Minimal and hybrid hydrogenases are active from archaea

Abstract

Microbial hydrogen (H₂) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H₂-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H₂. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H₂ catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H₂-metabolizing enzymes.

Keywords: anaerobic; archaea; eukaryogenesis; hydrogen; hydrogenase.

Graphical abstract

Figure 1

Phylogenetically and metabolically diverse archaea encode [FeFe] hydrogenases The left portion of the figure shows a maximum-likelihood phylogenomic tree (model LG + F + G4) based on the concatenated 15 ribosomal marker proteins of archaeal genomes that encode [FeFe] hydrogenases. Results are shown for the 118 (out of 130) genomes that are at least 60% complete, less than 5% contaminated, and contain at least 75% of the 15 syntenic proteins. Branches are color coded, encoding according to the respective phylum. Black circles indicate bootstrap support values over 80%. The middle portion shows the presence of key metabolic genes (in at least one genome) involved in different metabolic processes. Carbon fixation: ATP-citrate lyase beta subunit (AclB), acetyl-CoA synthase beta subunit (AcsB), 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA-delta-isomerase (AbfD), carbon monoxide dehydrogenase/ acetyl-CoA synthase (CODH/ACS) complex subunit delta (CdhD), CODH/ACS complex subunit gamma (CdhE), anaerobic CODH catalytic subunit (CooS), type II/III ribulose-bisphosphate carboxylase (RbcL II/II), and type III ribulose-bisphosphate carboxylase (RbcL III); respiration: reductive dehalogenase (RdhA), formaldehyde activating enzyme (Fae), formate dehydrogenase subunit alpha (FdhA), and reversible succinate dehydrogenase and fumarate reductase flavoprotein (SdhA/FrdA); ATP synthesis: ATP synthase subunit alpha (AtpA) and ATP synthase subunit beta (AtpB); fermentation: 2-oxoacid:ferredoxin or pyruvate:ferredoxin oxidoreductase alpha subunit (OorA/PorA), L-lactate dehydrogenase (Idh), ADP-forming acetyl-CoA synthetase (AcdA), acetate kinase (Ack), phosphate acetyltransferase (Pta), acetyl-CoA synthetase (Acs), and formate C-acetyltransferase (PflD); fatty acid degradation: acyl-CoA dehydrogenase (ACAD); aromatics degradation: flavin prenyltransferase (UbiX); sulfur metabolism: sulfur dioxygenase (Sdo), sulfate adenylyltransferase (Sat), adenylylsulfate kinase (CysC), sulfate adenylyltransferase subunit 1 (CysN), and anaerobic sulfite reductase subunit A (AsrA). The right portion shows the diverse environments from where the archaeal genomes were retrieved. Note that the phylum QMZS01 was classified as Aenigmatarchaeota in GTDB R06-RS207, while Thermoproteota class EX4484-205 was proposed as Brockarchaeia. See also Figures S1–S3 and S6.

Figure S1

Genome statistics, hydrogenase maturases, and hydrogenase domain structure of 130 [FeFe] hydrogenase-encoding archaeal genomes, related to Figures 1, 2, 4, and 5 (A and B) Bar charts showing the size and completeness (CheckM) of [FeFe] hydrogenase-encoding archaeal genomes from GTDB R05-RS202 (77 in total), newly assembled metagenomes (40 in total), and PATRIC (13 in total). (C) Heatmap showing the detection of known [FeFe] hydrogenase maturase (HydEFG) homologs based on homology search (Table S3). Navy shading denotes the presence of the homolog. (D) Domain and iron-sulfur cluster organization of archaeal [FeFe] hydrogenase. Incomplete open reading frames are denoted by asterisks (^∗) next to arrows. Subclass (protein domain structure-based scheme) and subgroup (protein phylogeny-based scheme) classification of [FeFe] hydrogenases are based on Land et al. and Greening et al., respectively, with modifications proposed by the current study. H-cluster, catalytic domain of [FeFe] hydrogenase; hyhS, [NiFe] hydrogenase small subunit/iron-sulfur domain; (His)[4Fe4S], (Cys)₃His-ligated [4Fe4S] cluster binding domain; [2Fe2S], [2Fe2S] cluster binding domain; [4Fe4S], [4Fe4S] cluster binding domain; 2[4Fe4S], bacterial ferredoxin-like 2[4Fe4S] cluster binding domain; 6Cys, putative iron-sulfur cluster binding domain. Note that the phylum QMZS01 was classified as Aenigmatarchaeota in GTDB R06-RS207, while Thermoproteota class EX4484-205 was proposed as Brockarchaeia.

