NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP⁺ with reduced ferredoxin (Fd_red) and the endergonic reduction of NADP⁺ with NADH in a reversible reaction: Fd_red²⁻ + NADH + 2 NADP⁺ + H⁺ = Fd_ox + NAD⁺ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H₂ (reaction 1) and in deriving a large (30%) portion of its cell carbon from CO₂. Both the energy metabolism and the pathways of biosynthesis have therefore been the subject of many investigations (for relevant literature, see references 12 and 27). (1)During growth of C. kluyveri on ethanol and acetate, approximately five ethanol and four acetate molecules are converted to three butyrate molecules and one caproate molecule (reaction 1a), and one ethanol molecule is oxidized to one acetate⁻, one H⁺, and two H₂ (reaction 1b) molecules (23, 31). How exergonic reaction 1a is coupled with endergonic reaction 1b and with ATP synthesis from ADP and P_i (ΔG^o′ = +32 kJ/mol) has remained unclear for many years. (1a) (1b)We recently showed (12) that, in Clostridium kluyveri, the exergonic reduction of crotonyl-coenzyme A (crotonyl-CoA) (E_o′ = −10 mV) with NADH (E_o′ = −320 mV) involved in reaction 1a is coupled with the endergonic reduction of ferredoxin (Fd_ox) (E_o′ = −420 mV) with NADH (E_o′ = −320 mV) involved in reaction 1b via the recently proposed mechanism of flavin-based electron bifurcation (7). The coupling reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase-EtfAB complex (reaction 2) (12): (2)The reduced ferredoxin (Fd_red²⁻) is assumed to be used for rereduction of NAD⁺ via a membrane-associated, proton-translocating ferredoxin:NAD oxidoreductase (RnfABCDEG) (reaction 3), and the proton motive force thus generated is assumed to drive the phosphorylation of ADP via a membrane-associated F₁F₀ ATP synthetase (reaction 4): (3) (4)The novel coupling mechanism represented by reactions 2 and 3 allowed for the first time the possibility of formulating a metabolic scheme for the ethanol-acetate fermentation that could account for the observed fermentation products and growth yields and thus for the observed ATP gains (27). One issue, however, remained open, namely, why the formation of butyrate from ethanol and acetate in the fermentation involves both an NADP⁺- and an NAD⁺-specific β-hydroxybutyryl-CoA dehydrogenase (16), considering that, in the oxidative part of the fermentation (ethanol oxidation to acetyl-CoA), only NADH is generated (8, 9, 13).The presence of a reduced ferredoxin:NADP⁺ oxidoreductase was proposed based on results of enzymatic studies performed 40 years ago. Cell extracts of Clostridium kluyveri were found to catalyze the formation of H₂ from NADPH in a ferredoxin- and NAD⁺-dependent reaction (34). The results were interpreted to indicate that C. kluyveri contains a ferredoxin-dependent hydrogenase and an NADPH:ferredoxin oxidoreductase with transhydrogenase activity. H₂ formation from NADPH was strictly dependent on the presence of NAD⁺ and was inhibited by NADH, inhibition being competitive with the presence of NAD⁺, indicating that ferredoxin reduction with NADPH is under the allosteric control of the NAD⁺/NADH couple. The cell extracts also catalyzed the NADH-dependent reduction of NADP⁺ with reduced ferredoxin (21, 34). Purification of the enzyme catalyzing these reactions was not achieved, and no function in the energy metabolism of C. kluyveri was assigned.In this communication, we report on the properties of the recombinant enzyme that catalyzes the NAD⁺-dependent reduction of ferredoxin with NADPH and the NADH-dependent reduction of NADP⁺ with reduced ferredoxin and show that the cytoplasmic heterodimeric enzyme couples the exergonic reduction of NADP⁺ with reduced ferredoxin with the endergonic reduction of NADP⁺ with NADH in a fully reversible reaction. The transhydrogenation reaction is endergonic, because in vivo the NADH/NAD⁺ ratio is generally near 0.3 and the NADPH/NADP⁺ ratio is generally above 1 (2, 30). (5)NADP⁺ reduction is most probably the physiological function of the enzyme, which is why we chose the abbreviation NfnAB (for NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase).     

Growth parameters (K s, μmax,Y s) of Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum was grown on a mineral salts medium in a fermenter gassed with H₂ and CO₂, which were the sole carbon and energy sources. Under the conditions used the bacterium grew exponentially. The dependence of the growth rate () on the concentration of H₂ and CO₂ in the incoming gas and the dependence of the growth yield ( ) on the growth rate were determined at pH 7 (the pH optimum) and 65° C (the temperature optimum).The curves relating growth rate to the H₂ and CO₂ concentration were hyperbolic. From reciprocal plots apparent K _s values for H₂ and CO₂ and _max were obtained: app. = 20%; app. = 11%; = 0.69 h^-1; t (max)=1 h. was 1.6 g mol^-1 and almost independent of the growth rate, when the rate of methane formation was not limited by the supply of either H₂ or CO₂. The yield increased to near 3 g mol^-1 when H₂ or CO₂ were limiting. These findings indicate that methane formation and growth are less tightly coupled at high concentrations of H₂ or CO₂ in the medium than at low concentrations. The physiological significance of these findings is discussed. K _s: H₂ and CO₂ concentration supporting 0.5 _max; _max: specific growth rate at infinite substrate concentration; Y _s:growth yield (g dry weight/mol substrate); t : doubling time     

