Characterization of the RnfB and RnfG Subunits of the Rnf Complex from the Archaeon Methanosarcina acetivorans

Rnf complexes are redox-driven ion pumps identified in diverse species from the domains Bacteria and Archaea, biochemical characterizations of which are reported for two species from the domain Bacteria. Here, we present characterizations of the redox-active subunits RnfG and RnfB from the Rnf complex of Methanosarcina acetivorans, an acetate-utilizing methane-producing species from the domain Archaea. The purified RnfG subunit produced in Escherichia coli fluoresced in SDS-PAGE gels under UV illumination and showed a UV-visible spectrum typical of flavoproteins. The Thr166Gly variant of RnfG was colorless and failed to fluoresce under UV illumination confirming a role for Thr166 in binding FMN. Redox titration of holo-RnfG revealed a midpoint potential of −129 mV for FMN with n = 2. The overproduced RnfG was primarily localized to the membrane of E. coli and the sequence contained a transmembrane helix. A topological analysis combining reporter protein fusion and computer predictions indicated that the C-terminal domain containing FMN is located on the outer aspect of the cytoplasmic membrane. The purified RnfB subunit produced in E. coli showed a UV-visible spectrum typical of iron-sulfur proteins. The EPR spectra of reduced RnfB featured a broad spectral shape with g values (2.06, 1.94, 1.90, 1.88) characteristic of magnetically coupled 3Fe-4S and 4Fe-4S clusters in close agreement with the iron and acid-labile sulfur content. The ferredoxin specific to the aceticlastic pathway served as an electron donor to RnfB suggesting this subunit is the entry point of electrons to the Rnf complex. The results advance an understanding of the organization and biochemical properties of the Rnf complex and lay a foundation for further understanding the overall mechanism in the pathway of methane formation from acetate.     

A Bacterial Electron-bifurcating Hydrogenase

The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.     

Purification and characterization of a flavin reductase from the biodesulfurizing bacterium Mycobacterium goodii X7B

Dibenzothiophene (DBT) in fossil fuels can be efficiently biodesulfurized by a thermophilic bacterium Mycobacterium goodii X7B. Flavin reductase DszD, which catalyzes the reduction of oxidated flavin by NAD(P)H, is indispensable for the biodesulfurization process. In this work, a flavin reductase DszD in M. goodii X7B was purified to homogeneity, and then its encoding gene dszD was amplified and expressed in Escherichia coli. DszD is a homodimer with each subunit binding one FMN as cofactor. The K_m values for FMN and NADH of the purified recombinant DszD were determined to be 6.6 ± 0.3 μM and 77.9 ± 5.4 μM, respectively. The optimal temperature for DszD activity was 55 °C. DszD can use FMN or FAD as substrate to generate FMNH₂ or FADH₂ as product. DszD was coexpressed with DBT monooxygenase DszC, the enzyme catalyzing the first step of the biodesulfurization process. It was indicated that the coexpressed DszD could effectively enhance the DszC catalyzed DBT desulfurization reaction.     

Asymmetric Dimeric Structure of Ferredoxin-NAD(P) Oxidoreductase from the Green Sulfur Bacterium Chlorobaculum tepidum: Implications for Binding Ferredoxin and NADP

Ferredoxin-NAD(P)⁺ oxidoreductase (FNR) catalyzes the reduction of NAD(P)⁺ to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 Å resolution, which reveals a unique structure-function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by π-π stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.     

Structural and biochemical characterization of flavoredoxin from the archaeon Methanosarcina acetivorans

Flavoredoxin is a FMN-containing electron transfer protein that functions in the energy-yielding metabolism of Desulfovibrio gigas of the Bacteria domain. Although characterization of this flavoredoxin is the only one reported, a database search revealed homologues widely distributed in both the Bacteria and Archaea domains that define a novel family. To improve our understanding of this family, a flavoredoxin from Methanosarcina acetivorans of the Archaea domain was produced in Escherichia coli and biochemically characterized, and a high-resolution crystal structure was determined. The protein was shown to be a homodimer with a subunit molecular mass of 21 kDa containing one noncovalently bound FMN per monomer. Redox titration showed an E(m) of -271 mV with two electrons, consistent with no semiquinone observed in the potential range studied, a result suggesting the flavoredoxin functions as a two-electron carrier. However, neither of the obligate two-electron carriers, NAD(P)H and coenzyme F420H2, was a competent electron donor, whereas 2[4Fe-4S] ferredoxin reduced the flavoredoxin. The X-ray crystal structure determined at 2.05 A resolution revealed a homodimer containing one FMN per monomer, consistent with the biochemical characterization. The isoalloxazine ring of FMN was shown buried within a narrow groove approximately 10 A from the positively charged protein surface that possibly facilitates interaction with the negatively charged ferredoxin. The structure provides a basis for predicting the mechanism by which electrons are transferred between ferredoxin and FMN. The FMN is bound with hydrogen bonds to the isoalloxazine ring and electrostatic interactions with the phosphate moiety that, together with sequence analyses of homologues, indicate a novel FMN binding motif for the flavoredoxin family.     

