Cryo-EM reveals a composite flavobicluster electron bifurcation site in the Bfu family member NfnABC

Abstract

The BfuABC family is a diverse group of electron bifurcating enzymes that play key roles in anaerobic microbial metabolism. Previous studies have focused almost exclusively on the BfuABC-type hydrogenases but the mechanism and site of electron bifurcation remain unknown. Herein we focus on the Caldicellulosiruptor saccharolyticus (Csac) NfnABC-type Bfu enzyme that catalyzes the oxidation of NADPH and simultaneous reduction of NAD and the redox protein ferredoxin (Fd). Cryo-EM structures determined with and without NAD and Fd reveal seven FeS clusters and one FAD in NfnA, one FeS cluster in NfnC, and three FeS clusters, two Zn ions, and one FMN in NfnB. The Zn ions take the place of FeS clusters previously proposed in other Bfu family members. Csac Nfn for the first time defines the minimum bifurcation site as a flavobicluster consisting of FMN, a [4Fe-4S] (B1) cluster and a [2Fe-2S] (C1) cluster. Binding of NAD to the FMN triggers a series of conformational changes, crucial to the bifurcation of two electron pairs derived from NADPH by the [B1-FMN-C1] flavobicluster into low and high potential electrons that reduce Fd and NAD, respectively. The structures lay the foundation for investigations of the proposed reaction cycle common to all Bfu enzymes.

Fig. 1. Current status of the BfuABC family of electron bifurcating enzymes and the bifurcation activity of purified Csac NfnABC.

a In NiFe-ABCSL hydrogenase of A. mobile, five iron sulfur clusters (B1-B5) were modeled in BfuB and the BF-site was considered to be a composite of [B1-FMN-C1-B2-B5]. b In the three subsequent FeFe-ABC hydrogenases (T. maritima, T. kivui and A. woodii) and one NfnABC (S. ovata Stn), only four such iron sulfur clusters (B1, B3, B4 and B5) were mapped in BfuB with the B2 site occupied with a Zn ion and a composite BF-site of [B1-FMN-C1-B5]. c Csac NfnABC has only three iron sulfur clusters in NfnB (B1, B3 and B4) with two Zn ions occupying sites B2 and B5, leaving the flavobicluster [B1-FMN-C1] as the composite BF-site conserved in all resolved BfuABC family members. d SDS-PAGE gel of the purified enzyme showing the presence of all three subunits. e Bifurcating activity of purified NfnABC (~60 µg/ml) in the presence of Csac Fd (~25 µM, purified from Csac), NADPH (0.8 mM), and NAD (1 mM), which were added as indicated. The decrease in the absorbance at 425 nm indicates reduction of Csac Fd, which occurs only in the presence of all three substrates.

Fig. 2. Cryo-EM structures of tetrameric Csac NfnABC in the holoenzyme form and in the NADH bound form.

a Domain architecture of NfnA, NfnB and NfnC. b Plausible arrangement of NfnA, NfnB and NfnC and their associated cofactors. The three possible redox reaction sites in the NfnABC complex are also indicated. Two orthogonal views of the EM map (c) and atomic model in cartoons (d) of the tetrameric Csac NfnABC holoenzyme. Two orthogonal views of the EM map (e) and atomic model in cartoons (f) of the tetrameric Csac NfnABC bound to both FMN and NAD. The locations of two invisible C-term domains of NfnB and NfnC are marked by two shaded areas. In panels c-f, NfnA is colored in peach, gold, beige, and wheat in protomers 1 to 4, respectively. NfnB, NfnC, FMN, NAD, and FAD are in different colors.

Fig. 3. The distribution of electron transporting cofactors indicates that the individual protomer complex is functional.

a Cofactor organization in the tetramer of the Csac NfnABC complex. The nearest distances of cofactors between neighboring protomers are indicated. b Atomic model of one protomer of the NfnABC complex is shown in the cartoon with Fe-S clusters and zinc ion in spheres, and FMN and FAD in sticks. c Arrangement of cofactors in one NfnABC complex protomer. The distances in Å of both edge-to-edge and center-to-center (in parentheses) between two nearby cofactors are labeled. Superposition of the EM density of Zn²⁺ at the B5 site (d) and the 4Fe-4S cluster at the A7 site (e). EM map is shown as gray surface with 60% transparency, the coordinating residues are shown in sticks. f Detailed interactions in the FAD binding pocket in NfnA. FAD and residues within 4 Å are shown as sticks. The EM density of FAD is superimposed and shown in gray with 60% transparency. g Sequence alignment of NfnA reveals conserved Cys and Lys residues coordinating the A7 [4Fe-4S].

Fig. 4. Conformational changes triggered by NAD binding.

a Superposition of the NAD bound form with the Csac NfnABC holoenzyme. In the NAD bound form, the two CTDs of NfnB and NfnC are highly mobile. b Closeup of the FMN binding pocket. The NAD nicotinamide forms a pi-pi interaction with the FMN isoalloxazine ring to facilitate electron transfer, and the NAD adenine is trapped between and stabilized by Phe-159 and Phe-294. The EM densities of FMN and NAD are shown in gray surface with 60% transparency. c Detailed side chain interactions with FMN and NAD. FMN, NAD, and the protein interacting residues (within 4 Å) are shown as sticks. Conformers 1 (d), 2 (e), and 3 (f) captured by 3DVA analysis are shown in two views. The three key domains affected by NAD binding, NfnB CTD, NfnC CTD and the NfnB thioredoxin fold, are labeled in each conformer. The corresponding model of conformers 1 (g), 2 (h), and 3 (i) are shown in cartoons and superimposed on the EM maps rendered in transparent surfaces. Key cofactors are shown as insets on top. D1 and D2 are two [4Fe-4S] clusters in the Csac Fd. The edge-to-edge distances in Å between cofactors are labeled.

Fig. 5. NAD binding is proposed to trigger two conformation-change-coupled electron bifurcation processes.

The red heart shape in the resting oxidized enzyme (a) encircles the flavobicluster [B1-FMN-C1] that is proposed to mediate electron bifurcation. For the complete reaction cycle (Eq. 1) two NADPH are oxidized and the number of electrons on the various clusters and combinations of clusters are indicated. The first NADPH binds to NfnA and donates two electrons to FAD and the mid potential electron path (a). NAD then binds to FMN in NfnB and triggers a series conformational changes in the NfnBC core that enable the first of two electron bifurcation processes. In the first, the flavobicluster generates a high-potential electron that remains within the BF-site (B1/FMN^1-, Conformer 1, b), while the low potential electron on C1 (C^1-) after a conformational change reduces B3/B4 (B3/B4^1-, Conformer 2, c). C1 returns to form the [B1-FMN-C1] flavobicluster and NAD still remains bound to FMN. A second NADPH then binds and donates two more electrons to the bicluster (B1/FMN/C1^1-) to give a bicluster with three electrons (B1/FMN/C1^3-, d). A second bifurcation event then occurs and reduced C1 dissocates once more (e) to generate a site containing a second low potential electron (C1/B3/B4^2-). This state enables NfnB CTD, NfnC CTD and the NfnB thioredoxin domain to form a tripod, which expands to bind Fd (Conformer 3; (C1/B3/B4^2-, f). Two electron transfer events then occur (shown here simultaneously). Bound Fd is reduced by two low potential electrons and dissociates, and the two high potential electrons on the B1/FMN sub-site reduce NAD to generate NADH. NADH dissociates to generate the resting oxidized holoenzyme (a).