Time Course Analysis of Motor Excitability in a Response Inhibition Task According to the Level of Hyperactivity and Impulsivity in Children with ADHD

Short interval intracortical inhibition (SICI) of motor cortex, measured by transcranial magnetic stimulation (TMS) in a passive (resting) condition, has been suggested as a neurophysiological marker of hyperactivity in attention-deficit/hyperactivity disorder (ADHD). The aim of this study was to determine motor excitability in a go/nogo task at stages of response preparation, activation and suppression in children with ADHD, depending on the level of hyperactivity and impulsivity. Motor evoked potentials were recorded in 29 typically developing children and 43 children with ADHD (subdivided in two groups with higher and lower levels of hyperactivity/impulsivity; H/I-high and H/I-low). In the H/I-high group, SICI was markedly reduced in the resting condition and during response preparation. Though these children were able to increase SICI when inhibiting a response, SICI was still reduced compared to typically developing children. Interestingly, SICI at rest and during response activation were comparable, which may be associated with their hypermotoric behaviour. In the H/I-low group, response activation was accompanied by a pronounced decrease of SICI, indicating reduced motor control in the context of a fast motor response. In summary, different excitability patterns were obtained for the three groups allowing a better understanding of dysfunctional response activation and inhibition processes within the motor cortex in children with ADHD.     

p63 and p73: roles in development and tumor formation

The tumor suppressor p53 is critically important in the cellular damage response and is the founding member of a family of proteins. All three genes regulate cell cycle and apoptosis after DNA damage. However, despite a remarkable structural and partly functional similarity among p53, p63, and p73, mouse knockout studies revealed an unexpected functional diversity among them. p63 and p73 knockouts exhibit severe developmental abnormalities but no increased cancer susceptibility, whereas this picture is reversed for p53 knockouts. Neither p63 nor p73 is the target of inactivating mutations in human cancers. Genomic organization is more complex in p63 and p73, largely the result of an alternative internal promoter generating NH2-terminally deleted dominant-negative proteins that engage in inhibitory circuits within the family. Deregulated dominant-negative p73 isoforms might play an active oncogenic role in some human cancers. Moreover, COOH-terminal extensions specific for p63 and p73 enable further unique protein-protein interactions with regulatory pathways involved in development, differentiation, proliferation, and damage response. Thus, p53 family proteins take on functions within a wide biological spectrum stretching from development (p63 and p73), DNA damage response via apoptosis and cell cycle arrest (p53, TAp63, and TAp73), chemosensitivity of tumors (p53 and TAp73), and immortalization and oncogenesis (DeltaNp73).     

Control of Adipose Tissue Expandability in Response to High Fat Diet by the Insulin-like Growth Factor-binding Protein-4

Adipose tissue expansion requires growth and proliferation of adipocytes and the concomitant expansion of their stromovascular network. We have used an ex vivo angiogenesis assay to study the mechanisms involved in adipose tissue expansion. In this assay, adipose tissue fragments placed under pro-angiogenic conditions form sprouts composed of endothelial, perivascular, and other proliferative cells. We find that sprouting was directly stimulated by insulin and was enhanced by prior treatment of mice with the insulin sensitizer rosiglitazone. Moreover, basal and insulin-stimulated sprouting increased progressively over 30 weeks of high fat diet feeding, correlating with tissue expansion during this period. cDNA microarrays analyzed to identify genes correlating with insulin-stimulated sprouting surprisingly revealed only four positively correlating (Fads3, Tmsb10, Depdc6, and Rasl12) and four negatively correlating (Asph, IGFbp4, Ppm1b, and Adcyap1r1) genes. Among the proteins encoded by these genes, IGFbp4, which suppresses IGF-1 signaling, has been previously implicated in angiogenesis, suggesting a role for IGF-1 in adipose tissue expandability. Indeed, IGF-1 potently stimulated sprouting, and the presence of activated IGF-1 receptors in the vasculature was revealed by immunostaining. Recombinant IGFbp4 blocked the effects of insulin and IGF-1 on mouse adipose tissue sprouting and also suppressed sprouting from human subcutaneous adipose tissue. These results reveal an important role of IGF-1/IGFbp4 signaling in post-developmental adipose tissue expansion.     

Identification of 27-oxo-octacosanoic acid and heptacosane-1,27-dioic acid in Legionella pneumophila

A strain of Escherichia coli having elevated levels of cytochrome bo and lacking the cytochrome bd quinol oxidase was grown in chemostat culture at low copper levels. Such cells had lowered levels of copper and of total cytochrome b. Cytochrome o concentration was unchanged when assayed by conventional CO difference spectroscopy, but apparently diminished by 80% in copper-deficient cells as determined by photodissociation of bound CO at 193 K. This is attributed to depletion of copper in the oxidase of copper-deficient cells, causing rapid recombination of photodissociated CO to haem O. CO recombination was also more sensitive to low intensities of actinic light in copper-depleted oxidase. The results illustrate a further similarity between the active sites of o- and aa3-type terminal oxidases.