The RNF/NQR redox pumps: a versatile system for energy transduction in bacteria and archaea

Abstract

The Na⁺ (or H⁺)-translocating ferredoxin:NAD⁺ oxidoreductase (also called RNF, rhodobacter nitrogen fixation, complex) catalyzes the oxidation of reduced ferredoxin with NAD⁺, hereby generating an electrochemical gradient. In the reverse reaction driven by an electrochemical gradient, RNF provides reduced ferredoxin using NADH as electron donor. RNF plays a crucial role in the metabolism of many anaerobes, such as amino acid fermenters, acetogens, or aceticlastic methanogens. The Na⁺-translocating NADH:quinone oxidoreductase (NQR), which has evolved from an RNF, is found in selected bacterial groups including anaerobic, marine, or pathogenic organisms. Since NQR and RNF are not related to eukaryotic respiratory complex I (NADH:quinone oxidoreductase), members of this oxidoreductase family are promising targets for novel antibiotics. RNF and NQR share a membrane-bound core complex consisting of four subunits, which represent an essential functional module for redox-driven cation transport. Several recent 3D structures of RNF and NQR in different states put forward conformational coupling of electron transfer and Na⁺ translocation reaction steps. Based on this common principle, putative reaction mechanisms of RNF and NQR redox pumps are compared. KEY POINTS: • Electrogenic ferredoxin:NAD⁺ oxidoreductases (RNF complexes) are found in bacteria and archaea. • The Na⁺ -translocating NADH:quinone oxidoreductase (NQR) is evolutionary related to RNF. • The mechanism of energy conversion by RNF/NQR complexes is based on conformational coupling of electron transfer and cation transport reactions.

Keywords: Electrochemical proton gradient; Electrochemical sodium gradient; Electron transport; NQR; RNF; Respiration.

Fig. 1

Comparison of RNF (C. tetanomorphum) and NQR (V. cholerae) architecture and electron transfer pathways. A Left, RNF from C. tetanomorphum (pdb code 7zc6); right, NQR from V. cholerae (pdb code 8a1w). The ferredoxin domain of RnfB, which was not resolved in the cryo-EM map, was modeled here by AlphaFold (Jumper et al. 2021) to complete the structure. Five out of the six subunits of both complexes share high homology as indicated by similar coloring. RnfC/NqrA: blue, RnfD/NqrB: orange, RnfG/NqrC: green, RnfA/NqrE: cyan, RnfE/NqrD: magenta; RnfB: brown, NqrF: red. B The cofactors of the integral transmembrane subunits RnfD/NqrB, RnfA/NqrE, RnfE/NqrD, and RnfG/NqrC are strictly conserved and reside at almost exactly the same positions in both complexes revealing an identical transmembrane electron-transfer pathway in RNF and NQR. RnfB subunit with six [4Fe-4S] clusters is structurally not related to NqrF, which harbors a FAD and a [2Fe-2S] cluster. RnfC and NqrA are structurally closely related; however, NqrA lacks any redox cofactors. The function of RnfC in the RNF complex is to transfer electrons from riboflavin_RnfD to the electron acceptor NAD⁺, while in NQR the electrons are directly transferred to ubiquinone in the membrane. The grey arrows indicate the forward reaction from a low potential electron donor to a high potential electron acceptor

Fig. 2

Architecture of the RnfA-E/NqrE-D dimer coordinating a membrane-bound [2Fe-2S] cluster. A, Structure of NQR of V. cholerae indicating the localization of NqrE (blue/cyan) and NqrD (magenta/purple) in NQR. The membrane plane is indicated by a grey box. B, View of the NqrE-D subunits from the periplasm. The inner helices of NqrE (cyan) and of NqrD (purple) each provide a Cys (in total 4) to coordinate the [2Fe-2S] cluster. C, Close-up view of the inner helices coordinating the [2Fe-2S] cluster.

