Hydrogen gas-evolving membrane-bound hydrogenase (MBH) and quinone-reducing complex I are homologous respiratory complexes with a common ancestor, but a structural basis for their evolutionary relationship is lacking. Here, we report the cryo-EM structure of a 14-subunit MBH from the hyperthermophile Pyrococcus furiosus. MBH contains a membrane-anchored hydrogenase module that is highly similar structurally to the quinone-binding Q-module of complex I while its membrane-embedded ion-translocation module can be divided into a H- and a Na-translocating unit. The H-translocating unit is rotated 180° in-membrane with respect to its counterpart in complex I, leading to distinctive architectures for the two respiratory systems despite their largely conserved proton-pumping mechanisms. The Na-translocating unit, absent in complex I, resembles that found in the Mrp H/Na antiporter and enables hydrogen gas evolution by MBH to establish a Na gradient for ATP synthesis near 100°C. MBH also provides insights into Mrp structure and evolution of MBH-based respiratory enzymes.

Hyperthermophilic archaea contain a hydrogen gas-evolving,respiratory membrane-bound NiFe-hydrogenase (MBH) that is very closely related to the aerobic respiratory complex I. During growth on elemental sulfur (S°), these microorganisms also produce a homologous membrane-bound complex (MBX), which generates HS. MBX evolutionarily links MBH to complex I, but its catalytic function is unknown. Herein, we show that MBX reduces the sulfane sulfur of polysulfides by using ferredoxin (Fd) as the electron donor, and we rename it membrane-bound sulfane reductase (MBS). Two forms of affinity-tagged MBS were purified from genetically engineered (a hyperthermophilic archaea species): the 13-subunit holoenzyme (S-MBS) and a cytoplasmic 4-subunit catalytic subcomplex (C-MBS). S-MBS and C-MBS reduced dimethyl trisulfide (DMTS) with comparable (∼490 μm) and values (12 μmol/min/mg). The MBS catalytic subunit (MbsL), but not that of complex I (NuoD), retains two of four NiFe-coordinating cysteine residues of MBH. However, these cysteine residues were not involved in MBS catalysis because a mutant strain (MbsLC85A/C385A) grew normally with S°. The products of the DMTS reduction and properties of polysulfides indicated that in the physiological reaction, MBS uses Fd ( ' = -480 mV) to reduce sulfane sulfur ( ' -260 mV) and cleave organic (RS R, ≥ 3) and anionic polysulfides (S , ≥ 4) but that it does not produce HS. Based on homology to MBH, MBS also creates an ion gradient for ATP synthesis. This work establishes the electrochemical reaction catalyzed by MBS that is intermediate in the evolution from proton- to quinone-reducing respiratory complexes.

Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S0)-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters.

Electron bifurcation enables thermodynamically unfavorable biochemical reactions. Four groups of bifurcating flavoenzyme are known and three use FAD to bifurcate. FeFe-HydABC hydrogenase represents the fourth group, but its bifurcation site is unknown. We report cryo-EM structures of the related NiFe-HydABCSL hydrogenase that reversibly oxidizes H 2 and couples endergonic reduction of ferredoxin with exergonic reduction of NAD. FMN surrounded by a unique arrangement of iron sulfur clusters forms the bifurcating center. NAD binds to FMN in HydB, and electrons from H 2 via HydA to a HydB [4Fe-4S] cluster enable the FMN to reduce NAD. Low-potential electron transfer from FMN to the HydC [2Fe-2S] cluster and subsequent reduction of a uniquely penta-coordinated HydB [2Fe-2S] cluster require conformational changes, leading to ferredoxin binding and reduction by a [4Fe-4S] cluster in HydB. This work clarifies the electron transfer pathways for a large group of hydrogenases underlying many essential functions in anaerobic microorganisms.

Significance The diverse microorganisms contained within the human gut are known to have significant effects on human health. Herein, we show that genes encoding members of the tungsten oxidoreductase (WOR) family of enzymes and a tungstate-specific transporter are prevalent in the human gut microbiome and metagenome. We demonstrate that two model gut microbes assimilate tungsten into multiple WOR enzymes and that some of these enzymes catalyze the conversion of gut aldehydes to the corresponding acid, likely as a detoxification strategy to remove these reactive compounds.