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Energy Production in Anaerobic Organisms.

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additional structure aggregates (“clusters”) to be
formed by the water molecules. The problem is merely
to what extent the models assumed correspond to the
real systems.
4. Conclusions
An understanding of the solvation structure of dissolved particles depends mainly on the interpretation
of the anomalous entropy and energy effects. The difficulty of this problem is shown by the pressure dependence of the entropy of pure water in the region of
20°C. As the pressure is increased, the entropy (and
initially also the viscosity) decreases, though the general view is that H bonds are broken.
The observations associated with the motion of the
water molecules and the existence of solid hydrates
appear to lend partial support to the hypothesis of the
increase in structure, but the properties based directly
on the electronic structure of the water molecule d o
not readily fit this hypothesis. If an increase in the
structure of the water is in fact associated with the
dissolution process, it is of a different type from the
increase in structure that occurs when pure water is
cooled. The packing of the water molecules in the
solution is evidently different from that in pure water.
Received: January 7, 1969
[A 740 IE]
German version: Angew. Chem. 82, 91 (1970)
Translated by Express Translation Service, London
Energy Production in Anaerobic Organisms[**’
By K. Decker, K. Jungermann, and R. K. Thauer[*J
Biological redox processes are required for the synthesis of “energy-rich’’ compounds
which are in an enzyme-controlled equilibrium with the general energy carrier adenosine
triphosphate (ATP). The basic mechanisms of biological energy transformation are
substrate level (SLP) and electron transport phosphorylations (ETP). In anaerobic
catabolism the numerous nutrients are channelled into only a few substrate level phosphorylations; the occurrence in chemotrophic anaerobes of energy conservation coupled
to electron transport has not yet been demonstrated unequivocally. At present, the determination of the ATP turnover (YATP)in growing cells appears to be a promising method
of approaching this problem. The existing knowledge of oxygen dependent enzyme reactions and their molecular evolution provides the basis for a biochemical dejinition of
aerobic and anaerobic organisms.
1. Introduction
Biological energy production was believed by Lavoisier
to depend upon oxidations with atmospheric oxygen;
this view was held for more than a century [I]. It is now
known that about 3 x l o 9 years ago, when life first
appeared on earth [21, energy could not have been produced by combustion reactions, since our planet was still
surrounded by a reducing, oxygen-free atmosphereC2.31.
It is therefore probable that the first living organisms
were anaerobes.
[*I Prof. Dr. K. Decker, Dr. K. Jungermann,
and Dr. R. K. Thauer
Biochemisches Institut der Universitat
78 Freiburg, Hermann-Herder-Strasse 7 (Germany)
[**I Based on a lecture to the Freiburg-Sudbaden Branch of the
GDCh on February 2, 1968 in Freiburg/Breisgau (Germany).
[l] A . P . Lavoisier and P. S. de Laplace 1784: Oeuvres de
Lavoisier. Vol. 11, Paris 1864-1893; J . Loeb: Vorlesungen uber
die Dynamik des Lebens. Barth, Leipzig 1906, p. 30.
[Z] D . H. Welfe,Naturwissenschaften 57, 17 (1970).
[3] H. Urey: The Planets, their Origin and Development. Yale
University Press, New Haven 1952.
138
“Life without oxygen” still occurs on earth; it is found
wherever putrefactive processes or excessive oxygen
consumption by aerobic cells gives rise to niches of
oxygen deficiency. This anaerobic life is sustained
either by the radiant energy of thesun (phototrophy) or
by the chemical energy (chemotrophy) of oxygenindependent reactions. It is reasonable to assume that
the photosynthetic apparatus is a relatively late acquisition of anaerobes, and that the energy was originally provided by a chemotrophic metabolism. Thus,
the study of strict anaerobes has gained interest also
in connection with the evolution of cellular chemistry.
In this paper the principles of biological energy transformation will be discussed first. In view of the very
large number of potential nutrients, it will not be attempted to discuss all the known pathways of substrate utilization in anaerobes. Instead, some recently
elucidated examples will be presented t o show how the
numerous substrates (carbohydrates, amino acids) are
transformed and directed to a few substrate level
phosphorylations. Since electron transport phosphorylations have so far been definitely detected only
Angew. Chem. internat. Edit.
Vol. 9 (1970)1 NO. 2
Energy metabolism
in chemotrophic aerobes (respiratory chain [61) or in
phototrophic cells [91, the question whether they can
also occur in chemotrophic anaerobic metabolism is
of great interest at the moment, and is therefore also
an important aspect of the following discussions.
The importance of growth yield measurements for the
determination of the in vivo ATP turnover and hence
for an understanding of the relations between overall
metabolism and cell growth will also be discussed.
Finally, the molecular evolution of biological energy
transformations, and thus the position of the anaerobes
in phylogeny, will be considered. An attempt will be
made to provide a biochemical basis for the physiological definition of anaerobic organisms and their distinction from facultative and obligate aerobes.
Ana bob is m
Maintenance
and organization
energy
2. Biological Energy Transformation
Fig. I . The energy flux in metabolism.
The metabolism of living cells is an open system characterized by the constant input and output of matter
and energy, and whose chemical reaction chains are
in a steady state. It can be subdivided into catabolism
(“Energiestoffwechscl”), which provides the molecular
energy carrier adenosine triphosphate (ATP), and
anabolism (“Baustoffwechsel”), which uses the energy
of ATP for the synthesis of monomers, for poIymerizations, and for active transport (Fig. 1).
The energy metabolism is an exergonic system of
electron donating and accepting reactions or reaction
conserved in the form of “energy-rich” compounds 141,
which are in an enzyme-controlled equilibrium with
the ATP system [eq. (a)]
ATP40
(AG;
glucose
--&-+
=
-8.2 kcal/mole) &5aI
Electron acceptance
(hydrogenation)
2 pyruvatee
[*I
(a)
Energy is transformed via the ATP* system [**I in two
ways. Substrate level phosphorylations (SLP) convert
the free energy of hydrolysis (the “group potential”) 141
Electron donation
:dehydrogenation)
Fermentation
+ H 2 0 + ADP3Q + HPOie + H e
+ 2 pyruvate@ --+
+ 2 H e + 4 [HI
4 [HI
+- 6 H a +~ 24 [HI
24 [HI
2 lactate@
1
(kcal/reaction) [b]
overall process
AGL
- 47.5
ATP
Respiration
y
.
glucose
12 H2O
+
6 HCOse
+ 6 O2 --\-
ATP
ATP
(ETP)
(SLP)
Photosynthesis
HzO
hv
---f
1/2 0 2
+ 2 He + 2 ee
2eei
2 He
s
x
-+
I
2 [HI ( N C P P )
+
56.7
ATP
(ETP)
g
0
c
0
c
a
-680.2
1 2 H20
Chi
hV
--
-P
Chle
+ ee
eo + Ch18
---r
0
Chl f c p p t
[a1 S L P = substrate level phosphorylation. E T P - electron transport phosphorylation. NCPP, C P P = noncyclic andcyclic photophosphorylation.
[bl Without ATP’ formation and without radiant energy, see Tables 2 and 3.
ICI Chemosynthesis and chemotrophy are frequently used as synonyms in the English literature [8] and denote the biological production
of energy from chemical reactions alone. The term chemosynthesis is therefore a more comprehensive term than fermentation and respiration. I n the German literature, on the other hand, chemosynthesis refers only t o the production of energy from redox processes between
inorganic electron donors and electron acceptors, particularly oxygen. Chl = chlorophyll.
chains which are coupled by electron carriers. The
donating part of the process comprises the flow of
from the donor substrate to the first
carrier (usually NAD); the accepting part is the flow
from the first electron carrier to the final acceptor.
The fraction of the total energy released by the redox
processes that can be utilized by the cell is initially
Angew. Chem. internat. Edit.
1 Vol. 9 (1970) I No. 2
t41 F. Lipmann, Advances in Enzymol. I , 99 (1941).
[5] K. Burton, Ergebn. Physiol., biol. Chem. expt. Pharmakol.
49, 275 (1957).
[5a] R.C.Phillips, P.George, and R.J.Rutman, J. biol. Chemistry
244, 3330 (1969); R. A . Afberty, ibid. 244, 3290 (1969).
[*I AGA = AGOat pH 7 (all other reactants 1 M, 25 “C).
[**I The symbol A”* is used when only the turnover Or the
free energy of hydrolysis of the P,y-pyrophosphate bond of
adenosine triphosphate is meant.
139
Table 2. Dehydrogenations. Electron-donating, formally hydrogen-forming reactions of the energy metabolism.
Eq.
no.
1
Substrates
AG;
ATP*
formed/
(kcall
mole Hz) reaction
(SLP)
[a1
Products
Ref.
1. Carboxylic acids
+
acetate@ 4 H z 0 (citrate cycle)
2 acetate@ 2 H 2 0
(glyoxylate cycle)
acetate@ H2O
propionate@ 3 H z 0
propionatee 7 H20
butyratee
10 HzO
succinateze 8 HzO
butyratee 2 H2O
caproate@ 4 HzO
+
+
+
+
+
+
+
+
i
2HCO,Q+ + 4 H z
1 HCO,e
pyruvatee
+
+ 3 HZ
1 H C 0 , e + CHI
1H C O +
~ acetates + H@ + 3 HZ
6.4
+12.6
0
-2
6.8
i17.8
+43.3
+61.3
+38.1
t10.1
+i0.2
5.9
-i- 6.2
t 6.1
5.4
5.0
+ 3 H e + 10 HZ
+ 2 H @+ 7 Hz
+
+
+
+ H@+ 2 HZ
+ 2 H@+ 4 Hz
i~25.5
+37.7
1
151
151
-
+
2HQ+7H>
3HCOje-I
4 HCO3G
4HC03Q
2 acetatee
3 acetate@
1
+
5.0
CClOD
cc
151
1101
[101
[51
CClOD
BO
BO
151
151
-12.1
+13.5
- 8.3
- 8.2
OD
OD/CC
OD
OD
151
151
[51
-12.1
-10.9
-18.6
OD
OD
OD
[dl
-
OD
OD
lc1
2. a-Keto acids
+
2 HzO
pyruvatee
pyruvatee -- 6 HzO
glyoxylate@ 2 HzO
glyoxylatee 3 HzO
(dicarboxylic cycle)
a-ketobutyratee
2 H20
a-ketoglutarateze
2 HzO
oxalacetateze
3 HzO
+
+
+
+
+
+
+
+
lactate@ 2 HzO
malate20 3 H z 0
citrate3e
3 H20
isocitrate3Q 3 H20
@-hydroxyhutyrateO H20
crotonatea 2 HzO
acrylatee
3 Hz0
fumarateze 4 HzO
glycolate6 2 H z 0
+
+
+
+
+
+
+
acetatee -I- HCO,O -I H@ HL
3 HCO3e
2 He
5 Hz
formatee
HCO,@ 4- H@ Hz
2 HCO3@+ H a
2 Hz
+
+
+
+ 2.1
+
+
propionatee
HCO,G
H e -I- H2
succinateze HC03e
H@ H2
acetate@ 2 HCO,O
H@ HZ
+
+
+
+
+
+
+ +
+
+
+
+
+
l
1
1
-12.1
- 10.9
-18.6
acetates
HCO3O H@-t 2 H2
acetate@ 2 HCO3O -t He
2 HZ
succinateze 2 HCO3e
H@ 2 Hz
succmate28 -t 2 HCOse H@ 2 H:
2 acetatee
H e -I H2
2 acetatee
H e Hz
acetate8 HCO3O H@ 2 HZ
acetates 2 HCO,@ He
2 HZ
formatee HCOse
H e 2 HZ
+
i
- 8.3
- 4.1
+
+ +
+ +
+
+
I
-12.1
I
1
I
- 0.9
-
+
-
+
+ +
+ +
+ +
-
+
3.6
2.9
3.7
9.9
8.1
0.2
4.0
3.3
1
1
1
1
1
I
1.8
- 7.2
-
5.8
7.4
9.9
8.1
0.4
8.0
' 6.6
151
r51
[51
cc
cc
BO
BO
OD
CCIOD
OD
4. Aldehydes (aldoses, ketoses)
+
+
+
formaldehyde H 2 0
acetaldehyde HzO
glyceraldehyde
HzO
glyceraldehyde
glyceraldehyde
2 HzO
glyceraldehyde t 6 H20
3 ribose
ribose
glucose
glucose 4 H20
glucose 2 HzO
glucose 12 HzO
3 heptose
HzO
gluconatee
gluconatee
3 gluconatee
3 Hz0
6 gluconateQ 13 x eq. (37)
+
+ +
+ +
+ +
+
+
+
+
+
+
+
+
+
formatee H@i- Hz
acetate@ H a
HZ
glyceratee
H e HZ
pyruvate@ H e HZ
acetatee
HCO3e 2 H@ 2 Hz
3 HCO3@t 3 H@ 6 H z
5 pyruvatee
5 He
5 Hz
acetate@ pyruvatea
2 H e Hz
2 pyruvatee
2 H@ 2 Hz
2 acetate8 2 HCO3@ 4 H@ 4 Hz
acetatee
pyruvatee
HCO,@ 3 H@ -'- 3 HZ
6 HCO,@ 6 H@ 12 Hz
7 pyruvates
7 Ha 7 HZ
acetate@ pyruvatee
HC03e 2 Ha
2 Hz
2 pyruvatee
He
Hz HzO
5 pyruvate@ 3 HCOse
5 H@ 8 Hz
11 pyruvatee
3 HCO,e
8 H@ t I 1 Hz
+ (38)l
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + +
+
+
+
+
+
+
1
1
1
1
2
2
- 5.5
- 8.5
- 8.9
-15.5
-13.3
- 0.3
-13.6
-39.1
-13.4
-12.8
-13.0
0
-13.1
5
2
2
4
2
4
7
2
1
5
8
-15.1
-18.2
- 5.1
- 8.7
- 5.5
- 8.5
-
8.9
-15.5
--26.6
- 1.9
-67.8
-39.1
-26.8
-51.0
-39.0
0.3
-91.8
-30.3
-18.2
-42.0
-95.6
EM
EM
EMIOD
EM/CC
TTlEM
PK
EM
EMIOD
PKIEM
EMICC
TT/EM
PK/EM
ED
TT/EM
5. Alcohols
+
methanol
Hz0
methanol
2 Hz0
ethanol
H20
ethylene glycol
glycerol
glycerol 2 HzO
+
+
+
+ +
+
+ +
+ +
+ +
formatee
H a 2 Hz
HCOje
H e 3 Hz
acetate@ H e 2 Hz
acetatee H@ Hz
pyruvatee
H e 2 Hz
acetate@4- HCO,e
2He
+
+
+
1
1
2.6
-I- 1.8
0.8
-19.6
- 3.1
- 6.1
+
+ 3 Hz
1
I
1
2
+
5.2
5.4
-t 1.5
-19.6
- 6.3
-18.4
EM
EM/OD
-12.0
-10.9
1.0
1.0
OD
OD
St
St
+
6. Amino acids
+
+
+
+
+
2 glycine 4 H z 0
glutamatee
3 Hz0
alanine 3 HzO
leucine
3 HzO
cholinee
HzO
+
+ +
+
+ +
+
+ +
+
+ +
+
acetate@ 2 HCO3@ H@ 2 NHI@ 2 Hz
2 acetate@ HCO,@ Ha
NH4@ Hz
acetate@t HC03e
H@ NH4@ 2 H Z
isova1erateQ HCO3O H e NHI@ 2 HZ
acetatee
H@ (CH,),NH@ i Hz
+
+
+
+
1
1
1
1
1
- 6.0
-10.9
i0.5
4 0.5
+
+
151
r51
151
[il
7. Sulfonium compounds
(54)
140
1 propiothetine + 3 HzO
+ HC03@+ 2 H e + (CH,hS + 2 HZ
acetate@
I-
5.1
I
I
1 -10.1
I OD
I 1101
Angew. Chem. internat. Edit. 1 Vol. 9 (1970) J No. 2
Table 2 (continued)
Eq.
