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577th MEETING, OXFORD
It is also noteworthy that the maximum phosphorylation potential that may be
generated by the vesicles (approx. 53.8kJ/mol) (Kell et al., 1978~)was not altered when
the rate of NADH oxidation was inhibited by up to 60 %. The corollary of this result is
that if Ap is poised at equilibrium with the phosphorylation potential then, as the phosphorylation potential does not decline when NADH oxidation is inhibited, our finding
that A p is not decreased on addition of rotenone is to be expected.
The data presented above show that the resistance (or, more likely, the capacitance)
of the P. denitrificans membrane must be variable. Both these data, and those of other
workers (Casadio et al., 1978; Kupriyanov & Pobochin, 1978) would seem to point to
the need to consider more localized factors in biomembrane energy-transduction processes. In this regard it may be profitable to utilize membrane models containing 3 capacitors in series (e.g. Andersen et al., 1978; cf. Bockris & Reddy, 1970).
Andersen, O . , Feldberg, S., Nakadomari, H., Levy, S. & McLaughlin, S. (1978) Biophys. J . 21,
35-70
Bockris, J. O’M. & Reddy, A. K. N. (1970) Modern Electrochemistry,Plenum, New York
Casadio, R., Baccarini-Melandri, A. & Melandri, B. A. (1978) FEBS Lett. 87, 323-328
Chance, B. & Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17,65-134
Ferguson, S. J., John, P., Lloyd, W. J., Radda, G. K. & Whatley, F. R. (1976) FEBS Letr. 62,
272-275
Hinkle, P. C., Tu, Y. L. & Kim, J. J. (1975) in Molecular Aspects of Membrane Phenomena
(Kaback, H . R., Neurath, H., Radda, G. K., Schwyzer, R. & Wiley, W. R., eds.), pp. 222-232,
Springer, New York
Kell, D. B., John, P. & Ferguson, S. J. (1978~)Biochem. J . 174,257-266
Kell, D. B., John, P., Sorgato, M. C. & Ferguson, S. J. (19786) FEBSLett. 86,294-298
Kupriyanov, V . V. & Pobochin, A. S. (1978) Biochim. Biophys. Acta 501,330-348
Lee, C.-P., Ernster, L. & Chance, B. (1969) Eur. J. Biochem. 8,153-163
Mitchell, P. (1966) Biol. Rev. 41,445-502
Mitchell, P. (1977) FEBS Lett. 78, 1-20
Sorgato, M. C., Ferguson, S. J. & Kell, D. B. (1978) Biochem. SOC.Trans. 6, 1301-1302
Williams, R. J. P. (1978) FEBSLetf. 85,9-20
The Respiratory Chain and Proton Electrochemical Gradient in the
Alkalophile Bacillus pasteurii
MARK H. HODDINOTT, GRAEME A. REID and W. JOHN INGLEDEW
Department of Biochemistry, University of Dundee, Dundee DD1 4HN,
Scotland, U.K.
Bacillus pasteurii requires rather unusual growth conditions. It grows at alkaline pH
(up to 10.5) and requires the presence of relatively high concentrations of ammonium
salts. Although the growth of cells is optima1 at pH9.0, substrate oxidation byelectrontransport particles is ammonia-independent and maximal at pH7.5; this has been
interpreted to mean that the matrix pH is maintained near neutrality during growth in
alkaline medium (Wiley & Stokes, 1963). Therein lies a chemiosmotic dilemma. In the
equation
A p H + = AI// - ZApH
using standard notation, the ApH is positive, thus for ApH+to be, say, 240mV the AI//
will have to be greater than that value by an amount equal and opposite to ZApH.
Haddock & Cobley (1976) have demonstrated that in terms of vectorial H+ movement
there is nothing unusual about this bacterium; in oxygen-pulse experiments H+ ions are
pumped out from the matrix.
The respiratory chain of B. pasteurii contains cytochromes of the b, c and a types.
