Interprotein electron transfer biohybrid system for photocatalytic H2 production

  • Udita Brahmachari
  • P. Raj Pokkuluri
  • David M. Tiede
  • Jens Niklas
  • Oleg G. Poluektov
  • Karen L. Mulfort
  • Lisa M. UtschigEmail author
Original Article


Worldwide there is a large research investment in developing solar fuel systems as clean and sustainable sources of energy. The fundamental mechanisms of natural photosynthesis can provide a source of inspiration for these studies. Photosynthetic reaction center (RC) proteins capture and convert light energy into chemical energy that is ultimately used to drive oxygenic water-splitting and carbon fixation. For the light energy to be used, the RC communicates with other donor/acceptor components via a sophisticated electron transfer scheme that includes electron transfer reactions between soluble and membrane bound proteins. Herein, we reengineer an inherent interprotein electron transfer pathway in a natural photosynthetic system to make it photocatalytic for aqueous H2 production. The native electron shuttle protein ferredoxin (Fd) is used as a scaffold for binding of a ruthenium photosensitizer and H2 catalytic function is imparted to its partner protein, ferredoxin–NADP+-reductase (FNR), by attachment of cobaloxime molecules. We find that this 2-protein biohybrid system produces H2 in aqueous solutions via light-induced interprotein electron transfer reactions (TON > 2500 H2/FNR), providing insight about using native protein–protein interactions as a method for fuel generation.


Interprotein electron transfer Biohybrid Solar hydrogen Ferredoxin Ferredoxin–NADP+-reductase Solar fuel Photosynthetic electron transfer 



This work is supported by the U.S. Department of Energy Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, under Contract No. DE-AC02-06CH11357.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11120_2019_705_MOESM1_ESM.docx (781 kb)
Supplementary file1 (DOCX 782 kb)