Energy Conservation Model Based on Genomic and Experimental Analyses of a Carbon Monoxide-Utilizing,Butyrate-Forming Acetogen,Eubacterium limosum KIST612

Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na⁺ dependent. Consistent with the finding of a Na⁺-dependent Rnf complex is the presence of a conserved Na⁺-binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H₂ + CO₂. The energetic benefit may be one reason that butyrate is formed only from CO but not from H₂ + CO₂.     

Discovery and Characterization of the First Archaeal Dihydromethanopterin Reductase,an Iron-Sulfur Flavoprotein from Methanosarcina mazei

The microbial production of methane by methanogenic archaea is dependent on the synthesis of the pterin-containing cofactor tetrahydromethanopterin (H₄MPT). The enzyme catalyzing the last step of H₄MPT biosynthesis (dihydromethanopterin reductase) has not previously been identified in methane-producing microorganisms. Previous complementation studies with the methylotrophic bacterium Methylobacterium extorquens have indicated that an uncharacterized archaeal-flavoprotein-like flavoprotein (AfpA) from Methylobacillus flagellatus or Burkholderia xenovorans can replace the activity of a phylogenetically unrelated bacterial dihydromethanopterin reductase (DmrA). We propose that MM1854, a homolog of AfpA from Methanosarcina mazei, catalyzes the last step of H₄MPT biosynthesis in methane-producing microorganisms. To test this hypothesis, a six-histidine (His₆)-tagged version of MM1854 was produced. Bioinformatic analysis revealed the presence of one flavin mononucleotide (FMN)-binding site and two iron-sulfur cluster sites, consistent with an oxidoreductase enzyme. Purified His₆-MM1854 occurred as a homodimer of 29-kDa subunits, and the UV-visible spectrum of the purified protein showed absorbance peaks at 380 and 460 nm, characteristic of oxidized FMN. NAD(P)H was incapable of directly reducing the flavin cofactor, but dithionite eliminated the FMN peaks, indicating successful electron transfer to MM1854. An electron transfer system of NADPH, spinach NADPH-ferredoxin oxidoreductase, and ferredoxin could also reduce the FMN peaks. A newly developed assay indicated that dithiothreitol-reduced MM1854 could transfer electrons to dihydromethanopterin. This assay was also effective with a heat-stable DmrX analog from Methanocaldococcus jannaschii (MJ0208). These results provide the first biochemical evidence that MM1854 and MJ0208 function as archaeal dihydromethanopterin reductases (DmrX) and that ferredoxin may serve as an electron donor.     

The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and Is Reversibly Coupled to the Membrane Potential

The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na⁺-translocating electron transport chain that couples electron transfer from reduced ferredoxin to NAD⁺ with the generation of a primary electrochemical Na⁺ potential across its cytoplasmic membrane. In previous assays in which Ti³⁺ was used to reduce ferredoxin, Na⁺ transport was observed, but not a Na⁺ dependence of the electron transfer reaction. Here, we describe a new biological reduction system for ferredoxin in which ferredoxin is reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-reduced ferredoxin, NAD⁺ reduction was highly specific and strictly dependent on ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na⁺ dependence of this electron transfer reaction. The K_m was 0.2 mm. Na⁺ could be partly substituted by Li⁺. Na⁺ dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na⁺ as a coupling ion. Electron transport from reduced ferredoxin to NAD⁺ was coupled to electrogenic Na⁺ transport, indicating the generation of Δ$\tilde{\mu }$_Na⁺. Vice versa, endergonic ferredoxin reduction with NADH as reductant was possible, but only in the presence of Δ$\tilde{\mu }$_Na⁺, and was accompanied by Na⁺ efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes ferredoxin reduction at the expense of an electrochemical Na⁺ gradient. The physiological significance of this finding is discussed.     

The role of the invariant glutamate 95 in the catalytic site of Complex I from Escherichia coli

Replacement of glutamate 95 for glutamine in the NADH- and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5-4.8 times depending on the used electron acceptor. The apparent K_m for NADH was 5.2 and 10.4 μM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD⁺ on activity showed that the E95Q mutation caused a 2.4-fold decrease of K_i^NAD+ in comparison to the wild type enzyme. ADP-ribose, which differs from NAD⁺ by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K_i^ADP-ribose relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD⁺. The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from − 350 mV to − 310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.     

Sulfolobus tokodaii ST2133 is characterized as a thioredoxin reductase-like ferredoxin:NADP+ oxidoreductase

The putative gene (st2133) for ferredoxin:NADP⁺ oxidoreductase (FNR) from Sulfolobus tokodaii, a thermoacidophilic crenarchaeon, was heterologously expressed. About 90 % of the purified product was a homodimer containing 0.46 mol FAD/mol subunit, and showing NADPH:DCPIP oxidoreductase activity, V _max being 1.38 and 21.8 U/mg (70 °C) in the absence and presence of 1 mM FMN. NADPH was a much better electron donor than NADH with various electron acceptors, such as oxygen, hydrogen peroxide, DCPIP, cytochrome c, and dithiobisnitrobenzoate. Most of the reactions were activated by 15- to 140-fold on addition of FMN, while FAD was 5–10 times less effective. Ferredoxin (Fd) from S. tokodaii served as an electron carrier in both Fd-dependent NADPH formation and NADPH-dependent Fd reduction. ST2133 belongs to the thioredoxin reductase-like protein family, which is slightly distantly related to FNR family proteins from bacteria, plants and man. This is the first report on FNR from a crenarchaeon, providing a clue to the recycling of Fd during archaeal metabolism.     