Fig. 3

Conformational changes during electron transfer in RNF/NQR. A The unusual electron transfer in RNF/NQR through the membrane is initiated by the transfer of an electron to the ferredoxin-like domain of RnfB/NqrF (red). The RnfE-A/NqrD-E heterodimer (magenta/cyan) adopts an inward conformation that allows access of the intramembranous [2Fe-2S]_{RnfE-A/NqrD-E} cluster from the cytoplasmic side, whereas access for RnfG/NqrC (green) from the periplasmic/extracellular side is blocked. B The FeS cluster of the flexibly tethered ferredoxin-like domain of RnfB/NqrF can bind sufficiently close to [2Fe-2S]_{RnfE-A/NqrD-E} to rapidly transfer an electron. C The reduction of [2Fe-2S]_{RnfE-A/NqrD-E} triggers an inward-outward switch in subunits RnfE-A/NqrE-D, obstructing access to [2Fe-2S]_{RnfE-A/NqrD-E} from the cytoplasmic side, and facilitating access to [2Fe-2S]_{RnfE-A/NqrD-E} from the periplasmic/extracellular side. D RnfG/NqrC has shifted from RnfD/NqrB (yellow) to a position close to RnfE-A/NqrD-E and the electron is transferred from the [2Fe-2S]_{RnfE-A/NqrD-E} to FMN_RnfG/NqrC. E The oxidation of [2Fe-2S]_{RnfE-A/NqrD-E} triggers the outward-inward switch and the rotation of RnfG/NqrC towards RnfD/NqrB. Subsequently, rapid electron transfer proceeds from FMN_RnfG/NqrC to FMN_RnfD/NqrB, and from there to riboflavin (RBF) in RnfD/NqrB. From RBF_RnfD the electron is transferred to the proximal iron-sulfur cluster of subunit RnfC and from RBF_NqrB, to ubiquinone

Fig. 4

Proposed mechanisms of redox-driven Na⁺ transport by RNF and NQR. Conserved RNF/NQR subunits are presented with identical colors. In RNF operating in the forward electron transfer mode illustrated here, electrons are provided by reduced ferredoxin, while in NQR NADH serves as electron donor. Top, left: Proposed Na⁺ transport mechanism by RNF from C. tetanomorphum via a gated channel in RnfD. (I) The flexible reduced ferredoxin-like domain binds to the inward-facing state of RnfA-E, reduces its [2Fe-2S] cluster and fixes a Na⁺ in a strained state. (II) The RnfG shuttle moves to the outward-facing position of RnfA-E, thereby triggering pore opening at RnfD and Na⁺ passage. After reduction RnfG flips back to RnfD and induces a conformational change that leads to the release of Na⁺. The electron is transferred further to RnfC and NAD⁺. Top, right: Proposed Na⁺ transport mechanism by RNF from A. woodii via RnfA-E. (I) Na⁺ and the reduced RnfB ferredoxin-like domain bind to the inward-facing conformation. Upon [2Fe-2S]_RnfA-E reduction, an inward-to-outward switch translocates Na⁺ to the periplasm. The electron is shuttled via RnfG, RnfD and RnfC to NAD⁺. Bottom: Proposed mechanism for NQR from V. cholerae via NqrB (I) Na⁺ is bound in NqrB at the periplasmic half-channel, but release is blocked by NqrC. NqrD-E heterodimer opens to the cytoplasmic side. (II) An electron is transferred from NqrF to the membrane [2Fe-2S]_NqrD-E cluster. This triggers an inward-to-outward switch of NqrD-E and prompts Na⁺ binding to NqrB (III). NqrC moves from NqrB towards NqrD-E and Na⁺ is released from NqrB to the periplasm. (IV) NqrC switches back to NqrB. It is proposed that electron transfer to FMN_NqrB triggers translocation of the Na⁺ in NqrB. The electron is transferred via riboflavin_NqrB to ubiquinone. Starting again from state (I), the second electron derived from the FAD semiquinone in NqrF is injected into the core, and the cycle is repeated. Per NADH oxidized, one ubiquinol is formed and two Na⁺ are translocated