FEMS Microbiology Letters 30 (1985) 87-92
Published by Elsevier
87
FEM 02241
Respiration-dependent proton translocation and the mechanism of
protonmotive force generation in Nitrobacter winogradskyi
(Cytochrome oxidase (aa3); proton pumping; chemiosmosis; nitrite oxidation)
H e i n z - G e o r g W e t z s t e i n ab a n d S t u a r t J. F e r g u s o n a,
'1 Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham, BI 5 2TT, U.K. and
h InstitutfiJr Mikrobiologie der UniversitSt GOttingen, D- 3400 GOttingen, F.R.G.
Received 7 June 1985
Accepted 10 July 1985
1. S U M M A R Y
Proton translocation associated with electron
flow to oxygen has been observed with cells of
Nitrobacter winogradskyi in the presence of either
potassium ferrocyanide or isoascorbate plus
N,N,N',N'
tetramethyl-p-phenylenediamine. The
data are consistent with a proton pumping function for the terminal oxidase, cytochrome aa3, in
this organism as the mechanism for generating a
protonmotive force. The failure of previous work
with Nitrobacter [4] to detect proton translocation
linked to oxidation of nitrite, the physiological
substrate, is discussed.
2. I N T R O D U C T I O N
Bacteria of the genus Nitrobacter rely upon the
oxidation of nitrite by oxygen to provide energy
for growth. It has been generally accepted that
electron flow from nitrite to oxygen is linked to
* To whom correspondence should be addressed.
Abbreviations: FCCP. carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; TTFB, 4,5,6,7 tetrachloro-2'-trifluoromethyl benzimidazole.
the generation of a proton electrochemical gradient (protonmotive force) across the plasma membrane [1-3], although there are recent reports of
failures to observe the expected proton translocation linked to electron flow [4,5]. This gradient is
envisaged to drive both ATP synthesis and reversed electron transfer from nitrite to NAD(P) to
provide reducing power for growth. The site of
nitrite oxidation is believed to be on the cytoplasmic surface of the plasma membrane from
where electrons are passed via the nitrite oxidase
to cytochrome c at the periplasmic surface of the
membrane. Cytochrome aa 3 then transfers electrons from cytochrome c to reduce oxygen, possibly at the cytoplasmic surface of the membrane
[2-3], or alternatively, but equivalently in thermodynamic terms, at the periplasmic surface using
protons derived from the cytoplasm. The latter
point is discussed and explained in a thermodynamic context by Wikstr6m and Saraste [6]. Such
a description of electron flow from nitrite to oxygen
does not account for the generation of a protonmotive force because there is neither net inward
movement of electrons nor outward movement of
protons across the membrane [3].
A feature of the electron transport from nitrite
to oxygen in Nitrobacter spp. is that it is inhibited
under conditions in which the membrane potential
0378-1097/85/$03.30 © 1985 Federation of European Microbiological Societies
88
is dissipated [1-3]. This observation is interpreted
as meaning that the membrane potential (positive
outside the cell) drives the transfer of electrons
from nitrite to cytochrome c. Thus this reaction
step must be associated with the movement of net
negative charge across the membrane rather than
hydrogen (proton plus electron) movement. Two
schemes which take this consideration into account
have been suggested to explain the required generation of a protonmotive force. In the first of these,
Cobley [2] postulated that the passage of two
electrons from nitrite to cytochrome c is associated with the movement of one proton so that the
hydride anion would be effectively transferred
across the membrane. The second proposal is that
cytochrome aa 3, as in mitochondria and some
other bacterial membranes, has a proton pumping
function [3]. These two alternatives could in principle be distinguished by testing whether cytochrome oxidase translocates protons in whole cells.
The present paper describes experiments of this
kind using non-physiological susbstrates that
donate electrons to cytochrome c, and also discusses why previous work [4-5] might have failed
to detect respiratory chain-driven proton translocation with nitrite, the physiological substrate.