no.
I
Substrates
ATP‘
3.G:
formedl
(kcal/
mole Hz) reaction
(SLPJ
[a1
Products
AG;
(kcal/
reaction)
[a1
Route
Ibl
Ref.
8 Inorganic electron donors
2 NH.,~
NH4@ 2 H20
NH4@ 3 H20
NO20
HzO
H2S
HzS 4 HzO
S , 4HzO
S I O : ~’ 5 H20
SzO’S
3 HzO
2 F22c‘ { 2 H”
+
+
+
+
+
+
+
2 He
3 H2
Nz
NO20
3He
3 H2
NO,B
2 H e 4 H?
NO,@ Hz
S + H:
SOZe 2 H a -t 4 Hz
SOiO i- 2 H e + 3 HZ
2 S O : o i5 HZ
2SO:O .: 3 HI
2 Fe3a
Hz
+
+
+
+
+
+
+
-i 6.3
---35.0
+35.9
t38.6
2- 6.6
-L 9.2
i-10.1
.j 10.3
-1-18.4
-!-54.8
0
0
0
0
0
0
0
0
0
0
i 18.9
+105.0
-t143.5
I 38.6
i
6.6
36.9
t - 30.3
-I- 51.5
t 55.2
54.8
+
+
la1 The AG; values have been calculated from AGo(f) values and are given t o the first decimal place; they d o not include formation or consumption
Ibl E M
Embden-Meyerhof pathway; OD = oxidative decarboxylation: CC = tricarboxylic acid cycle; BO = 8-oxidation of fatty
acids; E D :. Entner-Doudoroff pathway; PK
phosphoketolase pathway; T T = transaldolase-transketolase pathway; St = Stickland reaction.
[cl Analogous t o eq. (8).
[dl Analogous to eq. (10).
[el AGo(f) of crotonate = -66.1 kcal/mole. calculated from AGo(f) of butyrate [51 and
[gl Calculated
EL of butyrate/crotonate 11 I].
[fl !iGo(f) o f acrylate - -68.4 kcal/mole. calculated from eq. (92) and AGp(f) of propionate [lo].
[ I 21.
[h] Calculated from the IC,(f) values (kcal/mole) of the following groups: -COOQ, -86.5;
by analogy with gl~ceraldehyde-3-P/3-P-glycerate
[i] Analogous t o eq. (51).
-CHO. -31.0; “CHOH, -37.0; -CHzOH, -40.5; ;CH2, 11.2; -CH,. -2.9.
of ATP’.
7
of an “energy-rich” bond into a pyrophosphate bond
of ATP; they occur in the electron-donating part of
the overall redox process. Electron transport phosphorylations (ETP) [ 6 , 7 .7a1 convert the electrochemical
potential between two redox partners in the electronaccepting part of the energy metabolism into an
“energy-rich” (pyrophosphate) bond (see Table 1).
only with substrate level phosphorylations (Table 1);
they are essentially the same for aerobic and for
anaerobic organisms. In electron acceptor processes,
aerobic organisms use oxygen as the terminal oxidant,
while anaerobes use both organic (e.g. pyruvate,
crotonate, acetaldehyde) and inorganic (e.g. H
HC038, S 0 : O ) [*I electron acceptors.
Only t h e complete elucidation of electron transport phosphorylation will show whether it is justified mechanistically t o
distinguish it from substrate level phosphorylation. Current
views [bl suggest that electron transport phosphorylation is
closely linked t o physical processes (ion gradients, membrane
potentials) a t particulate cell elements, whereas substrate
level phosphorylations a r e chemical processes catalyzed by
soluble enzymes.
The hydrogenation of oxygen in the respiratory chain
is associated with a very efficient type of ATP* synthesis (ETP). The reduction of the anaerobic electron
acceptors, on the other hand, does not appear t o allow
ATP* synthesis by ETP, since only a few electron acceptors have a sufficiently positive redox potential
(Table 5 ) .
2.1. Biological Redox Processes
The metabolic processes that occur in nature are characterized by the diversity of the substrates that can be
used for the production of energy. This is particularly
true of the electron donors, but also to a considerable
degree of the acceptors; they can be either organic or
inorganic. The formal division of the energy metabolism into electron donating (Table 2) and electronaccepting (Table 3) reactions allows assembly of the
many types of metabolism b y combination of the much
smaller number of partial processes.
Donor and acceptor processes differ in the mechanism
of energy conservation. Donor processes are associated
[6] G . Schatz, Angew. Chem. 79, 1088 (1967); Angew. Chem.
internat. Edit. 6, 1035 (1967).
171 M. E . Pullman and G. Schatz, Annu. Rev. Biochem. 36,539
(1967).
[7a] H . A . Lardy and S . M . Ferquson, Annu. Rev. Biochem. 38,
991 (1969).
[8] R. Y . Stanier, M . Doudorofl; and E. A . Adelberg: General
Microbiology. MacMillan, London 1963, p. 292.
[9] G . Hind and J. M . Olson, Annu. Rev. Plant Physiol. 19, 249
(1968); N . Pfennig, Annu. Rev. Microbiol. 21, 285 (1967);
H . Gest, A . Sun Pietro, and L . P. Vernon: Bacterial Photosynthesis
The Antioch Press, Yellow SpringsjOhio, USA, 1963.
Angew. Chern. internat. Edit. 1 Vol. 9 (1970)
/ NO. 2
Protons can also act as electron acceptors [eq. (113),
Table 31 yielding molecular hydrogen. This increases
the number of anaerobically possible electron donor
processes, since the electrons can be removed via the
hydrogenase system in any dehydrogenation. However, there is a thermodynamic price to pay for this
advantage. The endergonic formation of hydrogen
[eq. (113)] requires 4.6 kcal/rnole, and this must also
be supplied by the exergonic dehydrogenation step.
Hydrogen formation has never been observed as the
sole electron-accepting reaction of any energy metabolism.
Anaerobiosis also imposes restrictions on the electrondonating process. Thermodynamic considerations
[lo] E . R. Stadtman in N . 0. Kaplan and E . P. Kennedy:
Current Aspects of Biochemical Energetics. Academic Press,
New York 1966, p. 39.
[ l l ] J . G. Hauge, J . Amer. chem. SOC.78, 5266 (1956).
[12] H . R. Mahler and E . H . Cordes: Biological Chemistry.
Harper & Row, London 1967, pp. 207, 411.
[13] Landolt-Bornstein: Zahlenwerte und Funktionen aus Physik, Chemie, Astronomie, Geophysik und Technik. Vol. 11,
Part 7 11, p. 752, Springer, Berlin 1960.
[*I Nitrate and nitrite should not be regarded in this context a s
typical anaerobic acceptor substrates, since they were probably
formed geologically after the appearance of oxygen [15].
[I41 J . R . Pestgafe, Bacteriol. Rev. 29, 425 (1965).
[15] A . Nason, Bacteriol. Rev. 26, 1 6 (1962).
141
Table 3,
Eq.
no.
Hydrogenations. Electron-accepting, formally hydrogen-consuming reactions of the energy metabolism.
Substrates
Products
1. Carhoxylic acids
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+
+
+
+
+
+ +
+
+
formaldehyde HzO
methanol HZO
methane
2 H2O
ethanol H20
butyratee
2 HzO
2 H2O
valeratee
caproatee
2 H20
butanol
H2O
propanol
HzO
acetoin
2 H20
2,3-butanediol+ 2 H 2 0
acetone HC03Q
isopropanol HC03Q
formatee H e
HZ
formatee H a
2 H2
formatee H e
3 HZ
acetate@ H e 2 H2
2 acetatee
H e 2 Hz
H@ i2 H2
acetate@ propionatee
acetate@ butyratee
H e 2 Hz
butyratee
H e 2 Hz
propionatee
H e 2 H2
2 acetate@ 2 H e 2 H2
2 acetate8
2He
3 H2
2 acetate@
2 acetatee
He
HI
+
i5.5
- 2.6
+
+
+
+
+
+
+
+
+
-10.7
- 0.8
- 5.0
- 5.0
+
+
1
1
I(?)
1
1
1
- 5.0
1
-
1
1
2
2
I
1
2.0
- 1.2
6.3
2.0
+
+
+ 2.8
+
5.5
i51
5.2
32.2
151
151
-
1.5
-
151
10.1
10.1
10.1
3.9
2.5
12.5
4.0
8.8
151
Ibl
-
+
+
+
bl
[51
IS, 101
[cl
Icl
151
2. a-Keto acids
+
H2
pyruvatea
HzO -I- H1
pyruvatee
Hz
pyruvatee
pyruvatee
2 Hz
2 pyruvatee
2 HzO 1- Hz
2 pyruvateQ H20 2 HZ
2 pyruvatee
H ~ O 2 H~
pyruvatee acetate@ H2
pyruvatee
HC03Q H2
HCO3e
Hz
pyruvatee
pyruvate@ HC038
2 Hi
a-ketobutyrate@ 2 H2
2 HZ
oxalacetatee
acetoacetatee
H2
+
+
+
+
+
+
+
+
+
+
+
+
+
-12.3
-15.0
-15.6
- 6.9
propionatee
butyratee
succinateze
propionatee
HzO
butyratee
HzO
succinateZe
H2O
acetate@ HzO
He
(CH3)2S
propionatee
-18.2
-18.2
-20.5
-19.6
-20.0
-19.7
-18.8
-27.9
+
+
+
lactate@
ethanol HCOze
acrylatee HzO
propionatee
H20
2,3-butanediol+ 2 H C 0 3 e
2,3-butanediol
HCO,e
for.mate@
butanol 2 HC03Q
butyratee
HCO3e
malateze HzO
furnarate2e 2 H20
succinate20 2 H 2 0
H20
butyratee
succinate20
H20
P-hydroxybutyratee
+
+
+
+
+
+
+
+
+
+
+
+
-10.3
-13.6
-11.7
-15.0
-20.2
-10.2
-19.1
-22.2
+
+
- 5.0
- 4.0
-
10.3
13.6
11.7
29.9
- 20.2
- 20.4
- 38.1
- 22.2
- 5.0
- 4.0
- 24.6
- 29.9
- 31.1
- 6.9
3. a,@-Unsaturatedacids, hydroxy acids
+
acrylatee
HZ
crotonatee HZ
fumarateze H2
Iactatee H2
(3-hydroxybutyratee
HZ
malate20
glycolate@ H2
propiothetine H2
+
+
+
+
+
+
+
+
+
+
+
+
- 18.2
-
18.2
20.5
19.6
20.0
19.7
18.8
27.9
-10.7
-10.0
- 5.9
- 8.3
-
10.7
-
5.9
8.3
-27.0
-19.1
-21.1
-19.7
-
27.0
19.1
21.1
10.7
4. Aldehydes
+
+
+
1
methanol
ethanol
isopropanol
glycerol
formaldehyde Hz
acetaldehyde
HI
acetone H2
glyceraldehyde H2
+
- 10.0
-
5. Alcohols
+
+
+
+
methane
H20
ethane HzO
ethanol HzO
1.3-propanediol
methanol Hz
ethanol
H2
ethylene glycol + H2
glycerol H2
+
+
+ HzO
6. Amino acids
+
+
acetateQ NHP
8-aminovalerate
8-aminovalerate NHP
8-guanidinovalerate
NHd@
indolepropionate@ NHde
glycine H Z
proline H2
ornithinem Hz
argininee H z
HZ
tryptophan
+
+
+
+
+
-18.7
+
+
7. Inorganic electron acceptors
+
H e NAD(P)H
H2
HC03e
2 H2 H e
HCOpe
HCOne 3 HZ H e
HC03Q 4 H2 H e
2 HC03e 4 HZ H e
S + HZ
S HZ
S0ze
2 He
2 HZ
SO:e+2H@+3Hz
S0:e
Hz
SO:Q
2 He
3 Hz
S0i@+2He+4H~
S Z O : ~ 2 H@ 4 HZ
Sz0ge
2 Hb
2 Hz
+
+
+
+
+
+
+
+
+
+
+
142
+
+
+
+
+
+
+
+
+
+
H~ NAD(P)@
formatee HzO
formaldehyde
2 HZO
methanol
2 Hz0
methane 3 HzO
acetatee
4 HzO
HzS
HSQ H e
S 3 H20
HzS 3 Hz0
S0z0
HzO
S 4 HzO
H,S
4 H20
2 HzS 3 H20
2S+3HzO
+
,
+
+
+
+
+
+
+
+
+
+
+ 4.6
0.2
+ 2.7
+
+
1.8
8.1
- 6.4
- 6.5
- 6.6
- 16.0
-12.9
1.9
-10.1
- 9.2
-10.3
-14.1
-
-
+
Angew. Chem. internat. Edit.
