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Arch. Microbiol. 102, 53-57 (1975) 9 by Springer-Verlag 1975 Pyruvate Dehydrogenase Complex, Pyruvate"Ferredoxin Oxidoreductase and Lipoic Acid Content in Microorganisms H. BOTHE and U. NOLTEERNSTING Abteilung Biologic der Ruhr-Universit~it Bochum Received August 2, 1974 Abstract. Several microorganisms were examined for the content of lipoic acid by using a strain of Streptococcus faecalis deficient in this coenzyme. In comparison to this, the specific activity levels were determined for the pyruvate: ferredoxin oxidoreductase and the pyruvate dehydrogenase complex, which both catalyse the cleavage of pyruvate and coenzyme A to acetyl coenzyme A, CO~ and two reducing equivalents. Anabaena cylindrica, Chlorobium, Clostridium pasteurianum and kluyveri, where only the pyruvate: ferre9 doxin oxidoreductase can be demonstrated, were found to contain minute levels of lipoic acid. Thus lipoic acid does not appear to be a cofactor of the decarboxylation catalysed by the pyruvate: ferredoxin oxidoreductase. On the other hand, the amount of lipoic acid is at least ten times higher in Ankistrodesmus, Chlamydomonas, Anacystis, Micrococcus, Azotobacter and Escherichia coli which have the dehydrogenase complex. Two different reactions are known for the formation of acetyl coenzyme A and CO2 from pyruvate and coenzyme A. The first one is catalysed by the multienzyme complex of the pyruvate dehydrogenase. The role of the cofactors involved in this system, namely thiamine-pyrophosphate, lipoic acid, FAD and NAD + is well established (Reed and Cox, 1966). The second oxidative decarboxylation of pyruvate is catalysed by the pyruvate: ferredoxin oxidoreductase which has mainly been described in anaerobic bacteria. In this case, the remaining electrons are not transferred to NAD + but to ferredoxin (Raeburn and Rabinowitz, i97i b). In blue-green algae, the nature of the enzyme systems which catalyse the decarboxylations of ketoacids with concomitant formation of acyl coenzymes is uncertain. The pyruvate dehydrogenase complex has not yet been demonstrated (comp. Smith, 1973, Table 1.2). Recently, the pyruvate: ferredoxin oxidoreductase was found in the two Anabaena species variabilis (Leach and Carr, 1971) and cylindrica (Bothe eta/.., 1974). In the present communication, the activities of the two enzymes were tested and the lipoic acid content was determined in extracts from the blue-green algae Anabaena cylindrica and Anacystis nidulans, and this investigation was extended for comparison to several other microorganisms. The data reported here show a correlation between a high lipoic acid content and the presence of the pyruvate dehydrogenase complex on one side and a low lipoic acid content and the occurrence of the pyruvate: ferredoxin oxidoreductase on the other. Among the blue-green algae, Anabaena cylindrica has a low lipoic acid level and appears to cleave pyruvate to acety! coenzyme A and CO2 exclusively by the pyruvate: ferredoxin oxidoreductase. In contrast, the presence of the pyruvate dehydrogenase complex and a high lipoic acid content can be demonstrated in Anacystis nidulans. Key words: Lipoic Acid--Pyruvate : Ferredoxin Oxidoreductase-Pyruvate Dehydrogenase Complex - Ferredoxin. Materials and Methods Growth of Organisms In the brackets the first place indicates the source of the organism and the second one the growth condition: Azotobacter vinelandii (Czechoslovak Collection of Microorganisms at Brno, CCM 289; van Lin and Bothe, 1972), Microcoeeus denitrificans = Paracoeeus denitrifieans (Czechoslovak Collection, CCM 982; grown anaerobically on nitrate, Drews, 1968, p. 174), Ankistrodesmus braunii (Collection of the Institute of Plant Physiology, GSttingen, No. 202-7c; Kuhl, 1962), Chlamydomonas reinhardtii, wild type (batch kindly supplied by Dr. N. Amrhein, Institute of Plant Physiology, this university; Fischer and Amrhein, 1974), Anacystis nidutans (Collection G6ttingen No. t402-1 ; Bothe, I968), Anabaena cylindrica (Collection GSttingen No. 1403-2; Bothe and Loos, 1972), Chlorobiurn thiosulfatophilum strain Tassajara (stock kindly supplied by Professor N. Pfennig, GSttingen; Lippert