Acetogenesis and the Wood-Ljungdhal Pathway
Carbon dioxide (CO2) is one of the primary greenhouse gases on earth and its continuous emission is leading to a rise in atmospheric temperature, causing a global climate crisis with catastrophic consequences for mankind and the biodiversity of the planet. The utilisation of autotrophic organisms that can fix gaseous CO2 to generate fuel ethanol from abundant waste gas resources is one way to reduce the carbon footprint. Acetogens are a specialised group of strictly anaerobic bacteria that thrive on the formation of acetic acid from CO2 with electrons coming from molecular hydrogen (H2) or carbon monoxide (CO). Basically, the gas mixture of industrial exhaust gases making them a promising production platform for the production of biofuels or industrial bulk chemicals.
For CO2 reduction acetogens use the acetyl-coenzyme A or Wood-Ljungdahl pathway (WLP), the only pathway known to couple CO2 fixation with the generation of ATP. Thus, arguably the WLP is considered as one of the first or even the first biochemical pathway on earth. While there are some mechanistic insights in single enzymes of the WLP from methanogenic archaea, nearly nothing is known about the completely different enzymatic machinery of the acetogenic WLP.
I. The hydrogen dependent CO2 reductase - HDCR
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The direct reduction of CO2 by the hydrogen-dependent CO2 reductase (HDCR) is the first step of the WLP. It directly converts the volatile gases hydrogen and carbon dioxide to formate. A reaction that has aroused considerable interest as a liquid electron carrier for hydrogen, due to its increased volumetric energy density and lower hazard compared to molecular hydrogen. Whereas most catalysts generally struggle to reduce the thermodynamically stable CO2 molecule, HDCR class enzymes performs this reaction with a higher activity than any other bio- and chemical catalysts known today. However, the structural and mechanistic basis of this high catalytic turnover rate remains unknown.
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The HDCR complex contains four different subunits, two of them are small iron-sulfur proteins and two of them have catalytic activities. One catalytic active subunit has hydrogenase activity (HydA2) that oxidizes H2 to 2 H+ + 2 e- and the other has formate dehydrogenase / CO2 reductase activity (FdhF), that reduces CO2 + 2 e- + 2 H+ to HCOOH. Under physiological conditions the enzyme works in both directions: during lithotrophic growth it catalyses CO2 reduction and during growth on reduced C1 substrates such as formate or methanol it works as formate dehydrogenase. Hydrogen oxidation and CO2 reduction proceed spatially separated from each other in different proteins which begs the questions how the electrons manage to bridge the distance between the active sites. In many acetogens the HDCR forms long filaments but the physiological or biochemical advantage of filamentation is poorly understood A structure and detailed mechanistic study of this enzyme is urgently needed to understand its unsurpassed catalytic turnover rate and unlock its potential as powerful biocatalysts for CO2 reduction and hydrogen storage.
We teamed up with the Müller Lab (Goethe University Frankfurt) and were able ​to determine the 3.4 Å resolution structure of a short HDCR filament from T. kivui. The minimum repeating unit of the filament is a hexamer consisting of a formate dehydrogenase (FdhF) and two hydrogenases (HydA2) bound around a central core of one HycB3 and two HycB4. These small bacterial polyferredoxin-like proteins oligomerize via their C-terminal helices to form the backbone of the filament. By combining structure-directed mutagenesis with enzymatic analysis, we demonstrate that a network of [4Fe4S] clusters connects all catalytic centers along the HDCR filament. This central nanowire enables efficient electron transfer between the enzymatic reactions, explaining the unsurpassed catalytic activity of HDCR.
To investigate what role the filament of HDCR plays in-vivo we imaged native T. kivui cells with cryogenic electron tomography (cryo-ET). To our greatest surprise we found that HDCR filaments bundle to form large ring-shaped superstructures attached to the plasma membrane.​
HDCR bundling and membrane connection likely represent an adaption to extreme environmental conditions, which demand efficient capture of rare gaseous substrates such as H2 and CO2. These conditions resemble those of early Earth, where life evolved in the absence of oxygen, and inorganic gases were the only potential carbon and energy sources. Highly efficient CO2 fixation is required to sustain growth in environments with such limited available energy. Thus, the molecular connectivity and supramolecular architecture of HDCR filaments likely form the basis of an acetogenic carbon concentration mechanism (CCM). The investigation of this exciting new mechanism is a current study area in the lab.
