Cryo-EM structures define the electron bifurcating flavobicluster and ferredoxin binding site in an archaeal Nfn-Bfu transhydrogenase

Abstract

Flavin-based electron bifurcation couples exergonic and endergonic redox reactions in one enzyme complex to circumvent thermodynamic barriers and minimize free energy loss. Two unrelated enzymes designated NfnSL and NfnABC catalyze the NADPH-dependent reduction of ferredoxin and NAD. Bifurcation by NfnSL resides with a single FAD but the bifurcation mechanism of NfnABC, which represents the diverse and ubiquitous Bfu enzyme family, is completely different and largely unknown. Using cryo-EM structures of an archaeal NfnABC, we show that its bifurcation site is a flavobicluster consisting of FMN, one [4Fe-4S] and one [2Fe-2S] cluster where zinc atoms replace two additional clusters previously identified in other Bfu enzymes. NADH binds to the flavobicluster site of NfnABC and induces conformational changes that allow ferredoxin to bind between the C-terminal domains of NfnC and NfnB. Site-directed mutational analyses support the proposed mechanism that is likely conserved in all members of the Bfu enzyme family.

Keywords: catalytic mechanism; electron bifurcation; electron transfer; flavin; iron-sulfur cluster.

Figure 1

Current status of BfuABC family.A, both B2 and B5 sites contain FeS clusters in the [NiFe] hydrogenase HydABCSL from A. mobile. B, the B2 site contains Zn and the B5 site contains [2Fe-2S] cluster in the [FeFe]-hydrogenases from Thermotoga maritima, Thermoanaerobacter kivui and Acetobacterium woodii and the NfnABC from Sporomusa ovata. C, both B2 and B5 sites contain Zn in the NfnABC from Thermococcus sibiricus. D, schematic electron transfer pathway in the Nfn-BfuABC family of electron bifurcating enzymes in the bifurcating direction (NADPH oxidation). Fd_ox and Fd_red represent oxidized and reduced ferredoxin, respectively. Blue arrow indicates mid potential electron transfer pathway, purple arrow indicates high potential electron transfer pathway, while brown arrow indicates low potential electron transfer pathway. Created with BioRender.com.

Figure 2

Cryo-EM structure of the Nfn-BfuABC from Thermococcus sibiricus.A, domain architectures of the subunits NfnA, NfnB, and NfnC. Created with BioRender.com. B and C, two orthogonal views of the cryo-EM 3D maps of the holoenzyme (B) and NAD(H)/Fd-bound Tsi NfnABC (C), segmented and colored by subunits. D and E, two orthogonal views of the structures of the holoenzyme (D) and NAD(H)/Fd-bound Tsi NfnABC (E) in cartoons. Subunits are colored as in (B and C). F, the domains of the individual subunits of Tsi NfnABC are shown separately with the B2 and B5 sites occupied by zinc ions.

Figure 3

Cofactor organization in the Tsi NfnA subunit.A, overview of cofactor organization in the holoenzyme. The reduction of NAD⁺ takes place adjacent to the FMN, and Fd is reduced at the C-terminal domain of the NfnB. B, close-up of the FAD binding site in Tsi NfnA, with the cryo-EM density around the FAD superimposed and shown as transparent surface. C, key residues interacting with FAD are shown in sticks. Hydrogen bonds between the residues and cofactors are shown as dashed yellow lines. D, superimposition of the FAD binding sites among Tsi NfnABC, Tma NfnSL, Pfu NfnSL, and Sov NfnABC. Residues R187 in Pfu NfnL and R201 in Tma NfnL form hydrogen bond with N5 of FAD, while R234 in Tsi NfnA and R239 in Sov NfnA are far from the N5 atom of the FAD. E, the “Y-shape” constellation of [4Fe-4S] clusters at the boundary between NfnA and NfnB in Tsi enzyme. F, edge-to-edge distances (Å) of the cofactors in the holoenzyme. A possible electron path is indicated by solid lines from the FAD in NfnA to B4 in NfnB. In this structure, the distance between C1 and B3 cluster is beyond electron transfer. G, subunit organization of the Tsi NfnABC enzyme and their associated cofactors. Created with BioRender.com. H and I, close-up of the A3 and A7 [4Fe-4S] clusters in NfnA superimposed with their EM densities. J, sequence alignment of the [4Fe-4S] A7 coordinating residues in Tsi NfnA homologues. Pink represents identical residues, blue indicates the conserved lysine coordinating with the A7 cluster, and other colors represent conserved residues.

