Search

We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.

The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase.IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. The "normalization" of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe proteins) housing the same cofactor (M-cluster) allows for an effective assessment of the impact of the protein environment on the CO reactivity of nitrogenase. The results of this study provide a first look into the "weighted" contributions of protein environment and cofactor properties to the overall activity of CO reduction; more importantly, they establish a useful platform for further investigation of the structural elements attributing to the CO-reducing activity of nitrogenase.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin--the physiological donor of electrons in the acetyl-CoA pathway--with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe0), but not ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today. [ABSTRACT FROM AUTHOR]

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. Rnf reversibly reduces NAD+ by ferredoxin coupled with Na+/H+ pumping for microbial ion gradient or reduced ferredoxin formation. Here, a cryo-EM structure provides information about the participating iron and flavin cofactors, the electron transfer route and the putative Na+ passage. [ABSTRACT FROM AUTHOR]

The human pathogenic bacterium Clostridium difcile thrives by the fermentation of L-leucine to ammonia, C[O.sub.2], 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions (1). The reductive branch to 4-methylpentanoate proceeds by means [...]

Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxinldependent formation of [H.sub.2] from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyI-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyI-CoA to butyryl-CoA. The cytoplasmic butyryI-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin ([E.sub.0]' = -410 mV) with NADH ([E.sub.0]' = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA ([E.sub.0]' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 [NAD.sup.+] + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.

The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of A. woodii catalyzes [H.sub.2]-dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits [alpha] (EtfA) and [beta] (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NA[D.sup.+] oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential [Na.sup.+] pump. These data suggest the following electron transport chain: [H.sub.2] [right arrow] ferredoxin [right arrow] NA[D.sup.+] [right arrow] Etf [right arrow] caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NA[D.sup.+] reduction catalyzed by Rnf.

The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine thus the amino acid is both oxidized to isovalerate plus [CO.sub.2] and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxylsocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates.

The enzymes that contain radicals and/or catalyze reactions with radical intermediates and are present in anaerobes are described. The families of coenzyme [B.sub.12]- and S-adenosylmethionine (SAM)-dependent radical enzymes are the exceptions as some of them occur in aerobes.

Research has been conducted on the Acidaminococcus fermentans 2-hydroxyglutaryl-CoA dehydratase. The isolation of flavodoxin from A. fermentans cell-free extracts has been carried out and the results demonstrate the postulated one-electron transfer from flavodoxin to the homodimeric activator.

Results demonstrate the ability of Clostridium cochlearium to biosynthesize native 'complete' B(sub)12 cofactors and two other homologous corrinoids. Analysis of corrinoids shows three cyano corrinoids in a ratio of about 3:1:1 and all of which contained pseudovitamin B(sub)12.