II. The electron bifurcating hydrogenase - HydABC
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Electron bifurcation is a fundamental energy coupling mechanism, widespread in anaerobic microorganisms. It allows for coupling between endergonic and exergonic redox reactions within the same protein complex, and it forms a major biological energy transduction mechanism. The electron bifurcating hydrogenase (HydABC) is a multi-subunit enzyme complex essential for chemolitothrophic bacteria that grow on hydrogen and carbon dioxide, but also for fermenting bacteria that produce hydrogen gas from sugars. However, as compared to other electron bifurcating enzymes, this unique hydrogenase employs only a single flavin (FMN) cofactor to catalyse the electron transfer along both the high and low potential pathways which is in strict contrast to other bifurcating enzymes. Since its discovery over a decade ago, the electron bifurcation mechanism of this hydrogenase has remained enigmatic and a major challenge for bioenergetics.
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To reveal the molecular basis of this novel electron bifurcation mechanism we determined the single particle structure of the HydABC complex from T.kivui. The structure revealed a dimeric architecture that has asymmetric conformational states responsible for the bifurcation process. HydABC comprises two arms that lead from the active site cofactor of [FeFe]-hydrogenase (the H-cluster) in HydA, responsible for the H2 oxidation, to the nucleotide binding sites in HydB/C. These active sites are connected by five iron-sulphur (Fe/S) centres (Fig. 1b) with edge- to-edge distances in the range of 6-12 Å, which is expected to support rapid electron transfer between the H-cluster and the FMN/NAD+ binding site. HydA contains a typical M3-type [FeFe]-hydrogenase-fold, whereas the HydBC module binds the FMN cofactor and is responsible for the bifurcation reaction.
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Responsible for the bifurcation reaction is the active site flavin and its neighbouring 2Fe2S clusters, located in the N-terminal domain of HydB (B1) and in HydC (C1), respectively. The enzymes has a novel electron bifurcation mechanism in which two subsequent steps, carry out 2e- transfer steps. The first two electrons are bifurcated to the B1/C1 cluster and than to Fd in the endergonic transfer mode. The second electron pair is transfered to NAD+ in form of a hydride transfer. The flavin (FMN) active site is responsible for switching between these two electron transfer modes. At a molecular level this is achieved by modulation of the nucleotide binding affinity via reduction of a nearby iron-sulphur centres. The dissociation of the reduced NADH transduces free energy, which induces conformational changes that kinetically block electron back-leak reactions and thus thrive the endergonic reduction of Fd. Our novel mechanism is validated by structural evidence observed in asymmetric cryoEM reconstructions, molecular simulations as well as mutagenesis experiments.
III. Rnf: the ancient redox-driven sodium pump
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Aiming to dissect the molecular principles of the the Rnf complex, we integrated cryoEM studies with biochemical functional assays and atomistic molecular dynamics simulations, together with the Müller lab (Goethe University Frankfurt) and the Kaila lab (Stockholm University).
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Redox-controlled cryoEM samples were prepared from the Rnf purified complex on its apo form, as well as after the incubation with NADH or reduced ferredoxin. This way we were able to capture the complex into both the endergonic (NADH→Fd) and exergonic (Fd→NADH) electron transfer directions.
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The Rnf complex catalyses the oxidation of low-potential ferredoxin to reduce NAD+, transducing the redox energy into an electrochemical sodium ion gradient (or sodium motive force, SMF) across the membrane in order to drive the synthesis of ATP. In the acetogenic bacterium A. woodii, Rnf functions in reverse when growing on low-energy substrates (lactate, ethanol, methanol) to balance the redox pools and provide reduced ferredoxin for CO2 reduction in the Wood-Ljungdahl pathway. Despite these functional insights, such redox-driven Na+ translocation mechanism remains elusive and highly debated.
In comparison to other Rnf structures, we could resolve all the structural features and cofactor content in our high-resolution structure from A. woodi. Distinct intermediate states were identified in the Fd-reduced complex, although one of major significance is the 4Fe-4S (B8) from the Fd-like domain in RnfB coming closer to the 2Fe-2S (AE1) neighbouring cofactor in the transmembrane subunit RnfA.
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Even though experimental evidence of the conformational changes linked to the sodium ion translocation pathways remain elusive, we performed large-scale atomistic molecular dynamics (MD) simulations of our experimentally determined cryoEM structures embedded in a model biological membrane to probe how the different redox states may link the electron transfer process with the translocation of the sodium.
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​Together with kinetic predictions, and confirmed by site-directed point mutations of the residues directly involved in the electron-transfer pathway, our MD simulations enabled us to propose an efficient redox-driven sodium pumping model as the possible mechanism of the Rnf complex. The proposed redox-driven alternate access mechanism introduces a unique principle for ion pumping employed by the Rnf and related enzymes that appeared early on during evolution to facilitate survival under energy-limited conditions at the thermodynamic limit of life.