and Pfennig, 1969), Clostridium pasteurianum (ATCC 6013; Lovenberg et al., 1963), Clostridium kluyveri (stock obtained from E. Merck, 54 Arch. Microbiol,, Vol. 102, No. 1 (1975) Darmstadt; Thauer et al., 1968). Escherichia coli (batch purchased from Merck, grown aerobically). The axenic cultures were harvested in the late logarithmic phase and stored at --20 ~C. Growth of the Lipoic Acid Deficient Strain of Streptococcus faeealis The strain Streptococcus faecalis 10 CI, either kindly supplied by the Czechoslovak Collection of Microorganisms (No. CCM 1875) or purchased from the ATCC (No. 11700), was used for the assays of lipoic acid. In the beginning it was grown in exactly the same medium as described by Gunsalus a n d Razzell (1957). However, even after repeated passage into fresh medium, these cultures did not respond significantly to the addition of lipoic acid. Since vitamin free casein hydrolysates (purchased either from Difco or Merck) are not always free from lipoic acid (Stokstad et al., 1956), this component was left out from the medium. The growth rate of these cultures was reduced by approximately 20 but still provided enough material for the assays. These bacteria now consistently responded to lipoic acid in the assays. 10 hrs old cultures were harvested at 5000 g for 5 min, washed twice with 0.03 M phosphate buffer, pH 6.5, frozen in liquid nitrogen and stored at --20 ~C before use. Determination of Lipoic Acid Lipoic acid was quantitatively determined via the pyruvate dependent oxygen uptake catalysed by the pyruvate dehydrogenase compIex in Streptococcus faecalis lO CI (Gunsalus and Razzell, 1957). The complete reaction mixture described by these authors comprises thiamine hydrochloride, adenosine, riboflavin, MnSO4 and MgSO4. In agreement with Kamihara et al. (1966) we found that thiamine, riboflavin and MnSO4 were not absolutely necessary for maximal activity. Also in our hands omission of adenosin and MgSO4 did not result in a sigvificant loss of the overall activity. Thus our assay mixture in conventional Warburg vessels contained in a final volume of 2 ml: potassium phosphate buffer, pH 6.5, 100 gmoles; glutathione, pH 6.5, 10 vmoles, fleshly prepared; and rethawed Streptococcus faecalis cells, 0.4 ml, having an optical density of 10.0 per ml at 900 nm. The centre well contained 0.15 ml of 20 700KOH and the side arm 60 ~moles of sodium pyruvate and the extracts of the organisms or DL-c~-lipoic acid dissolved as described by Gunsalus and Razzell (1957). After tipping, the oxygen consumption was followed for 40 min under air. In malay experiments, a fairly high rate of oxygen consumption, probably due to an active respiration process, was observed even in the absence of pyruvate and lipoic acid. In order to reduce the amount of endogenous substrates, the cells were always aerated for 4--6 hrs before the experiments. This treatment reduced the blank rate--measured in the presence of pyruvate but absence of lipoic acid--to about 5 ~xlO~ consumed/15 rain, and saturating amounts of lipoic acid stimulated to 60 ~xl 02 consumed/15 rain. In the linear portions of the curves, there was a linear response to graded amounts of lipoic acid in the range of 0--5 ug. Initially, only the two procedures recommended by Gunsalus and Razzell (1957) were used to release lipoic acid from the biological material. The cells were boiled in water which should release only free lipoic acid and they were autoclaved in 6 N HC1 for 2 hrs at 120~ followed by evaporating almost to dryness three times. This latter procedure should cleave the covalent bond between lipoiC acid and the e-amino group of lysine. Control experiments with added lipoic acid showed that all of the coenzyme could be redetermined after boiling, whereas only 2--5 ~o was recovered after autoclaving in 6 N HC1, in agreement with the observation of Wagner et al. (1956). Since an extrapolation from 2 - 5 ~ to 100 ~ is unreliable, the cells were also desintegrated by autoclaving in 2 N HCI in the presence of 2 bovine serum albumin which protects lipoic acid against destruction (Wagner et al., 1956). Also in this case the obtained extracts were evaporated three times in order