-
-
+
-
4.6
0.2
5.4
5.4
32.3
25.6
6.5
6.6
32.1
38.6
1.9
30.3
36.8
41.3
28.2
1 Vol. 9
(1970)
No. 2
Products
qno.
Substrates
I
I
2 NHP
NZ 4 H20
NH4a 2 H 2 0
N020 1 H20
NH4a 3 H 2 0
N24 6 H z 0
+
Hi02
2 HzO
2 HzO
I
I
- 6.3
- 18.8
-63.7
-35.0
-38.5
-35.9
-53.6
-32.7
-80.7
-56.7
-191.1
-105.0
- 38.5
-143.5
-268.1
- 32.7
- 80.7
- 1 13.4
[fl Analogous
[el Analogous to eq. (81).
[d] Cf. [f], Table ?.
[c] CE. [h], Table 2.
[a] Cf. [a]. Table 2 .
[b] Analogous to eq. (69).
[h] 2 ATP’ are required for the activation of sulfate. whereas no ATP’ appears to he necessary for the reduction
[gl Cf. [el. Table 2.
t o e q . (93).
of sulfite 1141.
practically rule out saturated carboxylic acids as
donor substrates, since their dehydrogenation [Table
2, eq. (8) and (9)] is so endergonic that any coupling
with anaerobic acceptor processes with simultaneous
ATP* synthesis seems hardly possible (but see[16,171).
Similarly, the dehydrogenating cycles [see Table 2,
eq. (l),(2), and (13)] cannot serve as sources of energy
under anaerobic conditions. Furthermore, the endergonic tricarboxylic acid cycle does not allow a net
ATP* synthesis from C2 units (acetate) by substrate
level phosphorylation. Degradation of a substrate
beyond acetate is therefore feasible for energy production only if the oxidation of the reduced coenzymes
formed i n the cycle can be linked to electron transport
phosphorylation. This is not possible with the known
anaerobic acceptor systems.
If no hydrogen is liberated in the energy metabolism
of an anaerobic organism, the hydrogen balance must
be compensated internally resulting in further restrictions on the possible metabolic pathways. Whereas
aerobic metabolic processes are always intermolecular,
the anaerobic energy metabolism must often also be
intramolecular. In intermolecular redox processes the
electron-donating and the electron-accepting steps
(Table 1) are coupled only via the electron carrier,
whereas in the intramolecular redox processes both
the electron carrier and the electron acceptor act as
giucose
4
4
J
3 - phospho-
glyceraidehyde3-phosphate
I
2 [HI
I
\1
\1
coupling links (Fig. 2). Intramolecular redox processes
consequently lose one “degree of freedom”: the extent of possible dehydrogenations of the donor substrate and hence also the economic efficiency (Section
2 . 3 ) are limited by the need for compensation of the
hydrogen balance via an intermediate formed from the
donor (product coupling).
2.2. Energy-Rich Compounds
The “energy-rich’’ compounds formed in the redox
processes of the cellular metabolism are characterized
by their free energies of hydrolysis (“group potentials”), which lie in the range 6--75 kcal/mole. However, compounds leading to the formation of an “energy-rich’’ pyrophosphate bond but utilizing an electrochemical (with respect to a suitable electron acceptor)
rather than a hydrolysis potential must also be regarded as “energy-rich” in the biochemical sense; these
include substances such as NAD(P)H, FADH2,
FMNH2, certain hydroquinones, and reduced cytochromes. I n any case, the decisive factor is the ability
of these compounds to serve for ATP* formation
under the given conditions, i.e. that they exist in an
enzymatic equilibrium with the ATP* system.
Compounds important for substrate level phosphorylations in anaerobic metabolism are listed in Table 4,
while possible donors for electron transport phosphorylations are given in Table 5. It can be seen(particularly from Figure 3) that despite the large number of
nutrients available t o anaerobic organisms, there are
only six to eight substrate level phosphorylation steps
for energy conservation. Many anaerobic microorganisms must accomplish the entire ATP* formation
by a single substrate level phosphorylation, whereas
most aerobic cells have in addition to several substrate
level phosphorylations also the very effective electron
transport phosphorylation system of the respiratory
chain.
2.3. Efficiencies
Fig. 2. Product coupling in glycolysis; pyruvate as product
[16] T . C. Stadtman and H . A . Barker, J. Bacteriol. 61,67 (1951).
[171 T . C. Stadfman and H . A . Barker, J . Bacterioi. 61, 81 (1951).
Angew. Chem. internat. Edit. / Vol. 9 (1970)
1 No. 2
The production of “energy-rich’’ compounds, and
hence the possibility of energy transfer to the master
carrier ATP*, requires an overall metabolism that
143
- -*
Table 4. The “energy-rich” substrates of the ATP-synthesizing enzymes (substrate level phosphorylation)
ADP
substrate
P
ATP -b substrate (137)
ADP Pi substrate
X
ATP -L substrate X (138)
Pi = inorganic phosphate.
+
+ +
+
+
-
Type of compound
Substrate
P or substrate
in eq. (137) and ( I 38)
phosphoacyl anhydridi
acetyl phosphate
-
I
X
Eq.
carbamyl phosphate
3-phosphoglyceryl 1-phosphate
-14.9 [a] [I21
3-phosphoglycerate kinase (E.C.2.7.2.3.)
propionyl phosphate
-10.7 [hl
propionate kinase (E.C. 2.7.)
butyryl phosphate
-10.7
butyrate kinase (E.C. 2.7.)
[b]
phosphoenol ester
phosphoenol pyruvate
-13.9 [a] [I21
pyruvate kinase (E.C. 2.7.1.40.)
acyl thioester
succinyl-coenzyme A
- 9.0 151
succinate thiokrnase (E.C. 6.2.1.4.)
acyl anilide
NIo-formrltetrahydrofolate
- 6.2 [a] 1191
Nlo-formyltetrahydrofolatesynthetase
(E.C. 6.3.4.3.)
(137)
(I3’)
[a] Calculated from AG;, of ATP* - -8.2 kcallmole [eq. (a)] and the AG; values of the kinase reactions
[bl Analogous to acetyi phosphate.
Table 5 . Possible “energy-rich‘’ compounds for electron transport
pbosphorylation.
The redox potential of “energy-rich” compounds must be so much
more negative than that of the respective electron acceptor at pH = 7
that the synthesis of one mole of ATP’ becomes energetlcally possible.
This means that in a two-electron transport LIE; must he at least
200 mV (AG; = -9.2 kcallmole) between two adjacent redox partners
in an electron transDort chain.
Electron donor
Electron acceptor
acetatelacelaldehyde
ferredoxin ox./red
NADQINADH
+ H@
FADIFA DHz Ibl
FMNiFMNHz [b]
-600
-460
-420
-420
-420
-320
-280
-280
-280
-240
-220
-220
-200
-I90
-190
-140
- 70
- 10
- 15
+
+
+
30
+I70
cytochrome c FeJ@IFeZ@ 220
340
f420
f815
+970
(formulated with the concentration of the exogenous
substrates and of the products [*I) is sufficiently
exergonic r* *I [eq. (b)]:
AG’
=
AG6-t 1.4 log ([products]/[exogenous substrates])
I n order to synthesize an ATP* a free energy of -8.2
kcal/mole is needed for a reaction when carried out
reversibly. The thermodynamic efficiency -q [eq (c)] of
such an idealized in-vitro process is 100 %.
Ref.
n
S/HzS
aceloacetyl-CoA/
P-hydroxybutyryl-CoA
HCO3elacetate
HCO,QICH~
=
moles of ATP* formed per overall process
So high an efficiency cannot be reached in living cells,
which are open systems with steady state equilibria,
since not all the individual processes are reversible or
even quasi-reversible. Moreover, the stoichiometric
coupling of energy-supplying reactions and ATP*
formation leads to a “quantization” of the energy
transfer, with the result that only packets of 7-9 kcal/
mole can be utilized for energy conservation. Exergonic processes yielding substantially smaller amounts
of free energy in fact reduce the efficiency of the overall
process; this is also true of the part of the energy of
strongly exergonic reactions that exceeds 7-9 kcal/
mole (Fig. 4).
SOZBIH,S
aretaldehyde/ethanoI
pyruvarellactate
SO:@/HzS
s0:qs
g!vcine/acetatea + N H ~ @
crotonyl-CoAl
butyryl-CoA
fumaratelsuccinate
methanollmethane
NO~QINH~
NO,OINO,Q
021H20
NO~~IN,
E,, (standard conditions) against the H electrode at pH
[ a ] E:
and 25 “ C Calculated from the AG; values of Tables 2 and 3.
(b)
7
[bl The redox potentials of flavin enzymes may differ by as much as
200 mV from the values for the free coenzymes.
A theoretical derivation of the upper efficiency limit
for metabolic processes in living organisms has not yet
been provided.
Experience has shown, however, that q values of more
than 50% for overall processes are very unlikely. The
~_
[*I They include the synthesized cell material which, in the case
1181 P . P. Cohen in P . D . Boyer, H . Lardy, and K . Myrback: T h e
Enzymes. Academic Press, New York 1962, Vol. 6, p. 479.
[19] R . H . Himes and J . C. Rubinowitr, J. biol. Chemistry 237,
2903 (1962), 237, 2915 (1962).
[20] R. C. Vulentrne, Bacteriol. Reviews 28, 497 (1964).
1211 F. Lynen and 0.Wielund,Methods in Enzymol. I , 566(1955).
[22] W. M . C/ark: Oxidation Reduction Potentials of Organic
Systems. The Williams and Wilkins Comp., Baltimore 1960, p.
444.
144
of anaerobes, can usually be disregarded a s a product with little
loss of accuracy (see Table 7)
[**I In microbiological cultures, the concentration ratio of products to exogenous substrates is generally > 10-2 at t h e beginning
of a growth phase and < 102 towards t h e end. T h e effectively
utilizable energy of a metabolic process AG’ thus deviates by a t
most ‘2.8 kcal/mole from its standard free energy AG;. I t is
therefore permissible to use AGL as the mean of AG&al and
AGhnal for the assessment of the thermodynamics of an overall
metabolism
Angew. Chem. internat. Edit. J Val. 9 (1970)
No. 2
lmethanoll
glycol
I
hornocysteine valine
aspartate
IHDP-pathway]
alanine
rnethionine
glycine
gluconate-6-P
serine
cysteine
fructose-1.6-PP
tryptophan
dimethylxylulo - 5 - P
propiothetine
7 - -
crotonate rnalate
y -amino- f urnarate
butyrate lsuccinate)
citrate
purines
pyrimidines
alanine
ophan
r
1
giycerate-1,3-~~
PGK
glycerate-3-P
I
i
phosphoenol-
V
V
3-ketoacyl- CoA
carbamyl-P
I
1
formyl-
FH‘
CAK
JI
hvdrooen
1
3-alkylpyruiate
3- arylpyrljate
2-arylacetyl-
butyryl-CoA
acetyl-CoA
2-alkylacetyl-
butyrylphosphate
acetylphosphate
2-alkyl2-arylacetylacetylphosphate phosphate
i
FTS
i
formate
/BK
IAK
butyrate
acetate
2-alkylacetate
carbinate
2-arylacetate
Fig. 3. Participation of the ATP*-synthesizing reactions in the catabolism (energy metabolism) of carbohydrates, amino acids, carboxylic acids.
alcohols, and purine bases in anaerobes. H D P (hexose diphosphate) pathway; H M P (hexose monophosphate) pathway; C A K = carbamate
kinase; FTS = formylletrahydrofolate synthetase; A K = acetate kinase; P K
propionate kinase; B K ~ = - butyrate kinase; AAK - alkyl (aryl)
acetate kinase; P G K = phosphoglycerate kinase; PyrK = pyruvate kinase.
2
I
I
a
r
of a cell considerably, improved (e.g. fermentation of
glucose y -= 2, uj = 34 %; respiration of glucose y < 38,
r, < 46%).
