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An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism.

ABSTRACT: Microorganisms utilize electron bifurcating enzymes in metabolic pathways to carry out thermodynamically unfavorable reactions. Bifurcating FeFe-hydrogenases (HydABC) reversibly oxidize NADH (E'∼-280 mV, under physiological conditions) and reduce protons to H₂ gas (E°'-414 mV) by coupling this endergonic reaction to the exergonic reduction of protons by reduced ferredoxin (Fd) (E'∼-500 mV). We show here that HydABC homologs are surprisingly ubiquitous in the microbial world and are represented by 57 phylogenetically distinct clades but only about half are FeFe-hydrogenases. The others have replaced the hydrogenase domain with another oxidoreductase domain or they contain additional subunits, both of which enable various third reactions to be reversibly coupled to NAD⁺ and Fd reduction. We hypothesize that all of these enzymes carry out electron bifurcation and that their third substrates can include hydrogen peroxide, pyruvate, carbon monoxide, aldehydes, aryl-CoA thioesters, NADP⁺, cofactor F₄₂₀, formate, and quinones, as well as many yet to be discovered. Some of the enzymes are proposed to be integral membrane-bound proton-translocating complexes. These different functionalities are associated with phylogenetically distinct clades and in many cases with specific microbial phyla. We propose that this new and abundant class of electron bifurcating enzyme be referred to as the Bfu family whose defining feature is a conserved bifurcating BfuBC core. This core contains FMN and six iron sulfur clusters and it interacts directly with ferredoxin (Fd) and NAD(H). Electrons to or from the third substrate are fed into the BfuBC core via BfuA. The other three known families of electron bifurcating enzyme (abbreviated as Nfn, EtfAB, and HdrA) contain a special FAD that bifurcates electrons to high and low potential pathways. The Bfu family are proposed to use a different electron bifurcation mechanism that involves a combination of FMN and three adjacent iron sulfur clusters, including a novel [2Fe-2S] cluster with pentacoordinate and partial non-Cys coordination. The absolute conservation of the redox cofactors of BfuBC in all members of the Bfu enzyme family indicate they have the same non-canonical mechanism to bifurcate electrons. A hypothetical catalytic mechanism is proposed as a basis for future spectroscopic analyses of Bfu family members.

SUBMITTER: Schut GJ

PROVIDER: S-EPMC9304861 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Twelve evolutionarily unrelated oxidoreductases form enzyme complexes that catalyze the simultaneous coupling of exergonic and endergonic oxidation-reduction reactions to circumvent thermodynamic barriers and minimize free energy loss in a process known as flavin-based electron bifurcation. Common to these 12 bifurcating (Bf) enzymes are protein-bound flavin, the proposed site of bifurcation, and the electron carrier ferredoxin. Despite the documented role of Bf enzymes in balancing the redox state of intracellular electron carriers and in improving the efficiency of cellular metabolism, a comprehensive description of the diversity and evolutionary history of Bf enzymes is lacking. Here, we report the taxonomic distribution, functional diversity, and evolutionary history of Bf enzyme homologs in 4,588 archaeal, bacterial, and eukaryal genomes and 3,136 community metagenomes. Bf homologs were primarily detected in the genomes of anaerobes, including those of sulfate-reducers, acetogens, fermenters, and methanogens. Phylogenetic analyses of Bf enzyme catalytic subunits (oxidoreductases) suggest they were not a property of the Last Universal Common Ancestor of Archaea and Bacteria, which is consistent with the limited and unique taxonomic distributions of enzyme homologs among genomes. Further, phylogenetic analyses of oxidoreductase subunits reveal that non-Bf homologs predate Bf homologs. These observations indicate that multiple independent recruitments of flavoproteins to existing oxidoreductases enabled coupling of numerous new electron Bf reactions. Consistent with the role of these enzymes in the energy metabolism of anaerobes, homologs of Bf enzymes were enriched in metagenomes from subsurface environments relative to those from surface environments. Phylogenetic analyses of homologs from metagenomes reveal that the earliest evolving homologs of most Bf enzymes are from subsurface environments, including fluids from subsurface rock fractures and hydrothermal systems. Collectively, these data suggest strong selective pressures drove the emergence of Bf enzyme complexes via recruitment of flavoproteins that allowed for an increase in the efficiency of cellular metabolism and improvement in energy capture in anaerobes inhabiting a variety of subsurface anoxic habitats where the energy yield of oxidation-reduction reactions is generally low.

