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Evolution and diversity of oxidoreductases involved in redox balance and energy conservation

Nature Ecology & Evolution (2026)Cite this article

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Abstract

The regulation of redox balance and energy conservation is fundamental to life and relies on a large evolutionary network of oxidoreductases forming homologous protein complexes, collectively termed HORBEC (homologous oxidoreductase complexes involved in redox balance and energy conservation). These include hydrogenases, respiratory complex I and electron-bifurcating complexes, central to respiration, fermentation and methanogenesis. Despite their crucial role, a comprehensive investigation of the diversity and evolutionary history of HORBEC has been lacking. Here we exhaustively identified and analysed over 50 protein families representing all HORBEC components across thousands of bacterial and archaeal genomes. We propose a unified nomenclature and classification encompassing 31 complexes and provide an annotation tool. We highlight the extensive diversity of HORBEC, especially in Archaea. We provide information on overlooked systems and identify a new one probably acting as a cation transport platform. We show that HORBEC originated via extensive tinkering of ancestral modules, driven by strong evolutionary constraints. Finally, we infer the presence of respiratory complex I in the last universal common ancestor, opening questions on its potential role in early energy metabolisms. This work provides an evolutionary framework for HORBEC, representing a fundamental resource to predict and study redox metabolisms of ecological and biotechnological significance.

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Fig. 1: Roles and function of HORBECs.
Fig. 2: Diversity of HORBEC.
Fig. 3: Schematic representation of phylogenetic relationships between components among core HORBEC homologue families.
Fig. 4: Examples of modularity of HORBEC at different scales.
Fig. 5: Taxonomic distribution of HORBECs in Bacteria and Archaea.
Fig. 6: Phylogeny of Nuo, based on the concatenation of NuoB, NuoC, NuoD, NuoH, NuoJ and NuoK.

Data availability

The supplementary data are available via figshare at https://doi.org/10.6084/m9.figshare.30090436 (ref. 134). The metagenome-assembled genomes are available on NCBI (BioProjects number PRJNA692327 and PRJNA1112871).

Code availability

The script HORBEC-finder.py v.1 is available via figshare at https://doi.org/10.6084/m9.figshare.30090436 (ref. 134).

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Acknowledgements

We thank the EBMC team and R. Arias-Cartín for feedback and discussion. We thank K. Appler for genome repository at NCBI. We thank the computational and storage services (TARS cluster) provided by the IT department, particularly S. Creno, at Institut Pasteur, Paris. This work was supported by the French National Agency for Research (grant nos. Methevol ANR-19-CE02-0005-01 and ANR LBX-62 IBEID GRANT INTRA LABEX) and by the Moore-Simons Project on the Origin of the Eukaryotic Cell, Simons Foundation grant no. 73592LPI (https://doi.org/10.46714/735925LPI) (to B.J.B.) and a Simons Foundation investigator award LI-SIAME-00002001 (to B.J.B.).

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Authors and Affiliations

  1. Department of Microbiology, Unit Evolutionary Biology of the Microbial Cell, Institut Pasteur, Université Paris Cité, Centre National de la Recherche Scientifique UMR 6047, Paris, France

    Pierre Simon Garcia, Simonetta Gribaldo & Guillaume Borrel

  2. Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Institut Microbiologie Bioénergies et Biotechnologie, Aix-Marseille Université, Centre National de la Recherche Scientifique UMR 7283, Marseille, France

    Pierre Simon Garcia

  3. Department of Marine Science, Marine Science Institute, University of Texas at Austin, Austin, TX, USA

    Valerie De Anda & Brett J. Baker

  4. Department of Integrative Biology, University of Texas at Austin, Austin, TX, USA

    Valerie De Anda & Brett J. Baker

  5. Department of Functional and Evolutionary Ecology, Microbial Oceanography Unit, University of Vienna, Djerassiplatz, Vienna, Austria

    Valerie De Anda

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  1. Pierre Simon Garcia
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Contributions

S.G. and G.B. conceived and designed the experiments. P.S.G. performed the experiments. P.S.G. and G.B. analysed the data. V.d.A. and B.J.B. contributed materials/analysis tools. P.S.G., S.G. and G.B. wrote the paper.

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Correspondence to Pierre Simon Garcia or Guillaume Borrel.

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Extended data

Extended Data Fig. 1 Schematic representation of phylogenetic relationships between components among HORBEC homologue families.

Each schematic corresponds to a homologue family, from AA to BZ. AB, AC, AD, AH, AJ, AK, AL, AO, AP, AQ, AR families are represented in Fig. 3. The schematics are based on unrooted consensus of multiple trees and subtrees, inferred using Fasttree. Each triangle corresponds a component, colored by the HORBEC it belongs to. Light gray triangles correspond to non-classified sequences, dark gray to unrelated families. The size of triangles represents approximatively the size of the subfamilies. The length of branches is not proportional to real phylogenetic branches. The names in red indicate that several sequences are fused with another family, indicated between parentheses.

Extended Data Fig. 2 Alignment of the two hydrogenase [NiFe]-center coordination motifs (N-ter RxCGxCxxxH and C-ter DPCxxCxxH/R) from the AD family.

The alignment is split in three groups of AD representatives: A) those missing most of the conserved residues of the motifs (in particular the cysteines), B) those of membrane-bound hydrogenase complexes and C) those of cytosolic hydrogenase complexes. Conserved cysteines are in red, other residues mostly conserved in both membrane and cytosolic hydrogenase complexes are in blue, those mostly conserved in membrane-bound hydrogenase complexes are in yellow and those from cytosolic hydrogenase complexes are in pink.