If the pathway of an energy metabolism is known, the
-1values can be calculated from eq. (c). For anaerobic
lactic acid fermentation (Tables 2 and 3; eq. (34) + 2 x
eq. (78)] they are found to be 34.5 % (Embden-Meyerhof pathway, y = 2) and 17.3% (Entner-Doudoroff
pathway, y
1); the corresponding r, values for the
anaerobic alcohol fermentation [eq. (34) + 2 x eq.
(79)] are 30.4% and 15.2% respectively. The relations
between AGOof the catabolism, ATP* yield (y), and
cell synthesis, which are closely interrelated, will be
discussed in detail in Section 2.4.
~
a
CD
LL
h
substrates glucose
]products
ZLactate
Certain predictions concerning the course of in vivo
ATP* syntheses seem possible on the basis of thermodynamic data. The following rules are useful for the
formulation and evaluation of the energy metabolism:
1. The effectiveness of the cell metabolism depends on
its economic [y in eq. (d)], rather than its thermodynamic efficiency [q in eq. (c)].
majority of anaerobes work at efficiencies of 15 to
30%. With oxygen as an electron acceptor and a
functional electron transport phosphorylation, the
thermodynamic efficiency y1 is only slightly, the
economic efficiency y [eq. (d)]
Y
=
moles of ATP* formed
mole of substrate transformed
Angew. Chem. internat. Edit.
1 Vol. 9
(1970)
1 No. 2
2. The thermodynamic efficiency of the metabolism
of chemotrophic anaerobes does not exceed SO%, and
is usually between 15 and 35 %.
3. The transfer of energy by the ATP* system (substrate level and electron transport phosphorylations)
is stoichiometrically related to the substrate turnover
(“quantization” of energy).
145
4. If AG; of a catabolism allows the formation of only
one “energy-rich’’ compound and if a substrate level
phosphorylation is mechanistically feasible, it is more
likely than a conceivable electron transport phosphorylation.
The application of these rules will be discussed in
Section 3 in connection with some examples of anaerobic energy metabolism.
An average of 8 ATP* are required for the conversion of one
C3 unit into cell material.
An aerobic organism can make use of its full anabolic capacity by complete degradation [eq. (37) and (136)l of (8 x 30)/
38 = 6.3 c6 units for the supply of energy and 30/2 = 1 5 c6
units for the supply of anabolic precursors, i.e. using a total
of only about 20% of its catabolic capacity.
The anaerobe on the other hand would have to use [eqs. (34)
and (79)l (8 x 30)/2 = 120 c6 units for the production of
energy alone, and a further 15 for the anabolic substrate, i.e.
135 of its actual catabolic capacity.
2.4. ATP* Turnover in Growing Cells
2.4.2. G r o w t h y i e l d s ( Y s , YP, YATP)
2.4.1. L i m i t a t i o n o f G r o w t h by
Energy Metabolism
The ATP* turnover in a living cell can be measured,
if at all, only with great difficulty (e.g. by incorporation
of 1 8 0 from water into phosphate)122al. This important quantity must therefore be determined by measurement of the growth yields.
The growth yield Ys o r Yp is defined as the number
of grams 2 of cell material (dry mass) formed on
consumption of S moles of the energy substrate or
with formation of P moles of product.
The central position of ATP* in metabolism relates
the formation and consumption of this compound
closely to the total free energy turnover in the cell.
Since the intracellular concentration of ATP is of the
order of 1-5 mmoles/l, and so represents a stationary
ATP* energy of less than 0.04 kcal/g of fresh cells,
the adenylic acid system obviously has a catalytic
role. It can be calculated that ATP* must be consumed
and resynthesized at least 4000 times[*] during the
doubling of the cell mass in a growing bacterial
culture. The function of ATP* as a switchboard for
the energy turnover necessitates a kinetic equilibrium
between ATP* producing and consuming reactions:
“ATP* formation
v =
=
VATP*consumption
Vgrowth
reaction rate
The process with the smaller capacity thus becomes
the limiting factor for cell growth.
In aerobic organisms anabolism is generally ratelimiting. The energy-supplying processes can then be
matched if (a) the increased ATP concentration or the
decreased ADP (AMP) concentration have regulatory
functions; (b) ATP* energy is diverted to the syntheses
of storage material (polysaccharides, fats, poly-phydroxybutyric acid, etc.); (c) the energy-supplying
processes are uncoupled or short-circuited: either less
ATP* is formed or excess ATP* is split (“ATPases”)
with liberation of heat. In cases (b) and (c), which are
found in aerobic bacteria, there is no proportionality
between ATP* formation and cell synthesis.
In anaerobes the growth-limiting factor appears to be
mainly the generation of ATP* in catabolism. This
can be understood in view of the relation of the
catabolic to the anabolic substrate supply in aerobic
and anaerobic organisms.
This may be illustrated by the following example.
Consider an aerobic and a n anaerobic cell that have the same
enzymatic capacity and the same mechanisms (where relevant)
of anabolism and of catabolism (e.g. formation from glucose
of lactate or of COz); let the maximum anabolic capacity per
cell be arbitrarily 30 C3 units (e.g. pyruvate)/min, and the
maximum catabolic capacity 100 c6 units (e.g. glucose)/min.
[*I Assuming that 5 pmoles of ATP are present per g of wet cells
and that the average growth yield ( YATP) is 10 g of dry cells (corresponding to 50 g of wet cells) per mole of ATP*, 20000 prroles
of ATP* are required for the synthesis of 1 g of wet cells. The
ATP’ pool must therefore have been turned over at least 4000
times during the doubling or t h e cell mass.
146
If the metabolism of the substrate, and particularly
the phosphorylation reactions involved, is known, the
net gain y of ATP* (economic efficiency) can also be
expressed in relation t o the amount of substrate consumed S or product formed P.
?‘
=
moles of ATP* formed
x e o f substrate consumed
(d‘)
moles of ATP’ formed
-___
mole of product formed
(d”)
yp =
The ATP growth yield ( YATP) is thus found to be
This quantity indicates how many grams of cell material are synthesized at the cost of one mole of ATP*.
YATPis thus the coupling factor between the ATP*
turnover and the cell growth. If it were found to be a
biological constant, conclusions about unknown
metabolic pathways and in particular about the number of ATP*-forming steps (substrate level and electron transport phosphorylation) could be drawn from
yield measurements.
Significant results can be obtained from yields methods
only if very strictly defined experimental conditions
are observed E23-261. In particular, it is necessary to
~
[22a] P . D. Boyer in D. R . Sanadi: Current Topics in Bioenergetics. Vol. 2 . Academic Press, New York 1967, p. 99.
1231 W. W . Forrest in: Microbial Growth, 19. Sympos. of the
Society for General Microbiology, London 1969, Cambridge
University Press, Cambridge 1969, p. 65.
[24] T. BauchopandS. R . Elsden, J.gen. Microbio1.23,457(1960).
[25] I . C . Gunsalus and C . W. Shuster in I . C. Gunsalus and
R . Y. Stanier: The Bacteria. Academic Press, New Vork 1961,
Vol. 2 , p. I .
[26] J . C. Senez, Bacteriol. Reviews 26, 95 (1962).
Angew. Chem. internat. Edit. / Vol. 9 (1970)J No. 2
ensure that the growth is energy-limited (limited by
the energy-substrate), that the measurements are
carried out only during the active (logarithmic)
growth phase, that the metabolic balance of the energy
substrate is quantitatively obtained by measurement
of Ys and Yp, and that additives to the nutrient
medium, e.g. yeast extracts, are used for the anabolism rather than for the catabolism. The individual
differences of various organisms as to nature, structure, and composition of the proteins, nucleic acids,
and polysaccharides are compensated by the approximately equal ATP* requirements for their syntheses
(Table 6).
Y A T ~values of about 10.5 are obtained when monomers (this refers mainly to the amino acids) are supplied for cellular synthesis in the form of protein
hydrolyzates (complex medium). If no monomers are
added (minimal medium), additional energy is required for their synthesis from the energy substrate;
Table 6. ATP’ requirement of the anabolism of C.kluyveri
gy” [47-491(resynthesis of cell components in connection
with their “dynamic state”) r501, and “organization
energy” (negative entropy of cellular organization) 123,26,511.
It is assumed that the movement energy and in most
cases (particularly in non-active uptake of the energy
substrate) also the osmotic energy accounts for only a
small part of the total energy expenditure; similarly,
the turnover of the cell components in actively growing
cells appears to be unimportant. The question how
much energy a growing cell has to expend in the form
of negative entropy of organization has been treated
theoretically with varying results [23,261;little relevant
experimental material appears to be available.
Though the above-mentioned experimental conditions were only partly observed in most cases (Table 7),
the data obtained lead to the conclusion that a YATP
value of about 10.5 is generally valid for anaerobes
under energy-limited conditions and for growth on
[27,281
1
I
Contribution t o dry weight (%)
mmoles/100 mg fraction [bl
ATP*
required
I
mmoIes/100 mg dry cells
I
’
I
I
I
monomer syntheses [cl
I
monomer syntheses
I-
polymerizations
5.2
DNA
1
Lipids
1.4
5.9
-
I
i
Soluble
fraction [a]
20
0.6
0.7
7.2 Id]
5.0
-
the Y A T ~values found under these conditions are
consequently smaller (Table 7). The fact that the difference in the YATP values obtained on minimal
media is small as compared to complex media suggests
that ATP* is necessary for the production of monomers even with supplemented media.
Whereas the ATP* requirement for monomer syntheses and polymerization can be calculated (Table 6),
this is not possible a t present for the other ATP*consuming processes (Fig. l), such as movement
energy (particularly in ciliated cells), transport energy
(active uptake of moIecuIes or ions from the medium,
active transport within the cell), “maintenance ener[27] R. K . Thauer, K. Jungermann, and K . Decker, Europ. J.
Biochem. I , 482 (1967).
[28] R. K . Thauer, Dissertation, Universitat Freiburg 1968.
[29] H.H. MoustafaandE. B. Collins,J. Bacterio1.96,117(1968).
[30] W. W.ForrestandD. J. Walker,J. Bacterio1.89,1448(1965).
[31] R. W. Beck and L. R. Shugart, J.Bacteriol. 92, 802 (1966).
[32] A . J. Smalley, P. Jahrling, and P. J. Van Denmark, J.
Bacteriol. 96,1595 (1968).
1331 A . H . Stouthomer in J. R. Norris and D . W. Ribbons:
Methods in Microbiology. Academic Press, London 1969,
p. 644.
4.2
2.3
43.5
+ polymerizations (calc.)
total metabolism (exp.)
Vol. 9 (1970) No. 2
7.6
I
i
5.0
polymerizations (calc.)
Angew. Chem. internat. Edit.
4.0
RNA
i ~ l ~ l ~ l
total
monomer syntheses
I
‘
2.7
polymerizations
I
YATP
Protein
15.4
9.1
[34] M . S.Oxenburgh and A . M. Snoswell, J. Bacteriol. 89,913
(1965).
[351 W. R. Mayberry, G. J. Prochazka, and W. J . Payne, J.
Bacteriol. 96, 1424 (1968).
[36] R. E. Hungate, J. Bacteriol. 86,848 (1963).
[37] P. N . Hobson, J. gen. Microbiol. 38, 167 (1965).
[38] P. N . Hobson and R . Summers, J. gen. Microbiol. 47, 53
(1967).
[39] R.Twarog and R. S. Wove, J. Bacteriol. 86, 112 (1963).
[40] J. K . Hardman and T. C. Stadtman, J. Bacteriol. 85, 1326
(1963).
141 1 L . W. Gastonand E. R. Stadtman, J. Bacteriol.85,356 (1963).
1421 R. K. Thauer, K. Jungermann, H . Henninger. J. Wenning,
and K. Decker, Europ. J. Biochem. 4 , 173 (1968).
[43] T. C. Stadtman, Annu. Rev. Microbiol. 21, 139 (1967).
(441 A . H. Sfoutharner, Biochim. biophysica Acta 56, 19 (1962).
[45] L. P. Hadiipetrou, J. P . Gerrits, F. A . G.Teulings, and
,4. H. Stouthamer, J. gen. Microbiol. 36, 139 (1964).
[46] S.L . Chen, Nature (London) 202, 1135 (1964).
[47] A . G. Marr, E . H . Nilson, and D. J. Clark, Ann. New York
Acad. Sci. 102, 536 (1962).
[48] S.J. Pirt, Proc. Roy. SOC.(London), Ser. B 163,224(1965).
[49] N. van Uden, Arch. Mikrobiol. 58, 145 (1967); 62, 34
(1968); Annu. Rev. Microbiol. 23, 473 (1969).
1501 R . Schonheirner: The Dynamic State of Body Constituents.
Hafner, New York 1964,p. 3.
[51] H. J. Morowitz, Biochim. biophysica Acta 40, 340 (1960).
147
Table 7. Survey of methods and results of growth yield determinations f o r unicellular organisms (bacteria and yeasts) under anaerobic and
aerobic conditions.
Organism
Streptococcus
faecalis
Substrates
glucose
YS
20
Products
YP
?
Y
(theor.)
2/GI
glucose
arginine
ribose
22
10.5
21
?
glucose
glucose
arginine
33.5
30.6
18.3
?
?
measured
glucose
[a1
glucose
[bl
32
up t o 50 % SV!
T ( "C
Medium
Id1
semisynthetic
2/GI.
1 IArg.
2IRib.
1 % casein
2/GI.
2IGI.
l/Arg.
1 % casein
hydrolyzate
2 % yeast
1h1
Environ
ment
an
hydrolyzate
37
an
turb.
37
an
turb.
an
turb.
extract i
2 % peptone
glucose
mannitol
pyruvate
glycerol
58.2
64.6
15.5
24.7
80% as
4/G1.
acetate
4IMan.
