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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.
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