Kurt Jungermann

Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia

Abstract The physiological function of the clostridial NADH- and NADPH-ferredoxin oxidoreductases was investigated with Clostridium pasteurianum and Clostridium butyricum . The NADH-ferredoxin oxidoreductases are concluded to be catabolic enzymes required for the reduction of ferredoxin by NADH. The conclusion is based on the finding that during the entire growth phase the fermentation of glucose can be formally represented by the weighted sum of Eqns 1 and 2, Glucose + 2 H 2 O → 1 butyrate − + 2 HCO 3 − + 3 H + + 2 H 2 (1) Glucose + 4 H 2 O → 2 acetate − + 2 HCO 3 − + 4 H + + 4 H 2 (2) and that in these redox processes NADH rather than NADPH is specifically formed during glyceraldehyde phosphate dehydrogenation. This NADH can be consumed by substrate reduction in Process 1 only, while it must be reoxidized in Process 2 by the ferredoxin-dependent proton reduction to hydrogen which involves the NADH-ferredoxin oxidoreductases. The kinetic and regulatory properties of these enzymes are in line with their catabolic role: they are found with high specific activities typical for other catabolic enzymes; essentially they catalyze electron flow from NADH to ferredoxin only because the back reaction is very effectively inhibited by low concentrations of NADH. These enzymes have a key role in the coupling of the two partial processes and in regulating the overall thermodynamic efficiency of the fermentations. The NADPH-ferredoxin oxidoreductases are concluded to participate in anabolism; they are required for the regeneration of NADPH. The conclusion is based on the finding that in the two clostridia all catabolic oxidations-reductions are specific for NAD(H) and that the usual NADPH-producing processes such as the glucose 6-phosphate dehydrogenase or malate enzyme reactions are absent. The kinetic properties of the enzymes are in agreement with their anabolic function: the NADPH-ferredoxin oxidoreductases are found with sufficient specific activities; they preferentially catalyze electron transfer from reduced ferredoxin to NADP + .

Hydrogen formation from NADH in cell-free extracts of Clostridium kluyveri: Acetyl coenzyme A requirement and ferredoxin dependence☆

Recently cell-free extracts of Clostridium kluyveri were found to produce hydrogen from NADPH [ 1,2]. Proton reduction by NADPH was ferredoxin-dependent and effectively regulated by the NAD+/NADH redox couple, NAD+ being an activator and NADH an inhibitor. In this communication it will be shown that cellfree lysates of the organism also cqtalyze the formation of hydrogen from NADH. The NADH system is dependent on ferredoxin and has a strict requirement for acetyl CoA. The reduction of ferredoxin is not effected via an acetyl CoA/acetaldehyde shuttle as has been proposed [3]. Acetyl CoA appears to have a regulatory function in ferredoxin reduction by NADH.

The synthesis of one-carbon units from CO2 via a new ferredoxin dependent monocarboxylic acid cycle☆

COz was shown to be the predominant precursor of one-carbon units in Clostridium kluyveri [ 1,2] . For this COa assimilation, three mechanisms can be envisaged: (1) a direct COa reduction to formate, formally a reversal of one of the known formate dehydrogenases [3-61, followed by formate activation to formyl tetrahydrofolate; (2) a direct CO? reduction to formyl tetrahydrofolate without free formate as an intermediate, formally a reversal of a formyl tetrahydrofolate dehydrogenase [7] ; and (3) an indirect COa reduction via reductive carboxylation of acetyl CoA to pyruvate [8], pyruvate cleavage to acetyl CoA and formate and formate fixation into formyl tetrahydrofolate. In this communication data are presented indicating that the anabolic COa reduction and fixation into the one-carbon units is effected solely via mechanism (3): this new ferredoxin dependent pyruvate synthase-pyruvate formate lyase cycle is tentatively named reductive monocarboxylic acid cycle.

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg2+ dependent; Co2+ and Mn2+ but not Ca2+ could replace Mg2+ to some extent; the activation by Mg2+ was slightly antagonized by Ca2+. Even in the presence of Mg2+, Na+ or K+ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]0.5 v = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg2+. Solubilization, however, led to instability of the enzyme. The clostridial solubilized and membrane-bound ATPase showed different properties similar to the allotopic properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10(-4) M, led to 80% inhibition of the membrane-bound enzyme; oligomycin ouabain, or NaN3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment. Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation. A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prolaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.

Demonstration of NADH-ferredoxin reductase in two saccharolytic clostridia

NADH ferredoxin reductase has so far been demonstrated only in the non-saccharolytic Clostridium kluyveri (Thauer et al., 1969; Jungermann et al., 1971). The regulation of ferredoxin reduction with NADH by the acetyl-CoA/CoA couple pointed to a physiological function of this activity, for which a definitive role, however, was not established. In this communication evidence will be reported demonstrating t ha t a similar NADH-ferredoxin reduetase is present in high activi ty in the two saccharolytic C. pasteurianum and C. butyricum. Available fermentation balances (Wood, 1961; Daesch and Mortcnson, 1968) indicate t ha t in the catabolism of these organisms t t 2 is derived from NAD-dependent dehydrogenations. This led Mortcnson to postulate a NADH-ferredoxin reductase, for which, however, no experimental proof was presented (Mortenson, 1968).

