Bringing to Light the Mechanism of Flavin-Based Electron Bifurcation

As the demand for value-added products (such as fuels, drugs, and small molecules) increases, critical advances in the design of synthetic catalysts capable of driving energetically demanding reactions will become even more necessary. Enzymes serve as a point of inspiration for this because they can perform a variety of difficult reactions with high selectivity, fidelity and efficiency. An intriguing target is that of flavin-based electron bifurcation (FBEB), a unique biological mechanism where a thermodynamically unfavorable redox reaction is driven through direct coupling with an exergonic reaction [1]. Our work focuses on investigation of the FBEB enzyme NADH-dependent ferredoxin:NADP+ oxidoreductase (Nfn), which modulates the concentrations of three important cellular redox pools. Compared with other known FBEB enzymes, the energy landscape of Nfn is shifted into a significantly more negative regime. As a consequence of this, an extremely high-energy intermediate is formed at the bifurcating flavin site which must be precisely controlled in order to efficiently perform bifurcation while preventing unwanted reactivity [2]. To understand this aspect of the mechanism, we employed a suite of steady state and ultrafast biophysical techniques to specifically probe the properties of the electron bifurcating site and the initial electron transfer events [3,4]. Our recent results have allowed us to define the thermodynamic landscape and provided insight into emerging molecular determinants that facilitate the exquisite control over electron flow. This information is integral to understanding how energy is controlled and manipulated in complex biological systems; the insights of which will lead to design principles that accelerate the development of transformative energy technologies.

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1. Wise, C. E., Ledinina, A. E., Yuly, J. L., Artz, J. H., Lubner, C. E. The role of thermodynamic features on the functional activity of electron bifurcating enzymes, Biophys. Acta Bioenerg., 2021, 1862, 148377.
2. 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. Mechanistic insights into energy conservation by flavin-based electron bifurcation, Chem. Biol., 2017, 13, 655-659.
3. Wise, C. E., Ledinina, A. E., Mulder, D. W., Chou, K. J., Peters, J. W., King, P. W., Lubner, C. E. An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation, Natl. Acad. Sci. U. S. A., 2022, 119, e2117882119.
4. Wise, C. E.; Ledinina, A. E.; Lubner, C. E. Site-differentiated iron-sulfur cluster ligation affects flavin-based electron bifurcation activity, Metabolites, 2022, 12 (9), 823.