Extended Data Fig. 3 Diversity of genomic cluster organization of the different HORBEC.

For each system and for each organization, the total number of systems, the number of systems in Bacteria (B) and Archaea (A) and the number of systems found at the edge of a contig are indicated. For each system, the number of possible subunits is shown. The Shannon index that estimates the diversity of the system in terms of organization has been calculated. The pie charts show the distribution of the number of genomic clusters for each observed organization. Only the organizations that are more widespread and corresponding to the first half of the total number of genomic clusters are shown in detail.

Extended Data Fig. 4 The diversity of Ehi genme organization.

The theoretical model of protein complexes from the genomic information is represented. Each subunit is represented by shapes colored by the corresponding module. For each subunit, the top letters correspond to the protein family code (AA-BZ), the letters between parentheses indicates the name of the subunit.

Extended Data Fig. 5 Fusions across subunit datasets.

A. Graph of fusions between datasets of subunits. Each node corresponds to a subunit dataset. The number of shared sequences between datasets are indicated on the edges. The datasets that correspond to the same subunit but have been found in two homologue families are surrounded by a gray rectangle. B, C and D. Fusions mapped on the schematic structure of HORBEC. Only the main subunits are indicated (N-, Q-, P-, and Na- modules). B: membrane-bound HORBEC with oxidoreductase activity, C: cytosolic HORBEC, D: membrane-bound HORBEC without oxidoreductase activity.

Extended Data Fig. 6 Comparison of HORBEC and energetic metabolism distribution.

A. Taxonomic distribution of HORBECs and markers of several metabolisms in Bacteria and Archaea. The reference phylogenies have been inferred using IQ-TREE (See Methods) and are represented as cladograms. Each triangle corresponds to a clade (phylum/class/order/family). Dots at the branches indicate ultra-fast bootstrap suppports ≥95. The rectangles correspond to the presence of HORBEC/metabolic markers in each clade. For each clade, the size of the rectangles reflects the proportion of genomes having a given HORBEC/metabolic marker. The stars indicate cases where the genes from a given HORBEC are present but scattered, meaning they could not be identified based on the gene cluster method. The characteristics of each HORBEC in terms of localization, role and reaction is indicated in the table above. For Nqr and Qmo, the electron donors and acceptors are underlined. NAD: Nicotinamide, Fdx: ferredoxin, Q: quinone, MP: Methanophenazine, HDS: heterodisulfide. B. Cooccurrence analysis between HORBEC and metabolism markers in Bacteria and Archaea. Node represent HORBEC (blue) and metabolism markers (green). Edges represent cooccurrence, with a Pearson correlation higher than 0.3 (indicated on the edge).

Extended Data Fig. 7 Global evolutionary scenario of HORBEC.

Time goes from the left to the right. Each line corresponds to inheritance links, notably inferred from the phylogenetic analysis presented in Fig. 2 and Extended Data Fig. 1. The color of lines and components corresponds to the modules. When only a subset of components from a module are inherited, these are indicated in rounded rectangles on the line. Fusion between several lines indicates a coevolution of components. The unresolved relative placements are represented by multifurcated branches. The length of branch is not proportional to time. “a” before the name of a system or a subunit means “ancestral”. The circles indicate if the HORBEC emerged likely in the universal (LUCA), bacterial (LBCA), archaeal (LACA) ancestors, or during diversification of Bacteria (BAC) and Archaea (ARC), in Cyanobacteria (C.b.), or in Methanosarcinales (M.s.). Because of their complexity in terms of evolution, HdrABCDE have not been represented. Rnf and Nqr are also not represented because they do not share homologous subunits with HORBEC. The AI family has not been included because of the lack of phylogenetic signal. The multiple copies of subunits and the functional domain modularity have not been represented.

Extended Data Fig. 8 Detailed evolutionary scenario of Mbs.

Each arrow corresponds to inheritance links, notably inferred from the phylogenetic analysis presented in Fig. 2. The color of arrows and components correspond to the modules. The length of the arrows/branchs is not proportional to time. Specific components that are added to a system are indicated on the arrows. The red dashed lines on complex schematics delineate the system subparts that have a different origin.

Extended Data Fig. 9 Network of possible electron transfers and interactions between the major HORBEC components/modules observed in this study.

The circles correspond to major modules and squares to major protein families. The color of components/modules fits with color code of Fig. 2. Black arrows between components/modules show electron transfer, the arrow indicating the potential directions. Gray lines correspond to an interaction. Dashed lines correspond to a hypothetical electron transfer. The width of arrows/lines reflects the strength of evolutionary link discussed in the text. Reduced compounds are shown in green and oxidized one in red. Vertical arrows associated with AL and Na-module indicate translocation of cations across the membrane. For AV and Q-module, the list below the name indicates the possible inputs and outputs of electrons from protein partners. AV needs an input and an output of electron while the Q-module by its ability to perform several redox reactions itself exhibits several combinations. The redox potential of each redox couple is indicated in the table on the bottom left.

Extended Data Table 1 Criteria used to infer the origin of the HORBECs
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Garcia, P.S., De Anda, V., Baker, B.J. et al. Evolution and diversity of oxidoreductases involved in redox balance and energy conservation. Nat Ecol Evol (2026). https://doi.org/10.1038/s41559-025-02969-0

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