CO2 reductase from Clostridium pasteurianum: Molybdenum dependence of synthesis and inactivation by cyanide

The reduction of CO 2 to formate is an important reaction in the metabolism of many clostridia. It is the initial step both in the total synthesis of acetate from CO 2 in CI. thermoaceticum and CI. acidi urici [1,2] and in one-carbon unit synthesis from CO 2 in CI. pasteurianum [3, 4]. For many years a reduction of CO 2 to formate could not be demonstrated in vitro. All known formate dehydrogenases only catalyzed the oxidation of formate to CO 2 rather than the reverse reaction. It was not until 1970 that a direct CO 2 reduction to formate was discovered in cell free lysates of CI. pasteurianum [3]. The enzyme catalyzing the reaction was tentatively named CO 2 reductase. In the meantime CO 2 reductases have also been found in CI. thermoaceticum [5] and CI. acidi urici [6]. In the present investigation evidence is presented indicating that the CO 2 reductase from CI. pasteurianum most probably is a molybdoenzyme: It is synthesized only in the presence of molybdenum and is inactivated by low concentrations of cyanide, as are other molybdoenzymes. A dependence of CO 2 reductase synthesis on selenium, as has been reported for the formate dehydrogenase from Escherichia coli [7-11 ], is definitely ruled out.

POSSIBLE METABOLIC ZONATION OF LIVER PARENCHYMA INTO GLUCOGENIC AND GLYCOLYTIC HEPATOCYTES

With hepatocyte suspensions it was found that (a) in the C3 part of carbohydrate metabolism glycolysis can be shifted to gluconeogenesis, dependent only on substrate concentrations rather than on hormones, and that (b) in both the C6 and C3 part glycolysis and gluconeogenesis are catalyzed simultaneously. This mode of action of the liver led to the hypothesis that there are at least two types of metabolically different hepatocytes forming a gluconeogenic and a glycolytic organ zone. Histochemical studies and biochemical analysis after microdissection of liver tissue indicated that the key glucogenic enzyme glucose-6-phosphatase (G6Pase) is highly active in periportal, and hardly active in perivenous hepatocytes, and that in the post-absorptive period glycogen is degraded first in the G6Pase rich cells. These results together with findings reported by other investigators support the model of “metabolic zonation” of liver parenchyma.

NADH, a physiological electron donor in clostridial nitrogen fixation

Clostridial nitrogenase catalyzes the ATP dependent reduction of Ns to NHs with reduced ferredoxin as electron donor [l] . In cell-free extracts of N,-grown Clostridium pasteuriunum several systems have been used to regenerate the reduced ferredoxin required in the nitrogenase reaction: pyruvate and pyruvate-ferredoxin oxidoreductase [2] , hydrogen and hydrogenase [3] as well as formate and COZreductase [4]. Therefore pyruvate, hydrogen and formate are generally regarded as the physiological reductants in clostridial nitrogen fixation [5,6]. NADH has also been reported to reduce ferredoxin via a NADH-ferredoxin oxidoreductase in several clostridia, which were however NHs-grown and thus devoid of the nitrogenase system [7-lo]. Since the NADH-ferredoxin oxidoreductase has not been demonstrated in Ns-grown clostridia as yet, NADH does not appear to be accepted as an electron donor for clostridial Ns-reduo tion, although this was indicated by preliminary evidence [3] and theoretical considerations [l I]. In this communication it is shown that NADHferredoxin oxidoreductase is present also in cellfree extracts of Ns-grown Cl. pasteurianum and

Characterization of crotonate grown Clostridium kluyveri by its assimilatory metabolism.

SummaryConsiderable behavioral differences were observed during growth of Clostridium kluyveri on ethanol-acetate and on crotonate media. The identity of the crotonate grown Clostridium with the ethanol grown Clostridium kluyveri was therefore established by three characteristic biosynthetic routes: 1. ribose is synthesized from CO2 and acetate via pyruvate, triose phosphate and a non-oxidative pentose phosphate pathway, 2. reduced one-carbon units are formed predominantly from CO2 and not from serine as usual, and 3. glutamate biogenesis follows an atypical stereochemical course.

The Development of Functional Heterogeneity in the Liver Parenchyma of the Golden Hamster

SummaryPrenatal and postnatal stages of the development of golden hamsters were studied histochemically and biochemically. It was shown that, beginning with the 12th gestational day, the fetal liver starts to store glycogen, and that this process reaches its maximum a birth. Glycogen phosphorylase and glucose-6-phosphatase (G6Pase)-activity increased drastically in the last two days before birth, glycogen phosphorylase preceding G6Pase. As a histochemical characteristic, an even distribution of glycogen, glycogen phosphorylase and G6Pase activity is found in the liver parenchyma at birth. During the first two postnatal weeks typical heterogeneous patterns of distribution developed: glycogen depletion could be demonstrated predominantly in zone 1 of the liver acinus, this being at the same time the area of highest glycogen phosphorylase and G6Pase-activity. The periportal zone 1 thus became characterized as the primary site of glycogenolysis (glycogen phosphorylase) and gluco(neo)genesis (G6Pase). “Metabolic Zonation” is interpreted as the chemomorphological equivalent of the regulatory function of the liver as a glucostat.