The cytochrome a is spectrally similar to the aa3 terminal oxidase of mammalian systems
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BIOCHEMICAL SOCIETY TRANSACTIONS
and also binds CO. Haddock & Cobley (1976) have also noted the presence of a CObinding b-type cytochrome. Cells were grown aerobically as described by Haddock &
Cobley (1976) in a medium consisting of 2% yeast extract, 1 % (NH&S04 and 1 3 0 m ~ Tris, pH 9.0. Electron-transport particles were prepared by passing cells twice through a
French pressure cell followed by differential centrifugation. Redox titrations were
performed as described by Dutton (1971). The pH gradient across thecell membrane was
evaluated by studying the inhibition of oxygen uptake by azide as a function of the
external pH. This technique has previously been used to estimate the internal pH of the
acidophile Thiobacillusferrooxidans (Ingledew et al., 1977). It can be shown that
(Ci)couF.lsd
(Ci5O)noApH
where Ci50is the concentration of azide required to inhibit oxygen uptake by 50%. The
membrane potential (Aty) was estimated by following the transmembrane distribution
of 86Rb+ (in the presence of valinomycin) or [3H]dibenzyldimethylammonium+
(DDA+) in the presence of tetraphenylboron.
pH = log
The respiratory chain
Two cytochromes of the a-type were resolved by redox potentiometry of electrontransport particles (Fig. la). Their midpoint potentials at pH7.0 were +140mV and
+240mV, and the two components contributed equally to the spectral change at 600620nm. A titration of the wavelength pair 552--537nm indicates the presence of c-type
cytochromes with midpoint potentials of +130 and +240mV. The former component
contributes 85 % of the spectral change. A b-type cytochrome with a midpoint potential
of -10mV was also detected in electron-transport particles (not shown).
The results of a redox titration performed in the presence of CO are shown in Fig. 1 (b).
As is typical of cytochromes aa3, one of the components binds CO, increasing the
apparent midpoint potential to beyond the range of our titrations (> +400mV). The
remaining cytochrome a exhibits a midpoint potential of +225mV. The titration of
552-537nm indicates the presence of a CO-binding cytochrome c . The c-type component
at +240mV is unaffected by the presence of CO. However, only 60% of the absorbance
1
I
+ 300
I
t
200
I
+loo
I
0
4, (mV)
Fig. 1. Redox titrations of the cytochromes of’B. pasteurii
Redox titrations were performed in the absence (a) and presence (b) of CO. The degree
of reduction of cytochrome c ( 0 , 0)was monitored by the absorbance change at 552537nm, and cytochrome a (a,B) was measured at 600-620nm.
1978
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577th MEETING, OXFORD
change due to the fl20mV cytochrome c is apparent in the presence of CO. This indicates
that the apparently single +120mV component observed in the absence of CO is a
composite of two species, only one of which binds CO. The presence of a CO-binding
c-type cytochrome is confirmed by difference spectra.
During the preparation of electron-transport particles it was noted that the soluble
fraction contained a b-type cytochrome which bound CO exhibiting a typical cytochrome
o (CO-binding 6-type) difference spectrum.
The proton electrochemicalgradient
Fig. 2(a) shows that when cells are suspended in growth media, ApH is small. At pH7
ApH is negative, at pH8 almost zero, and above pH 8 becomes increasingly positive,
i.e. unfavourable in a chemiosmotic sense. Contrastingly when cells are suspended in
phosphate buffer, ApH is significantly greater, so much so that it was not possible to
obtain a point for pH 10 because the azide concentrations required were prohibitively
high. It seems likely that the ammonia is needed to help regulate the internal pH with
respect to the external. A high concentration of Tris ( 1 3 0 m ~ )may act as surrogate
ammonium.
Fig. 2(b) shows the variation of Av/, ApH and A p H + with pH for cells suspended in
growth media. Despite an unfavourable ApH term at high external pH this is partially
compensated by a concomitant rise in Av/. These trends are more dramatic with cells
suspended in growth media lacking NH4+ ions, and especially in cells suspended in
phosphate buffer (not shown).
Discussion
The cytochromes of B. pasteurii are not markedly different from those found in other
bacilli, except in the inclusion of a CO-binding c-type (e.g. B. subtilis; Chaix & Petit,
1956). We have measured a ApH+of 180mV (approx.) and in growth media a small
ApH. These findings differ slightly from those of Guffanti et al. (1978) in B. alcalophih.
We have used a novel method to measure ApH. Usually the distribution of methylamine
is used; this method gave anomalous results possibly because B. pasteurii has a NH,+
porter which can also carry methylamine. Such a system has been suggested for Escherichia coli (Stevenson & Silver, 1977).
10
7
8
9
External pH
10
-100
7
8
9
10
External pH
Fig. 2. Effect of extracellular p H on intracellular pH, b y , A@"+ and -ZApH
(a) Variation of intracellular pH with extracellular pH in cells suspended in growth
medium ( 0 ) or in phosphate buffer (m) .The straight line represents the theoretical case
where, at all values of pH, internal pH equals external pH. (6) Variation of A v (B),
ApH+(A) and -ZApH ( 0 ) with extracellular pH.
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BIOCHEMICAL SOCIETY TRANSACTIONS
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).
1978