Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe 4S] Fd activity

Lewis, Nathan M.; Kisgeropoulos, Effie C.; Lubner, Carolyn E.; Fixen, Kathryn R.

doi:10.1016/j.jinorgbio.2024.112521

Title: Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe 4S] Fd activity

Journal Article · 06 March 2024 · Journal of Inorganic Biochemistry

DOI: https://doi.org/10.1016/j.jinorgbio.2024.112521 · OSTI ID:2341856

Lewis, Nathan M. ^[1]; Kisgeropoulos, Effie C. ^[2]; Lubner, Carolyn E. ^[2]; Fixen, Kathryn R. ^[3]

Univ. of Minnesota, Minneapolis, MN (United States); University of Minnesota
National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Univ. of Minnesota, Minneapolis, MN (United States)

Ferredoxins (Fds) are small proteins which shuttle electrons to pathways like biological nitrogen fixation. Physical properties tune the reactivity of Fds with different pathways, but knowledge on how these properties can be manipulated to engineer new electron transfer pathways is lacking. Recently, we showed that an evolved strain of Rhodopseudomonas palustris uses a new electron transfer pathway for nitrogen fixation. This pathway involves a variant of the primary Fd of nitrogen fixation in R. palustris, Fer1, in which threonine at position 11 is substituted for isoleucine (Fer1^T11I). To understand why this substitution in Fer1 enables more efficient electron transfer, we used in vivo and in vitro methods to characterize Fer1 and Fer1^T11I. Electrochemical characterization revealed both Fer1 and Fer1^T11I have similar redox transitions (–480 mV and – 550 mV), indicating the reduction potential was unaffected despite the proximity of T11 to an iron-sulfur (Fe—S) cluster of Fer1. Additionally, disruption of hydrogen bonding around an Fe—S cluster in Fer1 by substituting threonine with alanine (T11A) or valine (T11V) did not increase nitrogenase activity, indicating that disruption of hydrogen bonding does not explain the difference in activity observed for Fer1^T11I. Electron paramagnetic resonance spectroscopy studies revealed key differences in the electronic structure of Fer1 and Fer1^T11I, which indicate changes to the high spin states and/or spin-spin coupling between the Fe—S clusters of Fer1. Finally, our data implicates these electronic structure differences in facilitating electron flow and sets a foundation for further investigations to understand the connection between these properties and intermolecular electron transfer.

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Research Organization:: Univ. of Minnesota, Minneapolis, MN (United States); National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Office of Workforce Development for Teachers & Scientists (WDTS); USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB)

Grant/Contract Number:: SC0020252; SC0014664; AC36-08GO28308

OSTI ID:: 2341856

Alternate ID(s):: OSTI ID: 2323366; OSTI ID: 2329406

Journal Information:: Journal of Inorganic Biochemistry, Journal Name: Journal of Inorganic Biochemistry Vol. 254; ISSN 0162-0134

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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