Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

What makes a bifurcase? Insights from a NADH-dependent reduced ferredoxin: NADP+ oxidoreductase (Nfn) and homologs (#566)

Syed Muhammad Saad Imran 1 , Michael E Dawson 1 , Carolyn E Lubner 1
  1. National Renewable Energy Laboratory, Golden, CO, United States

NADH-dependent reduced ferredoxin: NADP+ oxidoreductases1-4 (Nfn) are enzymes that perform the energy-conserving flavin-based electron bifurcation (FBEB) reaction5, 6. Nfn comprises one large (NfnL) and one small (NfnS) subunit. Thermoanaerobacterium saccharolyticum (Ts) is an anaerobic bacterium that – with known involvement of its Nfn7, 8 – produces ethanol in high, commercially viable concentrations9. We are investigating the activity and energetic landscape10 of TsNfn to determine how the enzyme effectuates FBEB.

TsNfnS, TsNfnL, and the partner ferredoxin (TsFd) were recombinantly expressed, purified, and reconstituted with iron-sulfur clusters and FAD cofactors. Electron paramagnetic resonance (EPR) was utilized for all proteins. Spectroelectrochemistry11 was performed with NfnL. Square-wave voltammetry11 was conducted on NfnL and Fd. Spectrophotometric activity was assayed for NfnL with or without NfnS.

TsNfnS can be stably isolated without requiring NfnL, unlike the Nfn from Pyrococcus furiosus (PfNfn) which our group also studies3, 12, 13. The energetic profile of FBEB in TsNfn is overall similar to that of PfNfn with some differences: (1) the proximal [4Fe-4S] cluster is at a lower potential (−782 mV vs −701 mV), (2) the bifurcating FAD is at a higher potential (−406 mV vs −436 mV), and (3) TsFd has two [4Fe-4S] clusters at −587 and −406 mV, unlike PfFd with a single cluster at ~−400 mV14. EPR results indicate that TsNfnS [2Fe-2S] cluster exists in two forms. Our recent work15 integrating NfnL and close homologs in a metabolic pathway is also discussed, where comparative bioinformatic analyses were used to functionally predict the differences between the two protein classes.

Our work builds upon the field of FBEB by demonstrating the similarity in energetic landscapes of distantly related16, 17 archael (Pf) and bacterial (Ts) Nfns, equipping us to understand design principles for FBEB and allowing us to modulate the process in vivo by protein- and genetic-engineering approaches for specific metabolic outcomes15.

  1. Wang, S.; Huang, H.; Moll, J.; Thauer, R. K. J Bacteriol, 2010, 192 (19), 5115-5123
  2. Demmer, J. K.; Huang, H.; Wang, S.; Demmer, U.; Thauer, R. K.; Ermler, U. J Biol Chem, 2015, 290 (36), 21985-21995
  3. Lubner, C. E.; Jennings, D. P.; Mulder, D. W.; Schut, G. J.; Zadvornyy, O. A.; Hoben, J. P.; Tokmina-Lukaszewska, M.; Berry, L.; Nguyen, D. M.; Lipscomb, G. L.; Bothner, B.; Jones, A. K.; Miller, A. F.; King, P. W.; Adams, M. W. W.; Peters, J. W. Nat Chem Biol, 2017, 13 (6), 655-659
  4. Nguyen, D. M. N.; Schut, G. J.; Zadvornyy, O. A.; Tokmina-Lukaszewska, M.; Poudel, S.; Lipscomb, G. L.; Adams, L. A.; Dinsmore, J. T.; Nixon, W. J.; Boyd, E. S.; Bothner, B.; Peters, J. W.; Adams, M. W. W. J Biol Chem, 2017, 292 (35), 14603-14616
  5. Buckel, W.; Thauer, R. K. Chem Rev, 2018, 118 (7), 3862-3886
  6. Peters, J. W.; Beratan, D. N.; Bothner, B.; Dyer, R. B.; Harwood, C. S.; Heiden, Z. M.; Hille, R.; Jones, A. K.; King, P. W.; Lu, Y.; Lubner, C. E.; Minteer, S. D.; Mulder, D. W.; Raugei, S.; Schut, G. J.; Seefeldt, L. C.; Tokmina-Lukaszewska, M.; Zadvornyy, O. A.; Zhang, P.; Adams, M. W. Curr Opin Chem Biol, 2018, 47, 32-38
  7. Lo, J.; Zheng, T.; Olson, D. G.; Ruppertsberger, N.; Tripathi, S. A.; Tian, L.; Guss, A. M.; Lynd, L. R. J Bacteriol, 2015, 197 (18), 2920-2929
  8. Hon, S.; Olson, D. G.; Holwerda, E. K.; Lanahan, A. A.; Murphy, S. J. L.; Maloney, M. I.; Zheng, T.; Papanek, B.; Guss, A. M.; Lynd, L. R. Metab Eng, 2017, 42, 175-184
  9. Zheng, T.; Olson, D. G.; Murphy, S. J.; Shao, X.; Tian, L.; Lynd, L. R. J Bacteriol, 2017, 199 (3), e00542-16
  10. Wise, C. E.; Ledinina, A. E.; Yuly, J. L.; Artz, J. H.; Lubner, C. E. Biochim Biophys Acta Bioenerg, 2021, 1862 (4), 148377
  11. Imran, S. M. S.; Wiley, S. A.; Lubner, C. E. Current Opinion in Electrochemistry, 2024, 47, 101536
  12. Wise, C. E.; Ledinina, A. E.; Lubner, C. E. Metabolites, 2022, 12 (9), 823
  13. Wise, C. E.; Ledinina, A. E.; Mulder, D. W.; Chou, K. J.; Peters, J. W.; King, P. W.; Lubner, C. E. Proc Natl Acad Sci USA, 2022, 119 (12), e2117882119
  14. Brereton, P. S.; Verhagen, M. F.; Zhou, Z. H.; Adams, M. W. Biochemistry, 1998, 37 (20), 7351-7362
  15. Poudel, S.; Dunham, E. C.; Lindsay, M. R.; Amenabar, M. J.; Fones, E. M.; Colman, D. R.; Boyd, E. S. Front Microbiol, 2018, 9, 1762