Figure S2

Genetic organization of 136 archaeal [FeFe] hydrogenases, related to Figures 1, 2, 4, and 5 Up to 10 genes upstream and downstream of the [FeFe] hydrogenase (hydA) are shown. Gene length is shown to scale. hydA, [FeFe] hydrogenase; hydS, [FeFe] hydrogenase small subunit; hydB, [FeFe] hydrogenase diaphorase subunit; hydC, [FeFe] hydrogenase thioredoxin subunit; hydD, [FeFe] hydrogenase nuoG-like conduit protein; hydF, [FeFe] hydrogenase H-cluster maturation GTPase; hyd6TM, uncharacterized 4 to 6-helix transmembrane protein associated with group A [FeFe] hydrogenases; hyhL/hoxH, group 3 [NiFe] hydrogenase catalytic subunit; hyhS/hoxY, group 3 [NiFe] hydrogenase small subunit; hyhD/hoxD, group 3 [NiFe] hydrogenase iron-sulfur subunit D; hyhB, group 3 [NiFe] hydrogenase diaphorase electron transfer subunit; hyhG, group 3 [NiFe] hydrogenase diaphorase catalytic subunit; hyaD, [NiFe] hydrogenase maturation protease; hypABCDEF, [NiFe] hydrogenase maturation factors; hdrABC, CoB-CoM heterodisulfide reductase subunits; oorAB/porAB, 2-oxoacid:acceptor or pyruvate:ferredoxin oxidoreductase alpha and beta subunits. Detailed information on loci, annotations, and amino acid sequences of each gene are available in Table S2.

Figure 2

Archaea encode genetically and structurally diverse [FeFe] hydrogenases Catalytic domain structure, genetic organization, and AlphaFold2-based structural modeling of representative [FeFe] hydrogenases encoded in archaeal genomes. (A) Group A1 [FeFe] hydrogenase from UBA95 sp002499405 (Micrarchaeota). (B) Group E [FeFe] hydrogenase from “Ca. Forterrea multitransposorum”. (C) Group B [FeFe] hydrogenase complex from “Ca. Prometheoarchaeum syntrophicum.” (D) Group A3 [FeFe] hydrogenase complex from DSAL01 sp011380095 (Altarchaeota). For each panel, the catalytic domain (H-cluster), iron-sulfur binding motifs, and amino acid sequence length are shown at the top. Genes encoding hydrogenase structural subunits are shown in their genetic context beneath, labeled and colored consistent with the corresponding subunit in the structural models. Predicted cofactors are positioned based on the structures of homologous proteins. A zoomed view of the H-cluster and conserved coordinating cysteine residues (C₁ to C₅) is shown for each group. For group B and A3 enzymes, FeS clusters within plausible electron transfer distance are connected by dashed lines. hydA, [FeFe] hydrogenase; hydB, diaphorase; hydC, thioredoxin; hydD, nuoG-like conduit protein; hyd6TM, uncharacterized 4- to 6-helix transmembrane protein associated with group A [FeFe] hydrogenases; (His)[4Fe4S], (Cys)₃His-ligated [4Fe4S] cluster binding domain; [2Fe2S], [2Fe2S] cluster binding domain; [4Fe4S], [4Fe4S] cluster binding domain; 2[4Fe4S], bacterial ferredoxin-like 2[4Fe4S] cluster binding domain; 6Cys, putative iron-sulfur cluster binding domain. ^∗ HydC protein in group A1 gene cluster was not predicted to form a complex with HydA. Surface structures are used for the multisubunit group B and A3 [FeFe] hydrogenases, with ribbon diagram versions provided in Figure S3. See also Figures S1–S4.