I/Pyr.
2/Gly.
complex
glucose
pyruvate
21.5
6.0
?
?
21G1.
complex
glucose
glucose
galactose
maltose
glucose
33.7
32
36.4
64.5
43.4 [cl
?
2/G1.
2IG1.
2/Gal.
4/Malt.
2/G1.
1 % peptone
i yeast
extract or
0.5 % casein
hydrolyzate
S . Iacfis
glucose
40
?
21GI.
32
an
S. cremoris
glucose
32
?
2/GL
32
an
Lactobacillus
delbrueckii
glucose
19.6
?
2iG1.
L. planfarum
glucose
18.8
Leuconosfoc
mesenferoides
glucose
arabinose
14.2
16.2
Micrococcus
lacti/yticus
lactate
10.1
?
Propionibacf.
penfosacezlm
glucose
glycerol
lactate
37.5
20
7.6
?
glucose
8.3
?
1 % casein
hydrolyzate
T 0.1%
yeast extrac
-t arginine
+ CO2
37
ae
ae
ae
ae
Desulfovibrio
desu/furicans
pyruvate+
sulfate
lactate
sulfate
pyruvate t
sulfite
Escherichia coli
Ruminococcus
+
glucose
cellobiose
14.5
16.1
15.5
12.4
an
an
32
[32]
10.7
gr.
an
turb.
b
-
complex
an
3 % additives
30
turb.
b
turb.
b I
ae
-
turb.
9.4
an
complex
b
an
0.5 % casein
hydrolyzate
1/G1.
30
an
30
an
minimal
30
ae
minimal
32
an
37
an
complex
~
23.5
217
[251
!
svs
acetate
l-dodecanol
10
~-
37
33
Pseudomonas
CizB
turb.
turb.
glucose
Pseudomonos
lindneri
b l
an
lactate
S . diacefilactis
Y A T ~ Ref.
k1
[el
-
21
2IGI.
Determinatioi
!
-
b l
1251
T
h
?
turb.
1241
-turb.
8.3
1241
+
gr.
turb.
9.4
9.9
?
9.5
IlPyr.
-~
25.8
102
neasured
considered
neasured
3/Gl.
11.2
[331
~-
9/Cb.
minimal
an
turb.
1.5 % yeast
extract
an
turb.
complex
an
turb.
11.3
1361
17.8
[381
albus
Selenomonas
ruminanfium
Bacferoides
amylopiiilus
glucose
62
maltose
glycerol
I60
20
Closrridium
retanomoruhui
glutamate
Clostridium
ominobutyricum
hydroxybutyrate
aminobutyrate
148
6.8
8.9
7.6
9IMalt.
?
2/GIyc.
jetermined
iuantitatively
lot determined
iuantitatively
considered
-I
10
-
0.621
Glu.
1 % yeast
0.5/Hb
0.4 % yeast
extract
37
gr.
10.9
1391
extract
an
17.8
O.S/Ab
Angew. Chem. internat. Edit.
1 Vol. 9
(1970)
I
/ No. 2
[401
Table 7 (continued)
Organism
Substrates
Clostridiirm s p .
dimethylpropiothetine
Products
YS
yp
1
:theor.)
Medium
1 1
EnvironT ( ^ C ) ment
[dl
not determined
quantitatively
I I
0.33/
Dpt.
ethylene
glycol
7.7
Clostridium
kluyveri
crotonate
4.8
determined
quantitatively
0.5/Eth.
_
acetate
butyrate
He
1.5
n s . [h]
Methanobacillus
omelianskii
ethanol/
Gluconobacter
liquefaciens
_
_
0.75xcasein
hydrolyzate
37
_
~
an
b
b
an
_
33
0.51
Crot.
l/H@
4.8
9.6
9. I
an
_
15.4
~
b
3.1
gr.
9.1
H2
HS
butyrate
caproate
methanol
l
Y A T ~ Ref.
37
Clostridium
glycolicum
Methanosarcina
barkeri
Deter;fation1
~~
~~
ethanol
acetate
E[fl
I
I
3.3
I I
Culture
[e 1
3.3
-I
?
0.25/
~
_ Meth.
_
_
1
?
fructose
21.0
?
Aerobacter
aeroxenes
glucose
fructose
26.1
26.7
determined
quantitatively
minimal
Saccharomyces
cerevisiae
glucose
21
?
1 ?< casein
hydrolyzate
nlucose
89
glucose
108
COZ
~~
Torulopsis urilis
2/Fr.
complex
37
I
30
I
an
Ial Logarithmically growing cells.
[bl Linearly growing cells.
[cl Ccrrected for maintenance energy and substrate incorporation. [dl a n
[el b = batch; c = continuous.
[f] E = substrate incorporation in mole % of the substrate turnover.
[g] Deteranaerobic; ae = aerobic.
mination of the cell mass. turb.
turbidity measurements; gr. = gravimetric.
[h] SVS = steam volatile substances; n s . = non-stoichiometric.
~
~
complex media. A YATP of about 15 has been calculated on the basis of a component analysis of C.
kluyveri (Table 6 ) and of the known biosynthetic
mechanisms [281; this value may be taken as the upper
yield limit when minimal media are used.
2.4.3. M e a s u r e m e n t o f YATP i n t h e
C r o t o n a t e M e t a b ol i s m o f Clostridium
kluyveri
The simplicity of the energy metabolism of C. kluyveri
with crotonate and hydrogen carbonate as the sole
carbon substrates [521 made this strict anaerobe [421 a
good subject for the determination of YATP. The experimental conditions mentioned in Section 2.4.2
could be maintained.
The anaerobic disproportionation of crotonate follows eq. (g) [sum of eqs. (22) and (93) in Tables 2 and
31:
2 crotonatee
(AG;
=
+ 2 H20
+ butyratee + 2 acetate0 + H a
-26.3 kcal/reaction)
(9)
It is clear from the scheme of the energy metabolism
(Fig. 5 ) that this process is stoichiometrically coupled
with a substrate level phosphorylation, and that one
ATP* is formed by the acetate kinase reaction [eq. (h)]
per proton produced.
acetyl phosphate
+ ADP +
acetate
+ ATP
(h)
1521 R. K . Thauer, K . Jungermann, J . Wenning, and K . Decker,
Arch. Mikrobiol. 64, 125 (1968).
Angew. Chem. internat.
Edit. 1 Vol. 9 (1970) J No. 2
Fig. 5. Scheme of the crotonate fermentation in Clostridium kluyvcri.
p. -.
'
- inorganic
phosphate.
y is thus 0.5 with respect to crotonate and 1.0 with
respect to H e . Identical Y A T ~values were calculated
from measurements of the cell yield (Table 8) related
Table 8. Y A T ~ values of the crotonate metabolism of Clostridiuni
kfuyveri [a].
Substrate
or product [bl
crotonate
butyrate
acetate
He
4.8
9.6
4.8
9.1
0.5
1.o
0.5
1 .o
9.6
9.6
9.6
9. I
[a] One He) is consumed during the reduction of acetate and COz via
pyruvate t o the average oxidation state of the cell material. By measuring the growth yield per mole of H e , therefore, one obtains only the
quantity of ATP* that is consumed in the synthesis of the cell material
starting from pyruvate. Owing t o the low incorporation (3.1 %. see
Table 71, the deviation lies within the limits of the experimental error.
[bl See eq. (g).
149
to substrate consumption ( Y s ) and to product formation ( Y p ) . The same result was obtained in corresponding experiments with ethanol + acetate -t
hydrogen carbonate as the growth substrates (Table 7).
3. The Energy Metabolism of Obligate Anaerobes
The most important growth substrates of anaerobic
organisms are listed in Figure 3 . Some classes of substances (e.g. saturated fatty acids, alkanes, and isocyclic aromatic compounds, see Section 2.1) cannot be
utilized for, the production of energy in the absence of
oxygen for thermodynamic and mechanistic reasons.
The nutrients of anaerobes are mainly alcohols or
acids with additional functional groups which permit
further dehydrogenation and hydrogenation reactions.
Substrate level (and possibly electron transport) phosphorylations are inserted into the metabolism of these
substrates in order to conserve their potential energy.
In this section typical energy conserving processes in
strict anaerobes are discussed (see Fig. 3 and Tables 2
and 3).
3.1. Substrate Level Phosphorylations
The number of enzymatic processes associated with
substrate level phosphorylation has been found t o be
surprisingly small (Table 5). From the various different
substratesf5331,afew “energy-rich” intermediates(acy1CoA, formyl-FH4, carbamyl phosphate) are formed.
In anaerobes the necessity to direct the growth substrates to one of these “energy-rich’’ metabolites with
compensation of the H balance (product coupling)
requires pathways of substrate conversion quite different from those found in aerobic organisms. Examples are the anaerobic degradation of glutamic
acid [39,55,561, glycine [57-591, lysine 160-631, and the
purines 164-671.
I531 W. A . Wood i n I . C . Gunsalus and R . Y. Stanier: The Bacteria. Academic Press, New York 1961, Vol. 2, p. 59.
[54] H. A . Barker in [53], p. 151.
1551 M . Sprecher, R . L . Switzer, and D . B. Sprinson, J. biol.
Chemistry 241, 864 (1966); R . L . Switzer and H . A . Barker,
ibid. 242, 2658 (1967).
[56] A . H . Blair and H . A . Barker, J. biol. Chemistry 241, 400
(1966).
[57] B. P . Cardon and H . A . Barker, Arch. Biochem. 12, 165
(1947).
[58] M . L . Baginsk-v and F. M . Huennekens, Biochem. biophysic.
Res. Commun. 23, 600 (1966); Arch. Biochem. Biophysics 120,
703 (1967).
[59] S. M . Klein and R . D . Sagers, J. biol. Chemistry 241, 197,
206 (1966); 242, 297, 301 (1967); J. Bacteriol. 83, 121 (1962).
[60] L . Tsai and T.C.Stadtman, Arch. Biochem. Biophysic 125,
210 (1968).
[61] T . C. Stadtman and P . Renz, Arch. Biochem. Biophysics
125, 226 (1968).
[62] E . E. Dekker and H . A . Barker, J. biol. Chemistry 243,
3232 (1968).
[63] R . C. Bray and T. C. Stadtman, J. biol. Chemistry 243,
381 (1968).
150
3.1.1. A c y l a t e K i n a s e s
The process catalyzed by the acylate kinases 139,65,
68-70] [eq. (i)] is the most important reaction for the
production of energy in anaerobes. It occurs in all
Clostridia that utilize amino acids, purine bases,
carbohydrates, and alcohols, as well as in sulfatereducing bacteria. Its occurrence in methanobacteria
has not been verified. The acylate kinases can therefore be regarded as typical enzymes of anaerobic life;
their central function, as well as that of the associated
a-keto acids (pyruvate), can be seen from Figure 3.
Acylate kinases are also found in facultative aerobes[711 (e.g. E. coli); they have never been definitely
detected in strictly aerobic cells (plants, animals) 169,721.
acyl phosphate
+ ADP
$
acylate
(AG;
+ ATP
-2.5 kcal/mole)
=
(i)
Acyl phosphates are formed mainly from acyl-CoA
by phosphotransacylation [eq. (j); Pi = inorganic
phosphate].
acyl-CoA
+ Pi
ii acyl phosphate+
(AG;
CoASH
=
f2.5 kcal/mole)
(j)
In systems whose purpose is the activation of an acid (acylate
thiokinases, arninoacyl-t-RNA synthetases), the direction of
the acyl-P (e.g. acyl adenylate) synthesis is therrnodynarnically favored since two pyrophosphate bonds [eq. (m)] of
the ATP are involved.
acylate
+ ATP +
PP+H20
acylate
acyl adenylate
+ PP
+ 2Pi
+ ATP + H20
+ acyl adenylate
+ 2 Pi
(m)
3.7.2. F o r m y l t e t r a h y d r o f o l a t e S y n t h e t a s e
The formyltetrahydrofolate synthetase reaction [19,67,
73-75’ converts the bond energy of the Nlo-formylFH4 into a pyrophosphate bond of ATP [eq. (n)].
formyl-FH4
+ A D P + pi
r-
+
+
formate ATP FH4
(AG; = f2.0 kcal/mole)
(n)
[64] R . D . Sagers and J . V . Beck, J. Bacteriol. 72, 199 (1956).
[65] R . D . Sagers, M . Benzirnan, and I. C. Gunsalus, J. Bacteriol. 82, 233 (1962).
[66] J. C. Rabinowitz and H . A . Barker, J. biol. Chemistry 218,
161 (1956).
[67] J . C. Rabinowitz and W. E . Pricer, J. biol. Chemistry 237,
2898 (1962).
[68] E. R. Stadtman and H . A . Barker, J. biol. Chemistry 184,
769 (1950).
[69] I. A . Rose in P . D . Boyer, H . Lurdy, and K . Myrback, T h e
Enzymes. Academic Press, New York 1962, Vol. 6, p. 115.
[70] J. Pawelkiewicr and A . B. Legocki, Bull. Acad. polon. Sci.
11, 569 (1963); Chem. Abstr. 60, 12291 (1963).
[71] I. A . Rose, M . Grunberg-Manago, S . R. Korey, and
S . Ochoa, J. biol. Chemistry 211, 737 (1954).
I721 K. Decker: Die aktivierte Essigsaure. Enke, Stuttgart 1959,
p. 151.
[731 B. K . Joyce and R . H . Himes, J . biol. Chemistry 241, 5716
(1966); 241, 5725 (1966).
[74] L . Jaenicke, Angew. Chem. 73, 449 (1961).