to reduce the acid concentration. This procedure gave recoveries of some 40--50~, which agrees with the observation of Wagner et al. (1956). Before use in the Warburg assays, the obtained extracts were in all cases adjusted to pH 6.5. Determination of the Enzymic Activities The pyruvate : CO2 exchange reaction and the pyruvate synthesis catalysed by the pyruvate: ferredoxin oxidoreductase were measured in exactly the same way as described in the previous paper (Bothe et al., 1974). The overall activity of the pyruvate dehydrogenase complex was followed by the formation of NADH, which was recorded in an Eppendoff photometer at 365 nm. Using the standard reaction mixture of Schwartz and Reed (1970), the reaction was linear only in the first one or two minutes with some of the organisms. The rates given in Table 2 are compiled from this initial phase. For the activity tests, Ankistrodesmus braunii and Chlamydomonas reinhardtii were broken twice in a Ribi cell fractionator at 4000 psi, and the two Clostridium species were incubated with lysozyme (Calbiochem., 2 mg per g cells) for 30 min at 30~C. All other organisms were broken by treatment with a Branson sonifier, 5 rain at power setting 5. As judged flora microscopic observation, far less than I 0 ~ of the cells remained unbroken in all cases. The enzymes were tested in the supernatants after centrifuging for 5 min at 20000 g. Protein in the supernatant was determined according to Lowry et al. (1951), and dry weight as described by Ruppel (1962). Results Determination of Lipoic Acid The data for the lipoic acid determinations done by three different procedures are given in Table 1. In boiled extracts, the amounts were Iow, indicating that only very small levels of free lipoic acid were present in the organisms. The figures for the lipoic acid content obtained in extracts treated by 2 N HC1 plus 2 ~ serum albumin were in all cases higher than those f o u n d in the cells desintegrated by autoclaving in 6 N HC1. After extrapolating to 100~, however, the extracts treated by 6 N HC1 gave the highest concentrations in all cases, indicating that not all of the covalently bound lipoic acid was released from the organisms by autoclaving in 2 N HC1 plus 2 ~ serum albumin. Thus it appears to be difficult to give correct data for the absolute amounts of lipoic acid in the organisms. It is, however, apparent from the relative differences in the results that the microorganisms tested can be 55 H. Bothe and U. Nolteernsting: Pyruvate Decarboxylations and Lipoic Acid Content Table 1. Lipoic acid content in several microorganisms Organism Escherichia colt Azotobacter vinelandii Micrococcus denitrificans Boiling Treatment by 2NHC1 4- 2yoBSA 6 N HCI measured extrapolated = 100700 measured =570 extrapolated = 40% = 1oo70 2.7 0.35 6.6 10.1 16.5 25 4.1 4.7 82 94 0.4 13.9 35 8.6 172 Ankistrodesmus braunii Chlamydomonas reinhardtii Anacystis nidulans 0.55 2.0 5 0.8 16 0.15 0.1 3.4 3.6 8.5 9 1.1 2.7 22 54 Anabaena eytindrica Chlorobium thiosulfatophilum Clostridium pasteurianum Clostridium kluyveri 0.05 0.3 0.75 0.1 2 0.05 0.3 0.75 0.1 2 0.01 0.01 0.25 0.55 0.6 1.4 0.05 0. t 5 1 3 The figures in this table are given in 10.6 g lipoic acid/g dry weight. divided into three groups according to their lipoic acid contents. The first one comprises Escherichia colt, Azotobacter and Micrococcus denitrificans which have high lipoic acid levels, and the second one the algae Ankistrodesmus, Chlamydomonas and Anacystis nidu(ans, where moderate amounts are present. The third group of organisms, consisting of Anabaena, Chlorobium, Clostridium kluyveri and pasteurianum, are found to contain minute but still unequivocally demonstrable levels of lipoic acid, of a lipoic acid derivative or of an unknown substance which may replace lipoic acid in this assay. Determination of the Enzyme Activities The activity of the pyruvate: ferredoxin oxidoreductase was assayed by the synthesis of pyruvate from CO2, acetyl coenzyme A and reduced ferredoxin. The extracts were also checked for the presence of the pyruvate: CO2 exchange reaction. Although the pyruvate dehydrogenase complex also shows this exchange reaction (Lowenstein, 1971), the