Figure S3

Analysis of AlphaFold2 models of archaeal [FeFe] hydrogenases, related to Figures 1, 2, 4, and 5 (A) Genetic organization and model of the group A1 [FeFe] hydrogenase from Ca. Iainarchaeum andersonii. (B) Genetic organization and model of the group E [FeFe] hydrogenase from CABMGN01 sp902385635 (Nanoarchaeota). (C) Model of the group B [FeFe] hydrogenase complex from Ca. Prometheoarchaeum syntrophicum. (D) Model of the group A3 [FeFe] hydrogenase complex from DSAL01 sp011380095 (Altarchaeota). (E) Model of the complete group F [FeFe] hydrogenase from Thermoplasmatota UBA147. (F) Model of the HydA-HyhS fusion from the group F [FeFe] hydrogenase from Thermoplasmatota UBA147. (G–I) The stability of protein-protein interfaces predicted by AlphaFold2 for each subunit of the group A3 Altarchaeota hydrogenase (G), the group B Ca. P. syntrophicum hydrogenase (H), and the group F Thermoplasmatota hydrogenase (I) shown with the relevant subunit displayed as a cartoon, with the rest of the complex shown as a surface view (top). PISA software package predicted parameters indicating the stability of each interface shown as a bulls-eye plot, with a larger blue area indicating that the complex is stable (bottom).

Figure S4

Conservation of active site residues in different classes of [FeFe] hydrogenases, sequence comparison of archaeal [FeFe] hydrogenases against bacterial [FeFe] hydrogenases, and structural comparison of the ultraminimal group E [FeFe] hydrogenase Fm, related to Figures 2 and 4 (A) Structural view of the active site of Clostridium pasteurianum [FeFe] hydrogenase (CpI, PDB: 4XDC) showing the H-cluster and interacting amino acid residues. (B) Normalized consensus logos of [FeFe] hydrogenase groups A–F generated in Jalview using a ClustalΩ sequence alignment of sequences retrieved from Greening et al. and this work. Coloring is based on the Clustal X color scheme. Numbering is based on CpI and black numbers are illustrated in the top panel. (C) Amino acid sequences of the archaeal [FeFe] hydrogenases heterologously expressed in this work. (D) A sequence alignment of archaeal [FeFe] hydrogenases expressed in this study with the prototypical [FeFe] hydrogenases from Chlamydomonas reinhardtii (CrHydA1) and Clostridium pasteurianum (CpI). Positions corresponding to the catalytic cysteines are highlighted in red. Gaps in the group E [FeFe] hydrogenases (Na and Fm) that contribute to the small size of this group are highlighted in blue. (E) Cartoon representations of archaeal minimal group E and A1 [FeFe] hydrogenases, and structurally characterized minimal, monomeric, and multimeric [FeFe] hydrogenases from bacteria. (F) A comparison of Fm with HydA1 from C. reinhardtii (PDB: 6GM5) with the location of connecting loops or structural elements with reduced size in Fm labeled L1 to L6 to aid comparison. (G) A comparison of Fm with Clp from C. pasteurianum, displayed and labeled as in (F).

