromatium tepidum compared to Allochromatium

vinosum and other Chromatiaceae. Photosynth. Res. 151, 125–142 (2022).

Link, T. A. et al. Comparison of the “Rieske” [2Fe-2S] center in the bc1

complex and in bacterial dioxygenases by circular dichroism spectroscopy and

cyclic voltammetry. Biochemistry 35, 7546–7552 (1996).

Link, T. A. In: Handbook of Metalloproteins. Messerschmidt A, Huber R,

Poulos T, Wieghardt K, editors. vol. 1. New York: Wiley; pp. 518–531 (2001).

Liu, G. et al. Heme biosynthesis depends on previously unrecognized

acquisition of iron-sulfur cofactors in human amino-levulinic acid

dehydratase. Nat. Commun. 11, 6310 (2020).

Maio, N. et al. Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA

polymerase are potential antiviral targets. Science 373, 236–241 (2021).

Iwata, S. et al. Complete structure of the 11-subunit bovine mitochondrial

cytochrome bc1 complex. Science 281, 64–71 (1998).

Kolling, D. J., Brunzelle, J. S., Lhee, S., Crofts, A. R. & Nair, S. K. Atomic

resolution structures of Rieske iron-sulfur protein: role of hydrogen bonds in

tuning the redox potential of iron-sulfur clusters. Structure 15, 29–38 (2007).

Watanabe, S. et al. Zinc regulates ERp44-dependent protein quality control in

the early secretory pathway. Nat. Commun. 10, 603 (2019).

Kalhor, P., Wang, Y. & Yu, Z. The structures of ZnCl2-ethanol mixtures, a

spectroscopic and quantum chemical calculation study. Molecules 26, 2498

(2021).

Harding, M. M. The geometry of metal-ligand interactions relevant to

proteins. Acta Crystallogr. D Biol. Crystallogr. 55, 1432–1443 (1999).

Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from

crystalline state. J. Mol. Biol. 372, 774–797 (2007).

Schmid, B. et al. Structure of a cofactor-deficient nitrogenase MoFe protein.

Science 296, 352–356 (2002).

Selbach, B. P. et al. Fe-S cluster biogenesis in Gram-positive bacteria: SufU is a

zinc-dependent sulfur transfer protein. Biochemistry 53, 152–160 (2014).

Fujishiro, T. et al. Zinc-ligand swapping mediated complex formation and

sulfur transfer between sufs and sufu for Iron-Sulfur cluster biogenesis in

Bacillus subtilis. J. Am. Chem. Soc. 139, 18464–18467 (2017).

Galeano, B. K. et al. Zinc and the iron donor frataxin regulate oligomerization

of the scaffold protein to form new Fe-S cluster assembly centers. Metallomics

9, 773–801 (2017).

Fox, N. G. et al. Zinc(II) binding on human wild-type ISCU and Met140

variants modulates NFS1 desulfurase activity. Biochimie 152, 211–218 (2018).

Li, J. et al. Zinc toxicity and iron-sulfur cluster biogenesis in Escherichia coli.

Appl. Environ. Microbiol. 85, e01967–18 (2019).

Atkinson, A. et al. Mzm1 influences a labile pool of mitochondrial zinc

important for respiratory function. J. Biol. Chem. 285, 19450–19459 (2010).

Yeung, N. et al. The E. coli monothiol glutaredoxin GrxD forms homodimeric

and heterodimeric FeS cluster containing complexes. Biochemistry 50,

8957–8969 (2011).

Bonomi, F., Iametti, S., Morleo, A., Ta, D. & Vickery, L. E. Facilitated transfer

of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange.

Biochemistry 50, 9641–9650 (2011).

O’Reilly, J. E. Oxidation-reduction potential of the ferro-ferricyanide system

in buffer solutions. Biochim. Biophys. Acta 292, 509–515 (1973).

Kabsh, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

Terwilliger, T. C. Maximum likelihood density modification. Acta Crystallogr.

D Biol. Crystallogr. 56, 965–972 (2000).

Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure

solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).

Adams, P. D. et al. PHENIX: building new software for automated

crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr.

58, 1948–1954 (2002).

Perrakis, A., Morris, R. & Lamzin, V. S. Automated protein model building

combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463

(1999).

Collaborative Computational Project, Number 4. The CCP4 suite: programs

for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763

(1994).

Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development

of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

Brünger, A. T. et al. Crystallography & NMR system: a new software suite for

macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr.

54, 905–921 (1998).

Vagin, A. & Teplyakov, A. (1997) MOLREP: an automated program for

molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

Chen, V. B. et al. MolProbity: all-atom structure validation for

macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66,

12–21 (2010).

The PyMOL Molecular Graphics System, Version 1.5.0.5 Schrödinger, LLC.

COMMUNICATIONS CHEMISTRY | (2023)6:190 | https://doi.org/10.1038/s42004-023-01000-6 | www.nature.com/commschem

ARTICLE

COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-023-01000-6

71. Shimizu, N. et al. Software development for analysis of small-angle X-ray

scattering data.”. AIP Conf. Proc. 1741, 050017 (2016).

72. Orthaber, D., Bergmann, A. & Glatter, O. SAXS experiments on absolute scale

with Kratky systems using water as a secondary standard. J. Appl. Crystallogr.

33, 218–225 (2000).

73. Yonezawa, K., Takahashi, M., Yatabe, K., Nagatani, Y. & Shimizu, N.

MOLASS: Software for automatic processing of matrix data obtained from

small-angle X-ray scattering and UV-visible spectroscopy combined with sizeexclusion chromatography. Biophys. Physicobiol. 20, e200001 (2023).

74. Franke, D. et al. ATSAS 2.8: a comprehensive data analysis suite for smallangle scattering from macromolecular solutions. J. Appl. Crystallogr. 50,

1212–1225 (2017).

75. Svergun, D. I. Determination of the regularization parameter in indirecttransform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503

(1992).

76. Manalastas-Cantos, K. et al. ATSAS 3.0: expanded functionality and new tools

for small-angle scattering data analysis. J. Appl. Crystallogr. 54, 343–355

(2021).

77. Franke, D. & Svergun, D. I. DAMMIF, a program for rapid ab-initio shape

determination in small-angle scattering. J. Appl. Crystallogr. 42, 342–346

(2009).

Acknowledgements

We thank Messrs. S. Taguchi, and Y. Tanaka for their help in this work. We also thank

the beamline staff at BL38B1 and BL41XU of SPring-8 for their help with the experiments (proposal nos. 2019A2560, 2020A2548 and 2021A2548 to K.T.). This work was

supported by the Takeda Science Foundation (to K.T.) and ISHIZUE 2022 of Kyoto

University (to K.T.). This research was also supported by Research Support Project for

Life Science and Drug Discovery (BINDS) from AMED (JP23ama121001).

Author contributions

K.T. and E.T. designed the experiments. E.T. established conditions for the expression,

purification and crystallization experiments. E.T., S.Ni., R.T., N.S., K.O. S.F. performed

biochemical analyses. E.T., S.Ni., N.S. prepared crystals. E.T., S.Ni. and K.T. performed

the X-ray diffraction data collections. S.Ni. and K.T. performed the crystallographic

analysis. H.A., S.Na. performed the SEC-MALS analysis. R.T., S.Na., H.S., K.T. performed

the SEC-SAXS analysis. All authors discussed the results. K.T. and S.Ni. wrote the initial

10

draft, and all authors revised it. All authors gave comments on the manuscript and

consented to submission of the final version.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s42004-023-01000-6.

Correspondence and requests for materials should be addressed to Kazuki Takeda.

Peer review information This manuscript has been previously reviewed at another

Nature Portfolio journal. The manuscript was considered suitable for publication without

further review at Communications Chemistry. A Peer review file is available.

Reprints and permission information is available at http://www.nature.com/reprints

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons

Attribution 4.0 International License, which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give

appropriate credit to the original author(s) and the source, provide a link to the Creative

Commons licence, and indicate if changes were made. The images or other third party

material in this article are included in the article’s Creative Commons licence, unless

indicated otherwise in a credit line to the material. If material is not included in the

article’s Creative Commons licence and your intended use is not permitted by statutory

regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder. To view a copy of this licence, visit http://creativecommons.org/

licenses/by/4.0/.

© The Author(s) 2023

COMMUNICATIONS CHEMISTRY | (2023)6:190 | https://doi.org/10.1038/s42004-023-01000-6 | www.nature.com/commschem

...