[75] H . R . Whiteley and F. M . Huennekens, J. biol. Chemistry
237, 1290 (1962).
Angew. Chem. internat. Edit. J Vol. 9 (1970)1 No. 2
The ATP* formation according to eq. (n) is an extremely specialized process confined to a few anaerobes
(e.g. C. acidi urici, C. cylindrosporum) [66,671. The
“energy-rich” formyl or formimino group is formed o n
degradation of purines, and possibly also from methanol (via methyl-FH4) [Section 3.2.41 and from histidine 1541.
Formyltetrahydrofolate synthetase activity is however f o u n d
not only in anaerobes b u t also in aerobic cells including t h e
liver o f animals 1761, where it fulfills a n anabolic function.
3.2. Does Electron Transport Phosphorylation
Occur in Chemotrophic Anaerobes
Though electron transport phosphorylation in chemotrophic anaerobes is theoretically feasible, its existence
has not yet been convincingly demonstrated. Possible
candidates are 1. C. kluyveri, C. sticklandii; 2. Methanobacillus omeiianskii and Methanosarcina barkeri;
3. Desulfovibrio species. An attempt will be made to
answer this question on the basis of the existing experimental data.
3.1.3. C a r b a m a t e K i n a s e
ATP* formation from carbamyl phosphate [18,77,781
[eq. ( o ) ]is associated with the degradation of arginine
( S . faecalis) 124,291 and possibly of pyrimidines (Zymobacterium oroticum) [541. However, this reaction has
not been found to be the sole ATP* source in any
organism.
+
carbamyl phosphate -t ADP
ATP
(AC;
=
+ carbamate
-2.0 kcal/mole)
(0)
As in the case of acylate kinases [eq. (i)] and of the
acyl thiokinases, a different pathway has also been
provided in nature for the anabolic formation of
carbamyl phosphate [eq. (p)].
T h e enzyme carbamyl phosphate synthetase requires Nacetyl glutamate a n d possibly biotin [79,79a] a s cofactors,
uses glutamine instead o f NH3, consumes t w o moles o f
ATP*, and so leads t o t h e practically irreversible formation
of carbamyl phosphate [791.
2 ATP
+ CO2 + glutamine + 2 H 2 0
carbamyl phosphate
i
.
+ glutamate + 2 ADP + Pi
(p)
3.1.4. P h o s p h o g l y c e r a t e K i n a s e a n d P y r u v a t e
Kinase
3.2.1. T h e E t h a n o l - A c e t a t e M e t a b o l i s m of
C l o s t r i d i u rn k l u y ver i. R e d u c t i o n 0 f
Crotonyl-CoA t o Butyryl-CoA
C. kluyveri grows on ethanol, acetate, and C 0 2 with
formation of butyrate, caproate, and hydrogen 1801.
The condensation of ethanol and acetic acid to form
butyric acid (and caproic acid) [eq. (45) + eq. (69) =
eq. (s), and 2 x eq. (45) + eq. (69) + eq. (71) = eq. (t)]
was long regarded as a process that should involve
electron transport phosphorylation, in view of the
absence of any recognizable substrate level phosphorylation [10,81,821.
ethanol
+ acetate
2 ethanol -6acetate
i
.
butyrate
(AC;
+ H20
-8.6 kcaiireaction)
(s)
+ caproate + 2 H2O
(AG; = -17.2 kcal/reaction)
(t)
=
Electron transport phosphorylation was thought to
be coupled t o the reductive formation of butyryl-CoA
[eq. (u>l.
crotonyl-CoA
+ H2
+ butyryl-CoA
(AGA = -18.6 kcal/reaction)
(u)
I n carbohydrate fermentations 1531 the enzymes phosphoglycerate kinase (PGK) and pyruvate kinase
(PyrK) participate in ATP* synthesis by substrate level
phosphorylations [eq. (9) and (r)]. Pyruvate kinase
does not lead to a “de n o w ” synthesis of ATE’*;
rather, the phosphate needed for carbohydrate activation (e.g. hexokinase reaction) and derived from
ATP* is transferred back to ADP.
+
1.3-phosphoenolpyruvate
ADP +
3-phosphoglycerate ATP tAC;
+
5
-6.7 kcal/mole)
(9)
-5.7 kcal/mole)
(r)
-
Phosphoenolpyruvate T A D P
pyruvate i ATP (AC;
=
Only these two enzymes of anaerobic carbohydrate
catabolism still have a n important role in aerobic
energy metabolism.
[76] L . Jaenicke and E. Brode, Biochem. 2. 334, 108 (1961).
[77] M . E . Jones and F. Lipmnnn, Proc. nat. Acad. Sci. USA 46,
1194 (1960).
[78] M . E. Jones, Methods in Enzymol. 5 , 903 (1962).
[79] V . P . Wellner, J . I. Santos, and A. Meister, Biochemistry 7,
2848 (1968).
[79a] R . B. Hkston and Ph. P. Cohen, Biochemistry8,2658 (1969).
Angew. Chem. internat. Edit.
i
Vol. 9 (1970) No. 2
The electrons could be transferred to a flavoprotein
via a pyridine nucleotide. Electron transport phosphorylation is theoretically conceivable in such a
sequence, since the reduction of flavoproteins by
pyridine nucleotides in the respiratory chain is also
associated with phosphorylation. The detection of
such a process would thus be of fundamental importance, particularly since the reduction of crotonylCoA has been found in a soluble enzyme systemt82JHowever, the discovery that the energy metabolism is
associated with the formation of He, H2 gas, and
ATP* in stoichiometric amounts [eq. (i) and (j)] and
that this ATP* synthesis is sufficient for growth (see
section 2.2) ruled out C. kluyveri as a candidate for
electron transport phosphorylation [42].
The quantitative data on the ethanol-acetate fermentation, together with the results of the dispropor[80] B.T. Bornstein and H. A . Barker, 3. biol. Chemistry 172,
659 (1948).
[81] H. A . Barker: Bacterial Fermentations. Wiley, New York
1956, p. 95.
[82] C . W . Shuster and I . C. Cunsnlus, Federation Proc. 17, 310
(1958).
151
Ix-yi
:1;‘
Ixtl)
acetaldehyde
NAoH N]AOH
acetaldehyde
=
[ , ~
J
12x1
~
t-NAOPH
121
NAOH
ix-11
-11
1
capronate
/ x i 1 1 ethanol t Ix-y-11 acetate%-lx-ZyI butyrateo+iyi c a p r ~ n a t e ~ t l x11-H,OtH@
t 2H,
lYl
1
Fig. 6. Scheme of the ethanol-acetate fermentation in Clostridrum klrryveri. The factors x and y take into account the non-stoichiometric
coupling of fatty acid and ATP’ synthesis and the growth phase dependent reIation of butyrate and caproate formation [42]. At the
beginning of the growth phase x % 5 and y % 1.
tionation of crotonate [eq. (g)], also allow an interpretation of the coupling mechanism between the
energy-supplying and the ATP*-synthesizing processes, as well as an explanation of the acetate requirement of C. kluyveri [42,82a1 (Fig. 6).
The dehydrogenation of acetaldehyde to acetyl-CoA
is the only individual reaction of the catabolism that
is linked to a de novo formation of an “energy-rich’’
bond. The right-hand part of Figure 6 shows, however,
that the greater part of the “active acetate” is consumed in the butyrate synthesis.
The ATP* synthesis by substrate level phosphorylation can only be associated with the process shown in
the left-hand part of Figure 6. However, there is no
organic hydrogen acceptor available in this reaction
sequence. The free energy of the dehydrogenation of
acetaldehyde can therefore be converted into ATP*
energy only if electrons react with protons to form
hydrogen. Formation of H2 from NADPH has been
demonstrated in a soluble system of C. kluyveri
(Fig. 8 ) [83,841.
It is not immediately obvious from Figure 6 why
C. kluyveri cannot grow with the reactions in the lefthand half of the diagram alone, without the need for
the extensive fatty acid formation. However, the
formation of acetate and hydrogen from ethanol
[eq. (45)] is endergonic, and alone cannot be asso[82a] S. Schoberth and G . Goitschalk, Arch. Mikrobiol. 65, 318
(1969).
[83] K . Jungermann, R . K . Thauer, E. Rupprecht, C. Ohrloff,
and K . Decker, FEBS Letters 3, 144 (1969).
I841 R . K . Thauer, K . Jungermann, E. Rupprecht, and K.Decker,
FEBS Letters 4, 108 (1969).
152
ciated with a net ATP* synthesis. The synthesis of
butyrate (or caproate) from ethanol and acetate
[eq. ( s ) and (t)], on the other hand, is exergonic, though
not sufficiently so, when the -q values are taken into
account (see Section 2.3), for the stoichiometric formation of one ATP* per butyrate formed. The fatty
acid synthesis must therefore be repeated several
times, like a “pump mechanism”, to provide sufficient free energy for an ATP* synthesis that is physiologically possible (3 < 35 %). The CoA transferase
system [eq. (v)] is responsible for this non-stoichiometric energy coupling.
butyryl-CoA
+ acetate +
butyrate
+ acetyl-CoA
(AG;
= 0)
(V)
The higher group potential of the butyryl-CoA resulting from its exergonic formation is transferred to
the acetyl-CoA pool by reaction (v) and so allows a
thermodynamically feasible ATP* synthesis [eq. (w);
cf eqs. (i) and (j)].
acetyl-CoA
+ ADP + Pi
$
ATP
+ acetate + CoA
(AGL = 0)
(w)
Since the different group potentials of the CoA-thioesters are reflected in their steady state concentrations
(Fig. 7), the non-stoichiometric relation of butyrate,
caproate, and ATP* syntheses can also be explained
by eq. (v). Under growth conditions, the “pump
mechanism” is repeated approximately five times per
mole of ATP*, leading to a thermodynamic efficiency
of about 20 %.
Angew. Chem. internat. Edit. 1 Vol. 9 (1970)/ N O .2
2
H,
butyrateQ
capionate@ butyrate@ acetate@ HQ
butyrate@
r
1.
A
I
f
t
t
~
60
7/////
30
0
h
I
I
2 ethanol
j
I
j
ethanol
acetate@
V
2 ethanol
acetate@
1
ethanol
acetate@
acetate@
metabolic fluxFig. 7. Energy profile of the metabolites of the ethanol-acetate fermentation of Clostridium kluyveri. Note the increase in the
group potential AG; of acetyl-CoA with each “cycle” of the fatty acid synthesis as result of the increase in the potential
difference A [AGo(f)] with respect t o ethanol.
The free energies of formation AGo(f) of coenzyme A, Pi. and HzO are accounted for in the calculation of the differences.
EtOH = ethanol; AcH =- acetaldehyde: Ac = acetyl; AcAc = acetoacetyl; HOBut -= P-hydroxybutyryl; Crot = crotonyl:
But = butyryl; Cap = caproyl. Overall metabolism:
6 ethanol i 3 acetateG + 3 butyratee i1 caproate@ -!- 4 HzO i- H@
2 H2.
+
The necessity of synthesizing C 4 and c 6 fatty acids
also explains why C. kfuyveri requires acetate as a
substrate and cannot grow on ethanol alone.
The main assimilatory reaction of C. kluyveriI52351 is
the ferredoxin (Fd)-dependent pyruvate synthesis 186,871
[eq. (x)] (see also Fig. 8).
acetyl-CoA
+ Fd:z + 2H@+ HCOF
+ pyruvate”
+ CoA + Fd,, + H 2 0
3.2.2. T h e A l a n i n e - G l y c i n e M e t a b o l i s m i n
C. s t i c k l a n d i i . R e d u c t i o n of G l y c i n e t o
A c e t a t e a n d NH3
Some clostridia can obtain their energy from a redox
process in which one mole of an amino acid is dehydrogenated and 2 moles of another are reduced
(Fig. 9) 1541.
(x)
This acetate and COz assimilation has no effect on the
total carbon balance (Table 7).
SL P
It is interesting to note that in C. kluyveri, the reduction of ferredoxin (Eh = -420 mV) is effected not by
electron donors with a more negative redox potential
but by pyridine nucleotides (Ei = -320 mV) (Fig. 8).
ATP ~
-
acetylphosphate
PATP
7
acetyl2 acetate
acetate
/b74291
NAOP @t H @
NAOH
pyruvate
Fig. 9. Scheme of a Stickland reaction (alanine-glycine metabolism of
Clostridium sticklandii).
SLP = substrate level phosphorylation: ETP = electron transport
phosphorylation.
The dehydrogenation is associated with a substrate
level phosphorylation. In Clostridium sticklandii and
Clostridium lentoputrescens
Fig. 8. Ferredoxin-dependent reactions in C . kluyveri. The reduction of
Fd by NADPH is activated by NADQ and inhibited by NADH, while
the reduction of Fd by N A D H is activated by acetyl-CoA.
[85] K . Julirngermann, R . K . Thnner, and K . Decker, Europ. J .
Biochem. 3, 351 (1968).
1861 I . G. Andrew and J . G. Morris, Biochim. biophysics Acta
97, 176 (1965).
1871 G . Gottschalk and A . A . Chowdhury, FEBS Letters 2, 342
(1969).
Angew. Chem. infernat. Edit.
1 Vol. 9
(1970)
No. 2
glycine
+ NADH + H@
--f
+
+
acetate0 NH,@ NAD@
(AG; = -14.4 kcal/mole)
(y)
the reducing system [eq. (y)] also appears t o be
coupled with an ATP* formation[8Bx891.
[W T. C . Stadtman, Biochem. Z . 331, 46 (1959).