Figure 4

Cofactor coordination in the bifurcating Tsi NfnBC core.A and B, close-up view of the two Zn binding site in NfnB superimposed with the EM densities in transparent surface view. C, close-up view of the NAD⁺/FMN binding site in NfnB superimposed their EM density in transparent surface view. D, key residues interacting with NAD⁺ and FMN are shown in sticks. Hydrogen bonds between the residues and cofactors are shown as dashed yellow lines. E, sequence alignment of the NAD⁺/FMN coordinating residues of the Tsi NfnB homologues. The conserved glycine residues are in pink. F, key NfnB loops coordinating FMN and NADH. These loops in FMN-bound state are superimposed with NAD(H)/Fd bound state to show conformation changes. The starting and end residues numbers of the five coordinating loops are labeled. G, overview of cofactor organization of NAD(H)/Fd-bound NfnABC, highlighting the NAD⁺/FMN site, the FAD site, the Zn sites, and the [4Fe-4S] site. Note that the CTD of NfnC is highly flexible and is not modeled in the NAD(H)/Fd-bound enzyme. H, edge-to-edge distances (Å) of the cofactors in the NAD(H)/Fd-bound NfnABC structure. CTD, C-terminal domain.

Figure 5

Three 3D Variability Analysis-derived conformations of the Tsi NfnABC enzyme in the presence of NADH and Fd. 3D models were generated using 3D variability analysis with particles low pass filtered to 8 Å. EM 3D maps (i) are colored by proximity to the subunit indicated as modeled using rigid body fitting (ii). A, in the NfnC-in state (conformer 1), NfnC CTD locates in between the NfnC NTD and the NfnB Fd-like domain, where C1 is close to FMN but far from B3/B4 (A-iii). B, in the NfnC-out state (conformer 2), NfnC CTD moves outward to a position between NfnC NTD and NfnB Fd-like domain, making C1 close to both B3 and B4 but farther away from FMN (B-iii). C, in the NfnC-open state (conformer 3), NfnC CTD moves outward which leaves space for Fd binding in between the NfnC CTD and the NfnB Fd-like domain. The dashed white shape highlights the gap size between NfnB and NfnC. The distance between B3/B4 to D1 is 12.8 Å (C-iii). CTD, C-terminal domain; NTD, N-terminal domain.

Figure 6

Putative electron bifurcation mechanism of the Tsi NfnABC enzyme. Binding and oxidation of NADPH initiate the reaction in NfnA. For the first round of NADPH oxidation, electrons travel along the [4Fe-4S]-clusters in NfnA (A7-A6-A3-A2-A1) to the electron bifurcation site consisting of B1-FMN-C1 (A). NAD binding adjacent to FMN in NfnB induces the first major conformational change in the NfnBC core (conformer 1) to bring the C1 cluster within electron transfer range of the FMN (B). The B1-FMN-C1 flavobicluster site donates two electrons as a hydride to the high potential acceptor NAD⁺, which also accepts electrons from the mid potential pathway formed by the [FeS] clusters in NfnA and transfers them via the C1 cluster to the B3/B4 clusters, the low potential acceptor, via the second major conformational change (conformer 2) (C). The second round of NADPH oxidation leads to a series of conformational switches between conformer 2 and conformer 1 allows C1 accept the second round of electrons from FMN (D) and transfer to the B3/B4 cluster (E). Subsequently, the third large redox-driven conformational change (conformer 3) leads to Fd binding and the low-potential electron transfer from the B3/B4 clusters to Fd (F). With the release of Fd, a new cycle starts. Panels were created with BioRender.com.