overall activity of it is negligible as compared to that catalysed by the pyruvate : ferredoxin oxidoreductase. Thus a rapid rate of the exchange reaction was suggested to indicate a catalysis by the pyruvate: ferredoxin oxidoreductase (Raeburn and Rabinowitz, 1971a). As expected, the pyruvate synthesis and the exchange reaction were present with high activities in Chlorobium, Clostridium kluyveri and pasteurianmn (Table 2). As described in detail in the previous paper (Bothe et aL, 1974), both activities can also clearly be demonstrated in the blue-green alga Anabaena cylindriea. There was a considerable rate of the exchange reaction as well as of the pyruvate synthesis both in Anacystis nidulans and Azotobacter vinelandii (Table 2). The significance of this finding is not yet clear in the moment. In all other organisms tested, no significant activity was found for either the exchange or the synthesis reaction. The pyruvate dehydrogenase complex was assayed by the NAD + reduction and was present with high activity in E. colt, Azotobacter, Micrococcus denitrificans and also in Anacystis nidulans (Table 2). This NADH-formation was completely blocked by 5 mM arsenite, which inhibits lipoic acid utilization. There was also a slow rate of an NAD + reduction in C. pasteurianum, Chlorobium and above all in C. kluyveri, but this reaction was completely insensitive to or only slightly affected by arsenite. Thus the NADHformation in these extracts is not catalysed by the dehydrogenase complex but by mechanisms which could involve the pyruvate : ferredoxin oxidoreductase and the NADH: ferredoxin oxidoreductase or the pyruvate: formate lyase and a formate: NAD + oxidoreductase. There were considerable difficulties in demonstrating the pyruvate dehydrogenase complex in the green algae Ankistrodesmus and Chlamydomonas, although the level of lipoic acid suggested its presence. No NAD + reduction was observed with low extract concentrations. More protein could not be checked due to the intense green colour of the algal extracts. Since the Arch. Microbiol., Vol. 102, No. 1 (1975) 56 Table 2. Pyruvate : CO2 exchange reaction, pyruvate synthesis and pyruvate dependent NAD + reduction in extracts from several microorganisms Organism Chlorobium thiosulfatophilum Clostridium kluyveri Clostridium pasteurianum Azotobaeter vhwlandii Anaeystis nidulans Anabaena cylindrica s eoli Microeoecus denitrificans Chlamydomonas reinhardtii Ankistrodesmus braunii Exchange reaction Synthesis NAD + reduction NAD + reduction + 5 mM arsenite 105 104 109 5.8 3.4 1.5 0.4 0.18 0.16 0.07 2.5 4.3 6.8 0.55 0.45 0.3 0.08 0.08 0.01 0.01 1.1 5.6 0.9 91.0 2.5 n.d. 35.6 20.6 n.d.* n.d.* 0.85 3.8 0.9 0.0 0.0 0.0 0.0 Rates are given in nmoles/min • protein. The preparations of the extracts and the tests are described under "Materials and Methods"; n.d. = not detected; for * see text. NAD + reduction may not be a suitable measure when an active N A D H oxidase is present in the extracts, other test systems were tried. The dismutation test for the pyruvate dehydrogenase complex (Reed and Willms, 1966) failed to give any significant activity for these two organisms. However, the pyruvate decarboxylase test using ferricyanide as the electron acceptor (Sanadi, 1969) was marginally positive. The pyruvate and ferricyanide dependent activity in these extracts was roughly 2 nmoles CO2 evolved/rain and mg protein in Chlamydomonas and 0.5 nmoles in Ankistrodesmus. No activity for the pyruvate dehydrogenase complex was found in Anabaena in either of the assays. Discussion The present communication attempts to find in several microorganisms a correlation between the lipoic acid content on one hand and the occurrence of those two enzymes which catalyse the cleavage of pyruvate and coenzyme A to CO2, acetyl coenzyme A and two electrons on the other. This attempt has to face the problem that the activity levels of the two enzymes are high in bacteria but low in the slowly growing algae--as is the case with many other enzymes. Nevertheless, it can be stated that in those organisms where the amount of lipoic acid is very low, that is in Clostridium pasteurianum and kluyveri (comp. also O'Kane, 