1891 T . C.Stadtman, Arch. Biochem. biophysics Z13, 9 (1966).
153
The details of this process, which involves several protein
fractions and cofactors, are not yet known. Since this phosphorylation is sensitive to uncoupling agents, the presence of
electron transport phosphorylation was postulated. If the
reductive process were coupled to an electron transport phosphorylation, which should proceed stoichiometrically, the
thermodynamic efficiency (q) of the catabolism would be
about 65 %. Yields as high as this make such a reaction unlikely. Y values have not yet been determined owing to the
complicated nutrient requirements of these organisms. It is
not yet possible to decide whether electron transport phosphorylation is involved in the reduction of glycine.
+ 2 acetateo
(AG;
=
+ Ha + CH4 + HxO
-29.3 kcal/reaction)
(2)
Since the available results[9oJindicate that acetate in eq. (45)
is not formed via acetyl-CoA, the course of the ATP* synthesis in this organism is unknown. Moreover, it has recently
been reported that t h e cultures described as M . omefianskii
are a symbiotic association of two species of bacteria, one of
which has a metabolism according to eq. (49,and the other
a metabolism according to eq. (117)[911. It is thermodynamically impossible, however, for an organism to maintain its
energy metabolism by means of the endergonic reaction eq.
(45) alone. Certain observations indicate that the organism
growing on COz and Hz also requires acetate for growth [911.
It is not possible at present to give an interpretation of the
metabolism of " M . omelianskii", and hence of the problem
of electron transport phosphorylation.
3.2.4. T h e M e t h a n o l M e t a b o l i s m i n
M e t h a n o s a r c i n a b a r k e r i.
R e d u c t i o n o f CH3OH t o CH4
Methanosarcina barkeri forms methane a n d C02 by a
disproportionation of methanol [3 x eq. (104) + eq.
(44) = eq. (aa)][431.
4 CHxOH
--t
3 CH4
+ HCOF + H e + H 2 0
(AG;
=
-75.5 kcal/reaction)
(aa)
O n t h e basis of an experimental Ys value of about 3.3,
corresponding t o y = 0.3, one mole of ATP* would
be formed per 3-4 moles of methanol consumed;
ATP* synthesis could most likely occur in the dehydrogenation of methanol via the formyltetrahydrofolate synthetase reaction [eq. (n)]. Since a n additional
electron transport phosphorylation in the reduction of
CH30H t o CH4 should give a y value of 1.0, there is
no reason t o postulate a n electron transport phosphorylation in this fermentation.
[90] W. J. Brill and R . S . Wolfe, Federat. Proc. 24, 233 (1964).
[91] M . P. Bryant, E. A . Wolin, M . J . Wolin, and R . S. W o v e ,
Arch. Mikrobiol. 59, 20 (1967).
154
+ SO:@
2 lactate@
4 pyruvate"
The metabolism of the methane-forming organisms
presents some mechanistically and thermodynamically
interesting problems. The formation of methane from
C02 and ethanol is, as a whole, a n exergonic process
[2 x eq. (45) + eq. (117) - = eq. (z)].
+ HCOY
In these organisms 126,921, the dehydrogenation of
lactate (or pyruvate) t o acetate is coupled with the
reduction of inorganic sulfate [2 x eq. (17) + eq.
(125) = eq. (bb) o r 4 x eq. (10) + eq. (125) = eq. (cc)].
+
+
2 acetate"
2 HCO?
H ~ S
(4G; = -40.6 kcal/reaction)
[bb)
4 H ~ O--f
4 acetates
4 HCOF H2S
(AG; = - 4 5 . 2 kcal/reaction)
(cc)
--f
or
3.2.3. T h e E t h a n o l - C O 2 M e t a b o l i s m i n
Methanobacillus omelianskii.
R e d u c t i o n o f C 0 2 t o CH4
2 ethanol
3.2.5. T h e L a c t a t e - S u l f a t e M e t a b o l i s m i n
D e s u 1f o v i b r i o O r g a n i s m s .
Reductions of Sulfate t o Sulfide
+ SO:"
+
+
+2 He
The eight electrons for the reduction of SO:' t o S2e
are provided by the dehydrogenation of lactate
(2 moles) or pyruvate (4 moles). The formation of
acetate is accompanied by a substrate level phosphorylation, so that 2 ATP*/mole of reduced SO:'
are formed with lactate as the electron donor, and
4 ATP*/mole of reduced SOY with pyruvate. The
reduction of the sulfate is preceded by its activation
[eq. (dd)] [141.
ATP
+ sulfate
+ adenosine 5'-phosphosulfate i- pyrophosphate
(dd)
Pyrophosphate is presumably hydrolyzed [eq. (l)] also
in these bacteria, so that the activation of SO:e is
associated with a net consumption of 2 ATP*. ATP*
synthesized by substrate level phosphorylation would
be available for the anabolic requirements of the
organism only in the presence of pyruvate, and not of
lactate. However, determination of Y A Twith
~
lactate
and pyruvate a s substrates gave similar values between
9 and 10 1261. Since Desulfovibrio has a cytochrome c3,
which takes u p the electrons from the dehydrogenation
of pyruvate, and since autotrophic growth has been
reported [931, electron transport phosphorylation has
been postulated t o occur. This hypothesis is supported
by reports 194,951 of a phosphorylation coupled t o the
reduction of sulfate in cell-free extracts of D.gigus and
its uncoupling by substances such as 2,4-dinitrophenol, which also inhibit oxidative phosphorylation.
Autotrophy, however, has been questioned 196-981;
moreover, all the substrates and products involved in
the energy metabolism have not been determined
simultaneously. The stoichiometry of eqs. (bb) a n d (cc)
[92] J. R . Postgare and L. L. Campbell, Bacteriol. Rev. 30, 732
(1966); P. A . Trudinger, Advances microbial Physiol. 3, 11 1
(1969).
[93] K . R . Butlin, M . E. Adams, and M . Thomas, J . gen. Microbiol. 3, 46 (1949).
1941 H . D. Peck, J. bioi. Chem. 235, 2734 (1960).
1951 H . D. Peck, Biochem. biophysic. Res. Commun. 22, 112
(1966).
[96] J . R . Postgare, 2. allg. Mikrobiol., Morphol., Physiol.,
Genetik 6 k o l . Mikroorganismen I, 53 (1960).
[97] B. J . Mechalas and S . C . Rittenberg, J. Bacteriol. 80, 501
(1960).
[98] Yu. 1. Sorokin, Nature (London) 210, 551 (1966).
Angew. Chem. internat. Edit. 1 Vol. 9 (1970) 1 No. 2
has thus not been experimentally verified. Though the
prevailing evidence points to electron transport phosphorylation in Desulfovibrio, conclusive proof has not
yet been presented.
4. Position of Anaerobes in Phylogeny
Considering important catabolic and anabolic mechanisms an attempt will be made to provide a biochemical
basis for the classification of anaerobes and aerobes
and for the definition of the principal types of metabolism.
4.1. Molecular Evolution
The first life foi.ms must have arisen at a time when
the earth was still surrounded by a reducing atmosphere r2,31. The nutrients available to these primordial anaerobes were simple abiogenic organic
compounds such as amino acids, alcohols, short-chain
substituted fatty acids, purine bases, and carbohydrates 1991. These chemoheterotrophic organisms
probably obtained their energy by substrate level
2
The change to the oxygen atmosphere of today is
generally explained by the biological photolysis of
water (of the plant photosynthesis type[*]). The most
recent mechanisms of biological energy transformation may therefore be considered to be the chemoheterotrophic and chemoautotrophic redox processes
with oxygen as the terminal electron acceptor (respiration, see below), and the aerobic organisms are therefore the most recent organisms.
Remarkable similarity exists between the energyconverting systems in respiration and i n photosynthesis; both have particulate electron transporting
cytochrome systems, which are coupled to ATP*
synthesis. It may therefore be assumed that they did
n o t develop independently, but that one arose from
the other. The evolution of the respiratory from the
photosynthetic apparatus can easily be pictured since
the photosynthetic electron transport effected by
cytochrome could in principle have been diverted to
oxygen by acquisition of a single autoxidizable enzyme, i. e. cytochrome oxidase (Fig. 10). Cytochrome
oxidase, Warburg’s “Atmungsferment”, must therefore be regarded as the specific enzyme of the aerobic
energy metabolism.
The enzyme class of oxygenases may be regarded as an
even more recent acquisition in the phylogenetic
scheme (Fig. TO). They are typical enzymes of aerobic
O x y g e n a s e s
i
though still in the anaerobic phase. The first photoautotrophic (still anaerobic) organisms then evolved
from these photoheterotrophic organisms.
Itrue) Oxygenases I E C i 13 1
Hydroxylases I mixed tunctlon oxygenases, E C 1 14 I
I,
\,COOH
.... ” .
0
(cosubstrate,,l
0 x i d a s e s
i
Cytochrome oxidases
IE C 1 9 3 1
Fig. 10. Reactions of oxygenases, hydroxylases, and oxidases. Oxygenases introduce both atoms, and hydroxylases only one atom
of molecular oxygen Into a substrate. Oxidases require molecular oxygen as a n electron acceptor; oxygen is not introduced into
the substrate.
phosphorylations (fermentations, see below). The
ability to utilize the light energy of the sun (photosynthesis, see below) via electron transport phosphorylation is believed to have developed later,
I991 A. 1. Oparin: Genesis and Evolutionary Development of
Life. Academic Press, New York 1968; S. W.Fox: T h e Origins
of Prebiologicai Systems. Academic Press, New York 1965.
Angew. Chem. internat. Edit. J Vol. 9 (1970)
1 No. 2
anabolism, but are also involved in catabolism ( e . g .
aromatic compounds) in a few cases[99al.
-___
[*I Water cannot act a s the electron donor in bacterial photosynthesis, and consequently n o oxygen is formed.
[99a] 0. Hayaishi, M . Kutugiri, and S . Rothberg. J. biol.
Chemistry 229, 905 (1957); 0. Huyuishi, Annu. Rev. Biochem.
38, 21 (1969).
155
4.2. Anaerobes-Aerobes
The usual physiological classification of organisms
into anaerobes and facultative and obligate aerobes
was based on the ability to grow in the absence and/or
in the presence of oxygen 11001. Current knowledge of
oxygen-dependent enzyme processes and their molecular evolution now provides a means of substantiating
these definitions on the basis of the metabolic chemistry.
Anaerobes are defined as organisms that do not possess
cytochrome oxidase and oxygenases. Facultative
aerobes have an oxygenase-independent metabolism,
and obtain their energy both by oxygen-dependent
(cytochrome oxidase) and by oxygen-independent
(pyridine nucleotide regenerating dehydrogenases or
reductases) redox processes. Obligate aerobes are
organisms that depend in their energy metabolism
always upon cytochrome oxidase, and usually in their
anabolism also upon oxygenases. This group includes
organisms whose anabolism is only indirectly oxygen
dependent. These organisms grow only when supplied
with substances that are biosynthesized in oxygenasecatalyzed reactions [100aI. Examples of such compounds
are polyenoic acids [loll, steroids [102,103J, and phycobilins[*J(1041, and for most organisms also monoenoic acids and pyridine nucleotides.
According to this definition, brewers yeasts (e.g.
Saccharomyces cerevisiae) are obligate aerobes, enterobacteria (e.g. Escherichia coli) are facultative aerobes,
and the lactobacilli (e.g. Lactobacillus plantarum) are
anaerobes. Saccharomyces can grow in the absence of
oxygen; under anaerobic conditions, however, it
requires the addition of steroids and unsaturated fatty
acids 1101 1051. “NO life without oxygen” is valid for
these organisms, though Pasteur’s experiments on
anaerobiosis with yeasts had helped to cast doubt on
this thesis. E. coli has no oxygenases [ l o l l , and can be
grown in the absence of oxygen. In the presence of
oxygen, a cytochrome oxidase dependent metabolism
is induced [*061, which allows higher growth yields and
a shorter generation time; E. coli must therefore be
regarded as a facultative aerobe. Lactobacilli have
[loo] R. H . McBee, C. Lamanna, and 0. B. Weeks, Bacteriol.
Reviews 19, 45 (1955).
[100a] D . E. Hughes and I. W. T.Wimpenny, Advances microbial
Physiol. 3, 197 (1969).
[loll J. Erwin and K . Bloch, Science 143, 1006 (1964).
[lo21 0. Hayaishi, Bacteriol. Reviews 30, 720 (1966).
[lo31 E. J . Corey, W . E. Russey, and P. R. Ortizde Montellano, J.
Amer. chem. SOC. 88, 4750 (1966).
[*I The biosynthesis of the phycobilins has not yet been fully
elucidated. It probably proceeds, like the synthesis of the bile
pigments, via a tetrapyrrole ring, which is opened in a n oxygenase-catalyzed reaction 11041.
[I041 R. Troxler and L . Bogorad in T. W . Goodwin: Biochemistry of Chloroplasts. Academic Press, London-New York 1967,
p. 421; R.Tenhunen, H . S . Marver, and R. Schmid, J. biol.
Chem. 244, 6388 (1969).
[lo51 A . Uhl in F. Re$, R. Kautzmann, H . Liiers, and M . Lindemann: Die Hefen. Verlag Carl, Niirnberg 1960, p. 252.
11061 J . W. T. Wimpenny and D . Lloyd in P. M . Meadow and
S . J . Pirt: Microbial Growth. Cambridge University Press,
Cambridge 1969, p. 161 and p. 299, respectively.
156
neither cytochrome oxidase nor oxygenases [1071, and
must be regarded as anaerobes; the same is true of the
few strains that are capable to some extent of a cytochrome-independent “flavoprotein respiration” catalyzed by constitutive or oxygen-inducible N A D H
oxidases and N A D H peroxidases [log].