  1. Alstrum-Acevedo JH, Brennaman MK, Meyer TJ (2005) Chemical approaches to artificial photosynthesis. 2. Inorg Chem 44:6802–6827PubMedCrossRefGoogle Scholar
  2. Bakac A, Espenson JH (1984) Unimolecular and bimolecular homolytic reactions of organochromium and organocobalt complexes. Kinetics and equilibria. J Am Chem Soc 106:5197–5202CrossRefGoogle Scholar
  3. Bakac A, Brynildson ME, Espenson JH (1986) Characterization of the structure, properties, and reactivity of a cobalt(II) macrocyclic complex. Inorg Chem 25:4108–4114CrossRefGoogle Scholar
  4. Binda C, Coda A, Aliverti A, Zanetti G, Mattevi A (1998) Structure of the mutant E92K of [2Fe-2S] ferredoxin I from Spinacia oleracea at 1.7 Å resolution. Acta Crystallogr Sect D Biol Crystallogr 54:1353–1358CrossRefGoogle Scholar
  5. Brown KA, Harris DF, Wilker MB, Rasmussen A, Khadka N, Hamby H, Keable S, Dukovic G, Peters JW, Seefeldt LC, King PW (2016) Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid. Science 352:448–450PubMedCrossRefGoogle Scholar
  6. Cammack R, Rao KK, Bargeron CP, Hutson KG, Andrew PW, Rogers LJ (1977) Midpoint redox potentials of plant and algal ferredoxins. Biochem J 168:205PubMedPubMedCentralGoogle Scholar
  7. Corrado ME, Aliverti A, Zanetti G, Mayhew SG (1996) Analysis of the oxidation-reduction potentials of recombinant ferredoxin-NADP+ reductase from spinach chloroplasts. Eur J Biochem 239:662–667PubMedCrossRefGoogle Scholar
  8. Diakonova A, Khrushchev S, Kovalenko I, Riznichenko GY, Rubin A (2016) Influence of pH and ionic strength on electrostatic properties of ferredoxin, FNR, and hydrogenase and the rate constants of their interaction. Phys Biol 13:056004PubMedCrossRefGoogle Scholar
  9. Faro M, Gómez-Moreno C, Stankovich M, Medina M (2002) Role of critical charged residues in reduction potential modulation of ferredoxin-NADP+ reductase. Eur J Biochem 269:2656–2661PubMedCrossRefGoogle Scholar
  10. Gould S, Strouse GF, Meyer TJ, Sullivan BP (1991) Formation of thin polymeric films by electropolymerization. Reduction of metal complexes containing bromomethyl-substituted derivatives of 2, 2'-bipyridine. Inorg Chem 30:2942–2949CrossRefGoogle Scholar
  11. Greenbaum E (1988) Interfacial photoreactions at the photosynthetic membrane interface: an upper limit for the number of platinum atoms required to form a hydrogen-evolving platinum metal catalyst. J Phys Chem 92:4571–4574CrossRefGoogle Scholar
  12. Grimme RA, Lubner CE, Bryant DA, Golbeck JH (2008) Photosystem I/molecular wire/metal nanoparticle bioconjugates for the photocatalytic production of H2. J Am Chem Soc 130:6308–6309PubMedCrossRefGoogle Scholar
  13. Hermoso JA, Mayoral T, Faro M, Gómez-Moreno C, Sanz-Aparicio J, Medina M (2002) Mechanism of coenzyme recognition and binding revealed by crystal structure analysis of Ferredoxin–NADP+ reductase complexed with NADP+. J Mol Biol 319:1133–1142PubMedCrossRefGoogle Scholar
  14. Hurley JK, Morales R, Martínez-Júlvez M, Brodie TB, Medina M, Gómez-Moreno C, Tollin G (2002) Structure–function relationships in Anabaena ferredoxin/ferredoxin:NADP+ reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography. Biochim Biophys Acta (BBA) - Bioenerg 1554:5–21CrossRefGoogle Scholar
  15. Ihara M, Nishihara H, Yoon K-S, Lenz O, Friedrich B, Nakamoto H, Kojima K, Honma D, Kamachi T, Okura I (2006) Light-driven hydrogen production by a hybrid complex of a [NiFe]-Hydrogenase and the cyanobacterial Photosystem I. Photochem Photobiol 82:676–682PubMedCrossRefGoogle Scholar
  16. Iwuchukwu IJ, Vaughn M, Myers N, O'Neill H, Frymier P, Bruce BD (2010) Self-organized photosynthetic nanoparticle for cell-free hydrogen production. Nat Nanotechnol 5:73–79PubMedCrossRefGoogle Scholar
  17. Kohler L, Niklas J, Johnson RC, Zeller M, Poluektov OG, Mulfort KL (2019) Molecular cobalt catalysts for H2 generation with redox activity and proton relays in the second coordination sphere. Inorg Chem 58:1697–1709PubMedCrossRefGoogle Scholar
  18. Kurisu G, Kusunoki M, Katoh E, Yamazaki T, Teshima K, Onda Y, Kimata-Ariga Y, Hase T (2001) Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP+ reductase. Nat Struct Biol 8:117–121PubMedCrossRefGoogle Scholar
  19. Lewis NS (2007) Toward cost-effective solar energy use. Science 315:798–801PubMedCrossRefGoogle Scholar
  20. Lewis NS, Nocera DG (2006) Powering the planet: Chemical challenges in solar energy utilization. Proc Natl Acad Sci USA 103:15729PubMedCrossRefGoogle Scholar
  21. Lubner CE, Grimme R, Bryant DA, Golbeck JH (2010) Wiring Photosystem I for direct solar hydrogen production. Biochemistry 49:404–414PubMedCrossRefGoogle Scholar
  22. Lubner CE, Applegate AM, Knörzer P, Ganago A, Bryant DA, Happe T, Golbeck JH (2011) Solar hydrogen-producing bionanodevice outperforms natural photosynthesis. Proc Natl Acad Sci USA 108:20988PubMedCrossRefGoogle Scholar
  23. Martínez-Júlvez M, Hermoso J, Hurley JK, Mayoral T, Sanz-Aparicio J, Tollin G, Gómez-Moreno C, Medina M (1998) Role of Arg100 and Arg264 from Anabaena PCC 7119 ferredoxin−NADP+ reductase for optimal NADP+ binding and electron transfer. Biochemistry 37:17680–17691PubMedCrossRefGoogle Scholar
  24. Medina M (2009) Structural and mechanistic aspects of flavoproteins: photosynthetic electron transfer from Photosystem I to NADP+. FEBS J 276:3942–3958PubMedCrossRefGoogle Scholar