Figure S5

Heterologous expression of archaeal [FeFe] hydrogenases, and isolation, reconstitution of the [4Fe-4S]⁺ cluster, FTIR difference spectra, and redox state kinetics of Fm, related to Figure 3 (A) Expression constructs with verified sequences were transformed in chemically competent E. coli BL21(DE3). Protein bands are shown from before induction with IPTG in lane B, after induction (name-subclass and with the expected kDa size in parenthesis), and lysate or supernatant after cell lysis and centrifugation in lane L. The bands in each after-induction lane corresponded well with the expected molecular weights in kDa. Both group A1 [FeFe] hydrogenases (Mu and Ia) had the highest expression and solubility levels. In contrast, the group F (Th1, Th2) and group B (Ps) enzymes exhibited moderate expression levels but poor solubilities in aqueous solutions. The group E (Na and Fm) enzymes exhibited high expression levels but poor solubility. (B) SDS-PAGE gel of purified Fm (33 kDa), obtained following expression in E. coli Origami B(DE3) and StrepTrap XT purification. Target protein indicated with horizontal arrow. (C) UV-visible spectra of Fm (205 μM) after reconstitution of [4Fe-4S]²⁺ cluster (blue spectrum), indicated by the absorbance at 405 nm. The Fe/protein content was 4.2 ± 0.4 after reconstitution, in agreement with the presence of a single [4Fe-4S] cluster. Upon addition of 20× excess sodium dithionite (NaDT, red spectrum), a decrease in absorbance at 405 nm is observable, indicating the reduction of [4Fe-4S]²⁺ cluster to [4Fe-4S]⁺. Spectra were collected in a 1-mm path length cuvette. (D) The full set of difference spectra of the photoreduction experiment shown in Figure 3B in the main text. The super-oxidized CO-inhibited H_sox-CO species (gray bands) depopulates in favor of the one-electron-reduced oxidized states H_oxH (cyan bands) and H_ox (blue bands). During continuous photoreduction, the oxidized species are further reduced to the [4Fe4S] cluster reduced state H_red′ (red bands). Illumination that facilitates photoreduction was applied for 88 s. (E) The summed delta peak area of each redox state of the difference spectra in (D) is plotted over the time course of the photoreduction experiment. The illumination period is indicated by the gray area. The depopulation of the super-oxidized CO-inhibited H_sox-CO species (black) is mostly completed within 44 s. At the same time the oxidized states H_oxH (cyan) and H_ox (blue) reach their maximum population during photoreduction. Subsequently during the illumination period H_red′ accumulates at the expense of the oxidized species (until 88 s). After photoreduction (88 s) H_red′ converts back into the oxidized species. Note that no re-population of H_sox-CO was detected.

Figure 3

Three classes of [FeFe] hydrogenases encoded by archaea are catalytically active (A) H₂ gas production monitored from cell lysates in E. coli BL21(DE3) cells expressing group A1, B, and E [FeFe] hydrogenases from archaea. All cell lysates, including the blank, were activated by addition of [2Fe]^adt. H₂ was measured by gas chromatography (GC) after addition of methyl viologen and dithionite to activated cell lysates, set to pH 6.8 with 100 mM KPi buffer. Activities are normalized for number of cells used (nmol H₂ min⁻¹ OD₆₀₀⁻¹) and error bars reflect standard deviation from biological triplicates. The strain expressing prototypical CrHydA1 was used as a positive control while “blank” represents the same strain but containing an empty vector. (B) FTIR spectra of the group E [FeFe] hydrogenase from “Ca. Forterrea multitransposorum” (Fm) after heterologous expression, semisynthetic maturation with [2Fe]^adt, and purification. The absorbance spectrum (top) indicates a CO-inhibited di-ferrous H-cluster state (H_sox-CO). The difference spectra (bottom) illustrates the transitions of Fm into catalytically active states through photoreduction (illumination after the addition of eosin Y as a photosensitizer and triethanolamine as a sacrificial electron donor). During illumination, bands associated with the highly oxidized CO-inhibited state decreased (gray bands), while new bands reflecting reduced and catalytically active H-cluster states appear, assigned to H_oxH (cyan), H_ox (blue), and H_red (red) (spectra arranged chronologically from top to bottom). (C) Cyclic voltammetry traces of immobilized Fm (orange) with H₂ oxidation current densities at high potentials and H⁺ reduction currents at low potentials. The 2H⁺/H₂ redox couple potential is indicated with a dashed line (E^o′_2H⁺/H2). Scan direction is indicated by black arrows. The blank trace (gray) represents the electrode without an immobilized enzyme film. The experiments were performed on two independent films for each enzyme at pH 7.0 (5 mM MES, 5 mM CHES, 5 mM HEPES, 5 mM TAPS, 5 mM sodium acetate [NaOAc], 0.1 M Na₂SO₄) and under 1 atm H₂. See also Figures S5.

