Mechanistic Implications of Excited High-Spin States, Spin-Spin Coupling, and Differential [2Fe–2S]+ Cluster Temperature Relaxations in the Electron-Bifurcating NfnABC from Thermococcus sibiricus

Abstract

Electron bifurcation (EB) is a mechanism of biological energy transduction in which multiple oxidation-reduction (redox) reactions are thermodynamically coupled within a single enzyme, enabling the enzyme to harness the excess free energy from an exergonic process to drive an endergonic process. Because of this unprecedented chemistry, there is interest to translate EB principles to artificial and bioengineered systems, but a hurdle is that knowledge pertaining to the fundamental design principles of EB enzymes remains scarce. Here, we investigated the fundamental physical and electronic properties of electron transfer sites from a spectroscopically uncharacterized member of the BfuABC family of EB enzymes, the NADH-dependent reduced-ferredoxin:NADP+ oxidoreductase from Thermococcus sibiricus (Tsi NfnABC). Cryo-EM structures of Tsi NfnABC previously demonstrated that it contains twelve redox cofactors: two flavins (one FAD and one FMN), eight [4Fe–4S] clusters, and two [2Fe–2S] clusters. The FMN, one [4Fe–4S] cluster, and one [2Fe–2S] cluster comprise the bifurcating active site termed the electron-bifurcating flavobicluster (BF-FBC), which is found in all BfuABC family members. By using electron paramagnetic resonance spectroscopy, herein we identified spectral signatures originating from interactions between the FMN radical and [4Fe–4S]+ cluster in the BF-FBC and observed temperature dependent behavior of the BF-FBC’s [2Fe–2S]+ cluster indicative of moderately slow spin-lattice relaxation. Additionally, we uncovered numerous spectral features corresponding to half-integer, S > ½ spin states of [4Fe–4S]+ clusters, including one attributable to the consequences of lysine-ligation of a [4Fe–4S] cluster unique to NfnABC. We contextualize these findings to electron transfer theory and NfnABC structure. Our insights further the understanding of how enzymes are designed to exert control over electron transfer to conduct thermodynamically challenging reactions.