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We are grateful to Dr. B. A. Haddock and Dr. M. D. Brand for many stimulatingdiscussions.
Chaix, P. & Petit, J. F. (1956) Biochim. Biophys. Acra 22,66-71
Dutton, P. L. (1971) Biochim. Biophys. Acfa 226,63-80
Guffanti, A. A , , Susman, P., Blanco, R. & Krulwich, T. A. (1978) J . Biol. Chem. 253,708-715
Haddock, B. A. & Cobley, J. G. (1976) Biochem. Soc. Trans. 4,709-711
Ingledew, W. J., Cox, J. C. & Hailing (1977) FEMS Letl. 2, 193-197
Stevenson, R. &Silver, S. (1977) Biochem. Biophys. Res. Commun. 75, 1133-1139
Wiley, W. R. &Stokes, J. L. (1963)J. Bacteriol. 86, 1152-1156
The Cytochrome Content of Escherichiu coli Grown with Different
Terminal Electron Acceptors
GRAEME A. REID and W. JOHN INGLEDEW
Department of Biochemistry, University of Dundee, Dundee DD1 4HN,
Scotland, U.K.
Escherichia coli is capable of altering the composition of its respiratory chain according
to environmental factors (Haddock & Jones, 1977). This organism can respire aerobically with oxygen as terminal electron acceptor, but when grown anaerobically on a
non-fermentable carbon source respiratory pathways to fumarate and nitrate may be
induced. Optical difference spectra of electron-transport particles from cells grown
either aerobically or anaerobically with (i) fumarate or (ii) nitrate show that the cytochrome content of each is very different. Aerobically grown cells have an absorbance
maximum at 556nm at 77 K with a shoulder at 563 nm, though five components have been
resolved spectrally by fourth-order finite-difference analysis (Shipp, 1972) of this complex alpha band. On the basis of the positions of their absorbance maxima, it has been
suggested that two of these components are c-type cytochromes and three are b-types.
The major c-type component is soluble (Fujita, 1966) and not a member of the main
respiratory pathway. Aerobically grown cells also contain small amounts of cytochromes
a1 and d, which increase in concentration as the culture approaches stationary phase
(Haddock &Jones, 1977).
When grown anaerobically with fumarate, a b cytochrome with an absorbance maximum at 558nm at 77K is synthesized, along with large amounts of cytochronies a , and
d . Difference spectra also show a cytochrome b absorbing maximally around 556nm,
and it has been presumed that this species is identical with that found in aerobically grown
cells. We show here, however, that these two cytochromes have different mid-point
potentials at pH 7.0. Escherichia coli grown anaerobically with nitrate plus glycerol are
spectrally quite similar to those grown on fumarate except that the peak at 556nm is
relatively much larger. This is due to the presence in these cells of cytochrome b223-,
which is specifically oxidized by nitrate (Haddock et al., 1976) and is considered to be
directly associated with nitrate reductase.
Two groups have attempted to characterize the cytochromes of E. coli grown under
aerobic conditions. By determination of the midpoint redox potentials of the b-type
cytochromes of membrane particles, Hendler et al. (1975) resolved three components,
with midpoint potentials of +220, +110 and -50mV at pH7. Pudek & Bragg (1976)
found only two major cytochromes b, titrating at +165 and +35mV. We have performed
similar redox titrations with E. coli particles and extended the method to cells grown
anaerobically with either fumarate or nitrate.
E. d i s t r a i n EMG-2 (prototroph) was grown in 20litre batches as described by Haddock et al. (1976). Redox titrations were performed essentially as described by Dutton
(1971).
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Aerobically grown cells
The results of a typical redox titration of the b cytochromes of electron-transport
particles derived from aerobically cells are shown in Fig. l(a). These particles were
washed with lm~-Tris/HCl,pH7.5, which we found removed a cytochrome b. In
unwashed particles this component was found to have a mid-point potential of -50mV,
corresponding to the low-potential cytochrome b described by Hendler et al. (1975).