1954), Chlorobium, the blue-green alga Anabaena cylindrica and also, as described by Peel (1960) in Peptostreptococcus elsdenii, only the pyruvate : ferredoxin oxidoreductase appears to be present. The low content of the coenzyme in these organisms suggests that it is not a cofactor of the pyruvate : ferredoxin oxidoreductase. Such a statement is not in accord with the opposite suggestion done by Sirevfig and Ormerod (1970) for the Chlorobium enzyme. Those organisms, where the levels of lipoic acid are at least ten times higher, appear to have the pyruvate dehydrogenase complex. The reaction can clearly be demonstrated in Escherichia coli, Azotobacter, Micrococcus and the blue-green alga Anacystis nidulans, and is likely present in the green algae Ankistrodesmus and Chlamydomonas. In conclusion, in those organisms tested, a low level of lipoic acid is a sufficient precondition for the occurrence of the pyruvate: ferredoxin oxidoreductase, whereas there is a one to one correspondence between the high amount of lipoic acid and the presence of the pyruvate dehydrogenase complex. However, it is not yet clear whether both enzymes are present in Anacystis and Azotobacter; a more detailed characterisation should clarify this point. Uyeda and Rabinowitz (1971) pointed out the similarities in the mechanisms of the reactions catalysed by the pyruvate dehydrogenase complex and the pyruvate: ferredoxin oxidoreductase. The replacement oflipoic acid and NAD + by a system comprising a protein bound iron--sulfur chromophore and ferredoxin has consequences for the reversibility of the pyruvate decarboxylation. The redoxpotential (Eo') of the couple pyruvate/acetyl coenzyme A is --550 mV, whereas that one of lipoic acid is --280 mV (Clark, 1960). Thus a synthesis of pyruvate from CO2, acetyl coenzyme A and dihydrolipoic acid catalysed by the pyruvate dehydrogenase complex has never been demonstrated. Since the potentials of ferredoxins are between --400 and --500 mV (Buchanan and Arnon, 1970), these carriers are electronegative enough to enable the reversibility of the pyruvate decarboxylation. Therefore, in the present communication, the synthesis of pyruvate was taken as the indicator reaction for the presence of the pyruvate :ferredoxin oxidoreductase. H. Bothe and U. Nolteernsting: Pyruvate Decarboxylations and Lipoic Acid Content As stated above, the level oflipoic acid is low in such organisms which have no pyruvate dehydrogenase complex. However, also in these organisms, some !ipoic acid or some of a lipoic acid replacing substance is consistently found in the assays with Streptococcusfaecalis. The function of the lipoic acid in these organisms is not understood. Since together with the pyruvate- also the ~-ketoglutarate dehydrogenase complex appears to be absent, one could envisage an essential function of this coenzyme in the oxidative decarboxylations occurring at the degradations of valine, leucine and isoleucine. However, having in mind that even E. coli contains a dihydrolipoamide transacetylase of unknown function (Bisswanger and Henning, 1973), one can imagine that also the above mentioned organisms "retain some secret corner in their metabolisms" (Bisswanger and Henning, 1973). Acknowledgements. The finan~cial support by the Deutsche Forschungsgemeinschaft, for the work herein reported, is gratefully acknowledged. References Bisswanger, H., Henning, U.: A new dihydrolipoamide transacetylase in Escherichia coli K 12. Biochim. biophys. Acta (Amst.) 321, 143 148 (1973) Bothe, H.: Ferredoxin und Phytoflavin aus Anacystis nidulans. Thesis, University of G6ttingen, p. 111 (1968) Bothe, H., Falkenberg, B., Nolteernsting, U. : Properties and function of the pyruvate: ferredoxin oxidoreductase from the blue-green alga Anabaena cylindrica. Arch. Microbiol. 96, 291--304 (1974) Bothe, H., Loos, E. : Effect of far red light and inhibitors on nitrogen fixation and photosynthesis in the blue-green alga Anabaena cylindrica. Arch. Mikrobiol. 86, 241 - 2 5 4 (1972) Buchanan, B.B., Arnon, D. I. : Ferredoxins: Chemistry and function in photosynthesis, nitrogen fixation and fermentative metabolism. Advanc. 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