Figure 4

[FeFe] and [NiFe] hydrogenases encoded by archaea are predicted to form unique complexes (A) Catalytic domain structure and predicted operon encoding a putative complex of a group F [FeFe] hydrogenase and group 3 [NiFe] hydrogenase in Thermoplasmatota UBA147 sp002496385. hydA, [FeFe] hydrogenase catalytic domain (fused with hyhS); hydB, diaphorase; hydC, thioredoxin; hydD, nuoG-like conduit protein; hyhL, group 3 [NiFe] hydrogenase catalytic subunit; hyhS, group 3 [NiFe] hydrogenase small/iron-sulfur domain (fused with hydA). (B) Predicted surface structure, cofactor composition, and electron flow through four potential arms in the hybrid hydrogenase complex. [FeS] clusters are numbered and labeled according to their subunit of origin (e.g., A1, A2, and A3 originate from the HydA subunit). (C) Atomic structure of the predicted [FeFe]- and [NiFe]-hydrogenase active sites in the hybrid enzyme. Distances between catalytic cluster and coordinating residues of less than 2.5 Å are shown as blue dotted lines. (D) H₂ gas production monitored from cell lysates in E. coli BL21(DE3) cells expressing the Th1 and Th2 [FeFe] hydrogenases from archaea. The cell lysates were activated by addition of [2Fe]^adt. H₂ levels were measured every 15 min for 2 h by gas chromatography after addition of methyl viologen and dithionite to activated cell lysates, set to pH 6.8 with 100 mM KPi buffer. Activities are normalized for number of cells used (nmol H₂ OD₆₀₀⁻¹) and error bars reflect standard deviations from two biological triplicates. Blank represents the same strain but contains an empty vector. See also Figures S1–S4 and S7.

Figure 5

[FeFe] hydrogenases are diverse and ancient in archaea An unrooted maximum-likelihood phylogenetic tree of the catalytic subunit (HydA) of [FeFe] hydrogenases and the hybrid hydrogenases. The tree was constructed based on 3,677 amino acid sequences using the LG + C20 + R + F model. The numbers at the branches indicate the aLRT (approximate likelihood ratio test) and ultrafast bootstrap (within bracket) support values, each with 1,000 replicates. The scale bar corresponds to the expected number of substitutions per site. Colored circles at the tip indicate sequences from eukaryotes and major archaeal groups. All other sequences are from bacteria. See also Figures S1–S3.

Figure S6

Phylogenetic trees of the three [FeFe] hydrogenase maturases (HydEFG), related to Figure 1 (A) HydE. (B) HydF. (C) HydG. Different colors show archaeal (red), bacterial (black), and eukaryotic (green) sequences. Evolutionary history was inferred using the maximum-likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model with 50 bootstrap replicates and midpoint rooting.

Figure S7

Phylogenetic trees of the [NiFe] hydrogenase large/catalytic subunit (HyhL) and iron-sulfur domain/small subunit (HyhS), with focus on group 3 [NiFe] hydrogenases, related to Figure 4 (A) HyhL. The subunits predicted to associate with [FeFe] hydrogenases are shown in red (for group F [FeFe] hydrogenases) and purple (for group G [FeFe] hydrogenases). (B) The domain that fuses with the group F [FeFe] hydrogenases is shown in red. The unfused subunit encoded downstream of group G [FeFe] hydrogenases is shown in purple. Evolutionary history was inferred using the maximum-likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model with 50 bootstrap replicates and midpoint rooting.