This cytochrome is probably that purified by Deeb & Hager (1964); its function is
unknown, but it cannot be directly involved in the main respiratory pathway since
particles depleted of it still oxidize NADH at high rates. The two major b cytochromes in
these washed particles have mid-point potentials of +260mV and +80mV. One of these
two components must be the terminal oxidase, cytochrome 0,described by Castor &
Chance (1959).
The titrations of cytochromes a, and din these particles are not represented here, but
the results were identical to those found with anaerobically grown cells (Fig. 2) where
these cytochromes were present in higher concentration. Our value of +280mV for the
mid-point potential of cytochrome dis close to that measured by Pudek & Bragg (1976),
+260mV. Whereas these authors described a single cytochrome a, component with a
midpoint potential at +147mV, we find two components, at +160mV and +260mV,
each contributing equally to the total spectral change. It is a common feature of cytochromes a, that where it is found, two distinct species are present (Ingledew, 1978).
Anaerobicall-vgrown cells
E. coli grown on glycerol with fiimarate appears green in colour due to the presence
of large amounts of the terminal oxidase, cytochrome d. Electron-transport particles of
these cells contain completely different cytochromes b compared to those synthesized
under aerobic growth conditions (Fig. l b ) . Two components with midpoint potentials of
+250mV and +140mV are found. Three b-type cytochromes are found in particles
derived from cells grown anaerobically on glycerol, with nitrate as terminal electron
acceptor. Two of these correspond to those found in fumarate-grown cells, and the other,
with a midpoint potential of +lOmV, is presumably cytochrome bgNP63-.
J
,350
I
I
I
1
I
4300 1250 +ZOO 1 1 5 0 ,100
I
t50
1
0
I
50
El, ( m W
Fig. 1 . Redox titrations of the b cytochromes of E. coli
Electron-transport particles were prepared as described in the text from cells grown
aerobically (a) or anaerobically with fumarate (b) or anaerobically with nitrate (c). The
degree of reduction of the b cytochrome was monitored by the absorbance change at
559nm with 576nm taken as the reference wavelength.
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BIOCHEMICAL SOCIETY TRANSACTIONS
I
+350
I
+300
I
I
+250
t200
I
t150
I
tlOO
El,( m V )
Fig. 2. Redox titrations of cytochromes al and d of E. coli
Electron transport particles from cells grown anaerobically with fumarate were prepared
as described in the text. The degree of reduction of cytochrome d ( a ) was monitored by the
absorbance change at 630nm with 610nm as the reference wavelength. The wavelength
pair 590-576nm was taken for cytochrome al (h).
Discussion
E. coli has the capability of regulating the composition of its respiratory chain according to changes in environmental conditions. This is particularly dramatic when the nature
of the terminal respiratory oxidant is varied. The induction of fumarate reductase and
nitrate reductase under conditions of oxygen limitation are the more obvious changes that
occur, but the electron-carrying pathways from quinone to oxygen (via cytochromes o
and d ) are also regulated. It would appear that the two cytochromes found in membranes
of aerobically grown cells constitute one pathway, and that a pathway to cytochrome d,
perhaps involving cytochromes b or a, or both, is induced when the availability of oxygen
becomes limiting.
Castor, L. N. & Chance, B. (1959) J . Biol. Chem. 234,1587-1592
Deeb, S . S. & Hager, L. P. (1964)J. Biol. Chem. 239, 1024-1031
Dutton, P. L. (1971) Biochim. Biophys. Acta 226.63-80
Fujita, T. (1966) J . Biochem. (Tokyo) 60,329-334
Haddock, B. A. &Jones, C. W. (1977) Bacteriol. Rev. 41,47-99
Haddock, B. A., Downie, J. A. & Garland, P. B. (1976) Biochem. J . 154,285-294
Hendler, R. W., Towne, D. W. & Schrager, R. I. (1975) Biochim. Biophys. Acta 376,42-62
Ingledew, W. J. (1978) in Functions of Alternative Oxidases (Degn, H., Lloyd, D. & Hill, G. C.,
eds.), pp. 79-87, Pergamon Press, Oxford
Pudek, M. R. & Bragg, P. D. (1976) Arch. Biochem. Biophys. 174,546-552
Shipp, W. S. (1972) Arch. Biochem. Biophys. 150,459-472
1978