Flavins in the electron bifurcation process.

The discovery of a new energy-coupling mechanism termed flavin-based electron bifurcation (FBEB) in 2008 revealed a novel field of application for flavins in biology. The key component is the bifurcating flavin endowed with strongly inverted one-electron reduction potentials (FAD/FAD•- ≪ FAD•-/FADH-) that cooperatively transfers in its reduced state one low and one high-energy electron into different directions and thereby drives an endergonic with an exergonic reduction reaction. As energy splitting at the bifurcating flavin apparently implicates one-electron chemistry, the FBEB machinery has to incorporate prior to and behind the central bifurcating flavin 2e-to-1e and 1e-to-2e switches, frequently also flavins, for oxidizing variable medium-potential two-electron donating substrates and for reducing high-potential two-electron accepting substrates. The one-electron carriers ferredoxin or flavodoxin serve as low-potential (high-energy) electron acceptors, which power endergonic processes almost exclusively in obligate anaerobic microorganisms to increase the efficiency of their energy metabolism. In this review, we outline the global organization of FBEB enzymes, the functions of the flavins therein and the surrounding of the isoalloxazine rings by which their reduction potentials are specifically adjusted in a finely tuned energy landscape.

Purification and structural characterization of the Na+-translocating ferredoxin: NAD+ reductase (Rnf) complex of Clostridium tetanomorphum.

Various microbial metabolisms use H+/Na+-translocating ferredoxin:NAD+ reductase (Rnf) either to exergonically oxidize reduced ferredoxin by NAD+ for generating a transmembrane electrochemical potential or reversely to exploit the latter for producing reduced ferredoxin. For cryo-EM structural analysis, we elaborated a quick four-step purification protocol for the Rnf complex from Clostridium tetanomorphum and integrated the homogeneous and active enzyme into a nanodisc. The obtained 4.27 Å density map largely allows chain tracing and redox cofactor identification complemented by biochemical data from entire Rnf and single subunits RnfB, RnfC and RnfG. On this basis, we postulated an electron transfer route between ferredoxin and NAD via eight [4Fe-4S] clusters, one Fe ion and four flavins crossing the cell membrane twice related to the pathway of NADH:ubiquinone reductase. Redox-coupled Na+ translocation is provided by orchestrating Na+ uptake/release, electrostatic effects of the assumed membrane-integrated FMN semiquinone anion and accompanied polypeptide rearrangements mediated by different redox steps.

A caffeyl-coenzyme A synthetase initiates caffeate activation prior to caffeate reduction in the acetogenic bacterium Acetobacterium woodii.

The anaerobic acetogenic bacterium Acetobacterium woodii couples the reduction of caffeate with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism using sodium ions as coupling ions, but the enzymes involved remain to be established. Previously, the electron transfer flavoproteins EtfA and EtfB were found to be involved in caffeate respiration. By inverse PCR, we identified three genes upstream of etfA and etfB: carA, carB, and carC. carA encodes a potential coenzyme A (CoA) transferase, carB an acyl-CoA synthetase, and carC an acyl-CoA dehydrogenase. carA, -B, and -C are located together with etfA/carE and etfB/carD on one polycistronic message, indicating that CarA, CarB, and CarC are also part of the caffeate respiration pathway. The genetic data suggest an initial ATP-dependent activation of caffeate by CarB. To prove the proposed function of CarB, the protein was overproduced in Escherichia coli, and the recombinant protein was purified. Purified CarB activates caffeate to caffeyl-CoA in an ATP- and CoA-dependent reaction. The enzyme has broad pH and temperature optima and requires K(+) for activity. In addition to caffeate, it can use ρ-coumarate, ferulate, and cinnamate as substrates, with 50, 15, and 9%, respectively, of the activity obtained with caffeate. Expression of the car operon is induced not only by caffeate, ρ-coumarate, ferulate, and cinnamate but also by sinapate. There is no induction by ρ-hydroxybenzoate or syringate.

Molecular and Low-Resolution Structural Characterization of the Na+-Translocating Glutaconyl-CoA Decarboxylase From Clostridium symbiosum.

Some anaerobic bacteria use biotin-dependent Na+-translocating decarboxylases (Bdc) of ß-keto acids or their thioester analogs as key enzymes in their energy metabolism. Glutaconyl-CoA decarboxylase (Gcd), a member of this protein family, drives the endergonic translocation of Na+ across the membrane with the exergonic decarboxylation of glutaconyl-CoA (ΔG 0' ≈-30 kJ/mol) to crotonyl-CoA. Here, we report on the molecular characterization of Gcd from Clostridium symbiosum based on native PAGE, size exclusion chromatography (SEC) and laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). The obtained molecular mass of ca. 400 kDa fits to the DNA sequence-derived mass of 379 kDa with a subunit composition of 4 GcdA (65 kDa), 2 GcdB (35 kDa), GcdC1 (15 kDa), GcdC2 (14 kDa), and 2 GcdD (10 kDa). Low-resolution structural information was achieved from preliminary electron microscopic (EM) measurements, which resulted in a 3D reconstruction model based on negative-stained particles. The Gcd structure is built up of a membrane-spanning base primarily composed of the GcdB dimer and a solvent-exposed head with the GcdA tetramer as major component. Both globular parts are bridged by a linker presumably built up of segments of GcdC1, GcdC2 and the 2 GcdDs. The structure of the highly mobile Gcd complex represents a template for the global architecture of the Bdc family.

The F420-reducing [NiFe]-hydrogenase complex from Methanothermobacter marburgensis, the first X-ray structure of a group 3 family member.

The reversible redox reaction between coenzyme F420 and H2 to F420H2 is catalyzed by an F420-reducing [NiFe]-hydrogenase (FrhABG), which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25MDa as recently revealed by high-resolution cryo-electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but is different because of the truncation of ca 160 residues that results in similar but modified H2 and proton transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of an N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F420-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°'≈-400mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.

De novo modeling of the F(420)-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy.

Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure. DOI:http://dx.doi.org/10.7554/eLife.00218.001.