  25. Miller M, Robinson WE, Oliveira AR, Heidary N, Kornienko N, Warnan J, Pereira IAC, Reisner E (2019) Interfacing formate dehydrogenase with metal oxides for the reversible electrocatalysis and solar-driven reduction of carbon dioxide. Angew Chem Int Ed 131:4649–4653CrossRefGoogle Scholar
  26. Morales R, Charon M-H, Kachalova G, Serre L, Medina M, Gómez-Moreno C, Frey M (2000a) A redox-dependent interaction between two electron-transfer partners involved in photosynthesis. 1:271–276Google Scholar
  27. Morales R, Kachalova G, Vellieux F, Charon M-H, Frey M (2000b) Crystallographic studies of the interaction between the ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena: looking for the elusive ferredoxin molecule. Acta Crystallogr Sect D Biol Crystallogr 56:1408–1412CrossRefGoogle Scholar
  28. Mosebach L, Heilmann C, Mutoh R, Gäbelein P, Steinbeck J, Happe T, Ikegami T, Hanke G, Kurisu G, Hippler M (2017) Association of Ferredoxin:NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii. Photosynth Res 134:291–306PubMedPubMedCentralCrossRefGoogle Scholar
  29. Mulfort KL, Utschig LM (2016) Modular homogeneous chromophore-catalyst assemblies. Acc Chem Res 49:835–843PubMedCrossRefGoogle Scholar
  30. Mulo P, Medina M (2017) Interaction and electron transfer between ferredoxin–NADP+ oxidoreductase and its partners: structural, functional, and physiological implications. Photosynth Res 134:265–280PubMedCrossRefGoogle Scholar
  31. Niklas J, Mardis KL, Rakhimov RR, Mulfort KL, Tiede DM, Poluektov OG (2012) The hydrogen catalyst cobaloxime: A multifrequency EPR and DFT study of cobaloxime’s electronic structure. J Phys Chem B 116:2943–2957PubMedPubMedCentralCrossRefGoogle Scholar
  32. Palma PN, Lagoutte B, Krippahl L, Moura JJG, Guerlesquin F (2005) Synechocystis ferredoxin/ferredoxin-NADP+-reductase/NADP+ complex: structural model obtained by NMR-restrained docking. FEBS Lett 579:4585–4590PubMedCrossRefGoogle Scholar
  33. Poluektov OG, Utschig LM, Dalosto S, Thurnauer MC (2003) Probing local dynamics of the photosynthetic bacterial reaction center with a cysteine specific spin label. J Phys Chem B 107:6239–6244CrossRefGoogle Scholar
  34. Quaranta A, Lagoutte B, Frey J, Sétif P (2016) Photoreduction of the ferredoxin/ferredoxin–NADP+-reductase complex by a linked ruthenium polypyridyl chromophore. Photosynth Res 160:347–354Google Scholar
  35. Silver SC, Niklas J, Du P, Poluektov OG, Tiede DM, Utschig L (2013) Protein delivery of a Ni catalyst to Photosystem I for light-driven hydrogen production. J Am Chem Soc 135:13246–13249PubMedCrossRefGoogle Scholar
  36. Soltau SR, Niklas J, Dahlberg PD, Poluektov OG, Tiede DM, Mulfort KL, Utschig L (2015) Aqueous light driven hydrogen production by a Ru–ferredoxin–Co biohybrid. Chem Commun 51:10628–10631CrossRefGoogle Scholar
  37. Soltau SR, Dahlberg PD, Niklas J, Poluektov OG, Mulfort KL, Utschig LM (2016) Ru–protein–Co biohybrids designed for solar hydrogen production: understanding electron transfer pathways related to photocatalytic function. Chem Sci 7:7068–7078PubMedPubMedCentralCrossRefGoogle Scholar
  38. Soltau SR, Niklas J, Dahlberg PD, Mulfort KL, Poluektov OG, Utschig LM (2017) Charge separation related to photocatalytic H2 production from a Ru–Apoflavodoxin–Ni biohybrid. ACS Energy Lett 2:230–237CrossRefGoogle Scholar
  39. Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55PubMedCrossRefGoogle Scholar
  40. Sun L, Berglund H, Davydov R, Norrby T, Hammarström L, Korall P, Börje A, Philouze C, Berg K, Tran A (1997) Binuclear ruthenium–manganese complexes as simple artificial models for Photosystem II in green plants. J Am Chem Soc 119:6996–7004CrossRefGoogle Scholar
  41. Utschig LM, Dimitrijevic NM, Poluektov OG, Chemerisov SD, Mulfort KL, Tiede DM (2011a) Photocatalytic hydrogen production from noncovalent biohybrid Photosystem I/Pt nanoparticle complexes. J Phys Chem Lett 2:236–241CrossRefGoogle Scholar
  42. Utschig LM, Silver SC, Mulfort KL, Tiede DM (2011b) Nature-driven photochemistry for catalytic solar hydrogen production: a Photosystem I–transition metal catalyst hybrid. J Am Chem Soc 133:16334–16337PubMedCrossRefGoogle Scholar
  43. Utschig L, Soltau SR, Tiede DM (2015) Light-driven hydrogen production from Photosystem I-catalyst hybrids. Curr Opin Chem Biol 25:1–8PubMedCrossRefGoogle Scholar
  44. Utschig LM, Soltau SR, Mulfort KL, Niklas J, Poluektov OG (2018) Z-scheme solar water splitting via self-assembly of Photosystem I-catalyst hybrids in thylakoid membranes. Chem Sci 9:8504–8512PubMedPubMedCentralCrossRefGoogle Scholar
  45. Wiegand K, Winkler M, Rumpel S, Kannchen D, Rexroth S, Hase T, Farès C, Happe T, Lubitz W, Rögner M (2018) Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production. Biochim Biophys Acta 1859:253–262CrossRefGoogle Scholar
  46. Woolerton TW, Sheard S, Reisner E, Pierce E, Ragsdale SW, Armstrong FA (2010) Efficient and clean photoreduction of CO2 to CO by enzyme-modified TiO2 nanoparticles using visible light. J Am Chem Soc 132:2132–2133PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© US Government 2020

Authors and Affiliations

  • Udita Brahmachari
    • 1
  • P. Raj Pokkuluri
    • 1
  • David M. Tiede
    • 1
  • Jens Niklas
    • 1
  • Oleg G. Poluektov
    • 1
  • Karen L. Mulfort
    • 1
  • Lisa M. Utschig
    • 1
    Email author
  1. 1.Chemical Sciences and Engineering DivisionArgonne National LaboratoryLemontUSA

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