Spinach Leaf Chloroplast CO(2) and NO(2) Photoassimilations Do Not Compete for Photogenerated Reductant: Manipulation of Reductant Levels by Quantum Flux Density Titrations

Potential competition between CO₂ and NO₂⁻ photoassimilation for photogenerated reductant (e.g. reduced ferredoxin and NADPH) was examined employing isolates of mesophyll cells and intact chloroplasts derived from mature `source' spinach leaves. Variations in the magnitude of incident light energy were used to manipulate the supply of reductant in situ within chloroplasts. Leaf cell and plastid isolates were fed with saturating CO₂ and/or NO₂⁻ to produce the highest demand for reductant by CO₂ and/or NO₂⁻ assimilatory processes (enzymes). Even in the presence of CO₂ fixation, NO₂⁻ reduction in intact leaf cell isolates as well as plastid isolates was maximal at light energies as low as 50 to 200 microeinsteins per second per square meter. Simultaneously, 500 to 800 microeinsteins per second per square meter were required to support maximal CO₂ assimilation. Regardless of the magnitude of the incident light energy, CO₂ assimilation did not repress NO₂⁻ reduction, nor were these two processes mutually repressed. These observations have been interpreted to mean that reduced ferredoxin levels in situ in the plastids of mature source leaf mesophyll cells were adequate to supply the concurrent maximal demands exerted by enzymes associated with CO₂ as well as with inorganic nitrogen photoassimilation.     

Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an α₃β₃ hexamer. The apparent K_m of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM⁻¹ min⁻¹ for 2NT and 1.2 μM⁻¹ min⁻¹ for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1 mol of CO₂ was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD⁺ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD⁺ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L⁻¹ of glucose was consumed to generate 12.08 g L⁻¹ of succinic acid. Furthermore, under optimized condition of CO₂ supply, the succinic acid productivity and the CO₂ fixation rate reached 223.88 mg L⁻¹ h⁻¹ and 83.48 mg L⁻¹ h⁻¹, respectively.     

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP⁺ with H₂ or formate and the reversible formation of H₂ and CO₂ from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO₂ reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H₂ formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E_0′ = −520 mV).     

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (Etf_Af) and butyryl-CoA dehydrogenase (Bcd_Af) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. Etf_Af contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The Etf_Af-NAD⁺ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH⁻ is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH⁻ by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD^⨪, which donates this electron further to Dh-FAD of Bcd_Af after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH^•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH⁻ that converts crotonyl-CoA to butyryl-CoA.     

Structure-function studies of glutamate synthases: a class of self-regulated iron-sulfur flavoenzymes essential for nitrogen assimilation

Glutamate synthases play with glutamine synthetase an essential role in nitrogen assimilation processes in microorganisms, plants, and lower animals by catalyzing the net synthesis of one molecule of L-glutamate from L-glutamine and 2-oxoglutarate. They exhibit a modular architecture with a common subunit or region, which is responsible for the L-glutamine-dependent glutamate synthesis from 2-oxoglutarate. Here, a PurF- (Type II- or Ntn-) type amidotransferase domain is coupled to the synthase domain, a (beta/alpha)8 barrel containing FMN and one [3Fe-4S]0,+1 cluster, through a approximately 30 angstroms-long intramolecular tunnel for the transfer of ammonia between the sites. In bacterial and eukaryotic GltS, reducing equivalents are provided by reduced pyridine nucleotides thanks to the stable association with a second subunit or region, which acts as a FAD-dependent NAD(P)H oxidoreductase and is responsible for the formation of the two low potential [4Fe-4S]+1,+2 clusters of the enzyme. In photosynthetic cells, reduced ferredoxin is the physiological reductant. This review focus on the mechanism of cross-activation of the synthase and glutaminase reactions in response to the bound substrates and the redox state of the enzyme cofactors, as well as on recent information on the structure of the alphabeta protomer of the NADPH-dependent enzyme, which sheds light on the intramolecular electron transfer pathway between the flavin cofactors.     

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association,Assembly, Maturation,and Function

Hydrogenases are metalloenzymes that catalyze 2H⁺ + 2e⁻ ↔ H₂. A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)⁺ as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.     

Purification and Properties of NADH-Dependent 5,10-Methylenetetrahydrofolate Reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the K_m for NADH is 13 μM, the K_m for CH₂-H₄folate is 0.8 μM, and the turnover number under V_max conditions estimated for the reaction is 1,800 mol of NADH oxidized min⁻¹ (mol of enzyme-bound FAD)⁻¹. NADPH also serves as a reductant, but exhibits a much higher K_m. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.