The anaerobic lactobacilli and propionibacteria occupy a n
exceptional position. Some of the lactobacilli form nonheme
catalases (pseudocatalases) or, in the presence of exogenous
porphyrins, heme catalases L107,1091. I n propionibacteria, in
addition to cytochromes and catalases 11101, one also finds all
the enzymes of the tricarboxylic acid cycle “111, which is responsible for the terminal oxidation of the carbon substrate
in aerobes. These properties, which are unusual for anaerobes,
suggest that the lactobacilli and the propionibacteria have
arisen from facultative aerobes by losses of different extent in
the cytochrome system, i.e. by reversion to an earlier stage of
development 11071.
Animals, higher plants, algae, protozoa, and fungi
require steroids and polyenoic acids for cell synthesis,
and the blue-green algae require polyenoic acids and
phycobilins. All eucaryonts and the blue-green algae
of the class of procaryonts are therefore obligate
aerobes.
Bacteria require neither steroids [I121 (but see also r1131)
nor polyenoic acids [1011 or phycobilins c1043, and form
pyridine nucleotides and monoenoic acids in oxygenase-independent reactions. Anaerobes and facultative aerobes can therefore occur only among the
bacteria. Even here, however, some obligate aerobes
are found which synthesize pyridine nucleotides
(Xanthomonas pruni) [I141 and monoenoic acids (Bacillus megaterium, Micrococcus lysodeikticus, Mycobacterium phlei, and Corynebacteria) 11153 only with
oxygenases (Table 9).
If lactobacilli and propionibacteria are considered to
be revertants, then the clostridia, the methaneforming, and sulfate-reducing bacteria, as well as the
purple and green sulfur bacteria, are the true anaerobes. Since autotrophy developed from heterotrophy
in evolution, the metabolism of the autotrophic sulfur
bacteria is hardly akin to that of the primordial anaerobes. The sulfate-reducing bacteria represent a
higher stage of development in view of their cytochrome content. There remain therefore the clostridia
and the methane-forming bacteria, whose metabolism
most closely resembles that of the primordial anaerobes.
[lo71 R . Whittenburj, J. gen. Microbiol. 35, 13 (1964).
[lOS] M . I. Dolin in I.C.Gunsalus and R . Y.Stanier: The Bacteria
Academic Press, New York 1962, Vol. 2, pp. 319, 425.
[lo91 M . A . Johnston and E. A . Delwiche, J. Bacteriol. 90, 347
(1965); 90, 352 (1965).
[110] C . W.Moss,V.R.DoweNjr., V.J.Lewis, and M.A.Schekter,
J. Bacteriol. 94, 1300 (1967).
[ l l l ] E.A. De1wicheandS.F. Carson, J. Bacteriol. 65, 318 (1953).
11121 K. Bloch, Angew. Chem. 77, 944 (1965).
[I131 K . Schubert, G. Rosa, H. Wachtel, C. Hiirhold, and
N . Ikekawa, Europ. J. Biochem. 5, 246 (1968).
[114] R. E . Saxton, V . Rochn, R. J . Rosser, A . J . Andreoli, M .
Shimoyama, A . Kosaka, J . L . R. Chandler, and R. K. Gholson,
Biochim. biophysica Acta 156, 77 (1968).
I1151 A. J. Fulco and K . Bloch, Biochim. biophysica Acta 63,
545 (1962).
Angew. Chem. internat. Edit.
Vol. 9 (1970)
NO. 2
Table 9. Occurrence of cytochrome oxidases and oxygenases in living organisms.
I
1
I
cytochrome
oxidases [a]
oxygenases [bl
I-
falcultative
aerobes
IT
cyanophyta
Eucaryonts
Anabolism
/ &
bacteria
algae
protozoa
fungi
higher
protista
Catabolism
I
obligate
aerobes
I
I
I
I+
I+
plants
animals
[a] Cytochrome oxidases (E.C. 1.9.3.).
[b] Oxygenases (E.C. 1.13. and l.I4), e.g. tryptophan
pyrrolase for NAD(P) synthesis, fatty acid hydroxylase for the synthesis of unsaturated fatty acids,
squalene epoxidase for steroid synthesis.
This view is supported by the observation that the
ability to metabolize all types of nutrients has been
retained only in this group of anaerobes, whereas even
the facultative aerobes can utilize only carbohydrates
and closely related compounds in the absence of
oxygen. This may be regarded as a consequence of a
change in the nutrient supply that paralleled the
change from a reducing to an oxidizing environment.
Whereas a large number of classes of substances were
still available to the primordial anaerobes, a large
stock of carbohydrates was produced together with the
oxygen by photosynthesis. The aerobes that developed
were therefore probably selected in the direction of
carbohydrate utilization.
The corrin ring can be synthesized by all bacteria,
whereas not all can synthesize the porphin ring, and
even fewer the phorbin ring. Moreover, the side
chains of the pyrrole rings are most completely retained in the corrin system in comparison with the
other tetrapyrroles. It may therefore be deduced that
the corrin ring system is phylogenetically the earliest
(Fig. 11).From this point of view the methane-forming
bacteria and the clostridia described in this article
are closest to the primordial anaerobes.
primordial anaerobes
+corrin 1
cytochrorne,’
‘oxidase
H
’
,
’ \
+ oxygenases
\
,’
+phorbin
-cyt
‘
oxidase
Pseudomonads,.
Corynebacteria
Enterobacteria ,
Athiorhodaceae
obligate
facultative
1
-\
4
aerotolerant
-obligate
Consideration of the molecular evolution of the tetrapyrrole compounds (Fig. 11) leads to the same result.
Corrin, porphin, and phorbin rings are synthesized in
the cells from porphobilinogen via common intermediates 11161. Since the biosynthesis of the phorbin
ring proceeds via the porphin system, the porphin ring
probably preceded the phorbin ring in the evolution.
11161 J.LasceZZes:Tetrapyrrole Biosynthesis and its Regulation.
Benjamin, New York 1964.
Vol. 9 (1970) 1 No. 2
I
Lactobacilli and
propionibacteria
A e r o b e s
Angew. Chem. internat. Edit.
\
,
a
Purple and green
sulfur bacteria
1
-3
Sulfate reducing
bacteria
I
i
Clostridia,
methane forming
bacteria
1
obligate
A n a e r o b e s
4.3. Fermentation, Photosynthesis, and Respiration
Closely related to the concepts of aerobiosis and anaerobiosis are the terms fermentation and respiration
which were mostly derived from physiological observations. These terms are frequently used inconsistently in the literature. Redox processes with organic
electron acceptors are usually referred to as fermen-
157
tations, while those with inorganic electron acceptors
(except 0 2 ) are described as anaerobic respiration.
Respiration occurs when oxygen acts as the terminal
oxidant. Terms such as “fumarate respiration” and “inorganic fermentation” are incompatible with this view.
A self-consistent definition of the basic metabolic
types, i. e. fermentation, respiration, and photosynthesis, is provided by the relation of electron donor
and acceptor systems on the one hand and the ATP*
synthesizing pathways on the other (Table 1). In
fermentations, ATP* is formed by substrate level phosphorylation only, whereas cell respiration is a redox
process in which ATP* is synthesized mainly by electron
transport phosphorylation. Photosynthesis is dominated by light-driven electron transport phosphorylations.
Inorganic fermentafion would thus be a redox process
with a n inorganic electron acceptor, in which ATP*
is formed exclusively by substrate level phosphorylation. Anaerobic respiration is an oxygen-free energy
metabolism with an ATP* synthesis coupled to electron transport.
A consistent and functionally logical definition is
obtained by assigning respiration to electron transport
phosphorylation and fermentation to substrate level
phosphorylation. The terms organic and inorganic
fermentation and aerobic and anaerobic respiration
then become unambiguous.
Received: May 14, 1969
[A 742 l E ]
German version: Angew. Chem. X2, 153 (1970)
Translated by Express Translation Service. London
COMMUNICATIONS
cr-Platinum(rr) Chloride and Platinum(II1) Chloride
By 0; Wiese, H . Schafer, H. G . v. Schnering, C . Brendel, and
K. Rinkec*I
Piatinum(r1) chloride is usually prepared by the route
HzPtC16 . 6HzO + PtC14 + PtClz at about 5OO0C in a
stream of Clz. The resulting “P-PtC12” has a molecular
lattice: Pt6C112 molecules contain a regular Pt6 octahedron,
the CI atoms being arranged in front of its edges[ll. This
structural type is encountered, in particular, in niobiumtantalum chemistry.
W e have now discovered a new modification, “a-PtCl2”,
which is formed when Pt6C112 is heated in a small ampoule
for 1-2 days at 500”C, or o n reaction of the elements in
quartz ampoules in a temperature gradient [Pt at 650OC;
a-PtCl2 deposition at 550 OC; p(Cl2, end) w 3 atm].
Like those of the P modification, crystals of a-PtC12 are almost black and exhibit a reddish luster; when powdered both
forms are yellowish brown.
a-PtC12 is characterized by the following interplanar spacings
(A), the intensities being given in parentheses; Guinier
photograph:
6.48 (10); 6.31 (10); 5.43 (5); 3.322 (1); 3.255 (1); 3.242 (1);
3.156 (3); 3.104 (8); 2.885 (3); 2.713 (3); 2.581 (1); 2.544 (3);
2.236 (5); 2.224 (3); 2.106 (3); 1.909 (1); 1.845 (5); 1.781 (3);
1.700 (1); 1.674 (1); 1.656 (5); 1.622 (3); 1.601 (5).
a-PtClz is not isotypic with the well known a-PdCl2, which is
built up of planar PdC14/2 chains.
Whereas P-PtCIZ dissolves in benzene to form a definite
Pt6Cllz-benzene adduct, a-PtClz is insoluble in this solvent.
The rate of conversion of P- into a-PtClz is so slow that both
modifications can be independently studied by thermal
analysis (thermobalance, 4 ‘C/min; 1 atm of argon): quantitative decomposition of a-PtClz to the elements is complete
at 570 ‘ C , that of P-PtC12 a t 550 ‘C. In a vacuum (thermobalance, 4 “Cimin), extensive volatilization in addition to decomposition is observed only with P-PtC12. Correspondingly,
gaseous platinum chloride is recorded only in the mass spectrum of P-PtCIz. Since the Pt6C1i2 ion has the lowest appearance potential, Pt6C112 clearly vaporizes.
We have obtained crystals of the known121 compound
platinum(Ir1) chloride (greenish black needles) on chlorination of the metal in a temperature gradient [quartz ampoule;
Pt at 600°C; other end of ampoule at 400OC; deposition
of PtC13 at intermediate temperatures; p(Cl2, end) = 10 atrn].
PtC13 forms trigonal rhombohedra1 crystals having a =
21.235 (9), c = 8.550 (10) A; cia = 0.403; space group
158
R3-C:i;
dX-ray = 5.39, dprk = 5.27 g/cm3. The compound
has the same structure as the recently described PtBr3 [31; thus
it contains [Pt6C1]2] and &[PtCl;Clb4,2] units. The average
bond lengths are: Ptrr-Cl(4x) = 2.32; PtIv-CP (2x) = 2.37;
PtIV-Clb ( 4 ~ =) 2.29 A.
Thermal degradation of PtC13 (thermobalance, 1 atm argon)
proceeds quantitatively, via a poorly defined P-PtC12 stage,
to the elements. In a vacuum, considerable volatilization of
PtgC112 accompanies decomposition, as is also apparent
from the mass spectrum.
Received: November 21, 1969
[Z 125 IE]
German version: Angew. Chem. 82, 135 (1970)
[*I Dip1.-Chem. U. Wiese, Prof. Dr. H. Schafer,
Prof. Dr. H. G. v. Schnenng, C. Brendel, and Dr. K . Rinke
Anorganisch-chemisches Institut der Universitat
44 Munster, Gievenbecker Weg 9 (Germany)
[I] K. Brodersen, G. Thiele, and H . G. v. Schnering, 2. anorg.
allg. Chem. 337, 120 (1965).
[2] L. Wohler and F. Martin, Ber. dtsch. chem. Ges. 42, 3958
(1909); L . Wohler and S. Streicher, ibid. 46, 1591 (1913).
[3] G. Thiele and P. Woditsch, Angew. Chem. 81, 706 (1969);
Angew. Chem. internat. Edit. 8, 672 (1969).
Oxidative Addition of the Azide Ion to Olefins.
A Simple Route to Diamines[ll [**I
By H . Schafer *I
Organic anions can be oxidatively added to non-activated
double b o n d s W We have now found that this type of reaction can also be extended to inorganic anions, e.g. to the
azide ion. I n the electrolysis of a 1.2 M solution of sodium
azide in glacial acetic acid vigorous evolution of gas is observed
at the anode thus indicating formation of azide radicals and
their decomposition to nitrogen 13~41.On addition of olefin to
the electrolyte (olefin: glacial acetic acid 1 : 3 v/v) the anodic
gas evolution is suppressed and azidoalkanes can be isolated
(see Table). The azidoalkanes thus formed were characterized
by C, H, N analysis, IR- and N M R spectroscopy, or by reductive elimination of nitrogen with Raney-Ni/hydrogen or
LiAlH4 to give amines which were then compared gas chromatographically with authentic substances.
The oxidative addition of the azide ion to olefins affords a
one-step synthesis of 1,2- and 1,4-diazidoalkanes, which can be
converted into 1,2- and 1,4-diamines respectively by reductive
elimination of nitrogen 151.
Angew. Chem. internat. Edit.
Vol. 9 (1970) No. 2
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