Project description:NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn) is an electron-bifurcating enzyme first discovered in the strict anaerobes Clostridium kluyveri and Moorella thermoacetica. In vivo, Nfn catalyzes the endergonic reduction of NADP+ with NADH coupled to the exergonic reduction of NADP+ with reduced ferredoxin. Most Nfn homologs consist of two subunits, although in certain species Nfn homologs are fused. In contrast to other electron-bifurcating enzymes, Nfn possess a simpler structure. Therefore, Nfn becomes a perfect model to determine the mechanism of flavin-based electron bifurcation, which is a novel energy coupling mode distributed among anaerobic bacteria and archaea. The crystal structures of Nfn from Thermotoga maritima and Pyrococcus furiosus are known, and studies have shown that the FAD molecule of the NfnB (b-FAD) is the site of electron bifurcation, and other cofactors, including a [2Fe2S] cluster, two [4Fe4S] clusters, and the FAD molecule on the NfnA subunit, contribute to electron transfer. Further, the short-lived anionic flavin semiquinone (ASQ) state of b-FAD is essential for electron bifurcation. Nfn homologs are widely distributed among microbes, including bacteria, archaea, and probably eukaryotes, most of which are anaerobes despite that certain species are facultative microbes and even aerobes. Moreover, potential evidence shows that lateral gene transfer may occur in the evolution of this enzyme. Nfn homologs present four different structural patterns, including the well-characterized NfnAB and three different kinds of fused Nfn homologs whose detailed properties have not been characterized. These findings indicate that gene fusion/fission and gene rearrangement may contribute to the evolution of this enzyme. Under physiological conditions, Nfn catalyzes the reduction of NADP+ with NADH and reduced ferredoxin, which is then used in certain NADPH-dependent reactions. Deletion of nfn in several microbes causes low growth and redox unbalance and may influence the distribution of fermentation products. It's also noteworthy that different Nfn homologs perform different functions according to its circumstance. Physiological functions of Nfn indicate that it can be a potential tool in the metabolic engineering of industrial microorganisms, which can regulate the redox potential in vivo.

Project description:A non-bifurcating NADH-dependent, dimeric [FeFe]-hydrogenase (HydAB) from Syntrophus aciditrophicus was heterologously produced in Escherichia coli, purified and characterized. Purified recombinant HydAB catalyzed NAD+ reduction coupled to hydrogen oxidation and produced hydrogen from NADH without the involvement of ferredoxin. Hydrogen partial pressures (2.2-40.2 Pa) produced by the purified recombinant HydAB at NADH to NAD+ ratios of 1-5 were similar to the hydrogen partial pressures generated by pure and cocultures of S. aciditrophicus (5.9-36.6 Pa). Thus, the hydrogen partial pressures observed in metabolizing cultures and cocultures of S. aciditrophicus can be generated by HydAB if S. aciditrophicus maintains NADH to NAD+ ratios greater than one. The flavin-containing beta subunits from S. aciditrophicus HydAB and the non-bifurcating NADH-dependent S. wolfei Hyd1ABC share a number of conserved residues with the flavin-containing beta subunits from non-bifurcating NADH-dependent enzymes such as NADH:quinone oxidoreductases and formate dehydrogenases. A number of differences were observed between sequences of these non-bifurcating NADH-dependent enzymes and [FeFe]-hydrogenases and formate dehydrogenases known to catalyze electron bifurcation including differences in the number of [Fe-S] centers and in conserved residues near predicted cofactor binding sites. These differences can be used to distinguish members of these two groups of enzymes and may be relevant to the differences in ferredoxin-dependence and ability to mediate electron-bifurcation. These results show that two phylogenetically distinct syntrophic fatty acid-oxidizing bacteria, Syntrophomonas wolfei a member of the phylum Firmicutes, and S. aciditrophicus, a member of the class Deltaproteobacteria, possess functionally similar [FeFe]-hydrogenases that produce hydrogen from NADH during syntrophic fatty acid oxidation without the involvement of reduced ferredoxin. The reliance on a non-bifurcating NADH-dependent [FeFe]-hydrogenases may explain the obligate requirement that many syntrophic metabolizers have for a hydrogen-using partner microorganism when grown on fatty, aromatic and alicyclic acids.

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An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism.

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