3. MATERIALS A N D M E T H O D S
N. winogradskyi (strain Engel) was grown at
30°C in the dark as 5-1 batch cultures in 20-1
carboys loosely stoppered with cotton wool. The
autotrophic growth medium described by Sundermeyer and Bock [7] was inoculated with 100 ml of
a growing pre-culture and stirred at approx. 100
rev./min. After 4-10 days the cultures were fed
with 20 mM NaNO 2, and 4-7 days later, at an
absorbance of 0.03 + 0.01 ()~ = 600 nm; pathlength = lcm; cell protein approx. 8 mg/1), the
cells were harvested by centrifugation at 10000 × g
for 60 min at 4°C. After washing in an anaerobic
buffer containing 1.5 mM glycyl-glycine pH 8.0,
100 mM KSCN, and 50 mM KCI, the cells were
resuspended to .4 ~< 50 in the same buffer and
stored under nitrogen on ice for not more than 8 h
before use. The buffer was degassed by boiling
and then cooled under a stream of nitrogen that
was purified from traces of oxygen by a passage
over a heated copper catalyst [8]. The purity of the
cultures was checked microscopically and by recording of redox-difference spectra of the cells.
pH Changes were measured with a Russell 757
microelectrode inserted into a water-jacketed glass
chamber of 4 ml volume. The electrode was connected to a Knick pH-meter linked to a Servogor
5B chart recorder via a back-off box [9]. The
contents of the chamber were maintained under
anaerobic conditions by a stream of watersaturated nitrogen that entered through an entry
port in the lid. The reaction mixtures contained, in
a final volume of 2.2 ml buffer, N. winogradskyi at
1.2-4.9 mg cell protein per ml. After a preincubation for at least 30 min at 30°C, the electron-donors
ferrocyanide or isoascorbate, plus N,N,N',N'-tetramethyl-p-phenylenediamine ( T M P D ) were
added. The pH was adjusted to between 8.2 and
8.4 with anaerobic N a O H or HCI. O x y g e n was
then added as increments of either 10 or 20 ~l of a
solution of 150 mM KC1, pH 8.2 that had been
equilibrated with air at 30°C. The electrode reading was calibrated by repeated additions of 20#1 of
a standard solution of 1 mM HC1 at the end of
each experiment. The concentration of 02 was
assumed to be 237 #M [10].
Respiratory activities were measured with an
oxygen electrode (Rank Bros., Cambridge, U.K.)
at 30°C in 50 mM H e p e s - N a O H , pH 7.8, with
either 2 mM NaNO 2 or the alternative electron
donors mentioned above.
Protein determination: Aliquots of cells were
dissolved by boiling in 4 M N a O H for 5 min.
After neutralization with HCI, protein was
determined according to Lowry [11]. A culture of
N. winogradskyi (Engel) was kindly provided by
Prof. E. Bock, University of Hamburg, F.R.G.
4. RESULTS
The measurement of respiration-driven proton
translocation requires lightly buffered reaction
conditions. When cells o f N. winogradskyi were
added to this type of medium, described in
MATERIALS AND METHODS, a drift towards alkaline
pH was always observed irrespective of whether an
89
exogenous reductant was added. Figs. 1 and 2
show typical examples of this behaviour. Conditions could not be found in which this steady drift
could be eliminated. The basis for the drift was
not elucidated.
When small volumes of an oxygen saturated
solution were introduced to an anaerobic suspension of cells in the presence of isoascorbate plus
T M P D there was an acidification of the external
medium (Fig. 1). Addition of pulses of oxygensaturated buffer after the uncoupler 4,5,6,7 tetrachloro-2'-trifluoromethyl benzimidazole (TTFB)
did not result in an acidification but rather led to
an alkalinisation (Fig. 1). The phase of acidification was also not observed if the permeant anion
S C N - was omitted from the reaction medium.
Qualitatively these observations indicate that electron flow from isoascorbate to oxygen is linked to
proton translocation in N. winogradsky.
The oxidation of isoascorbate by electron carriers such as T M P D or cytochrome c will result in
the release of 1H + per 2e transferred [12]. Hence
in experiments of the type shown in Fig. 1 proton
translocation by the electron transfer chain must
raise the stoichiometry of proton release above
1 H ÷ / O . Values for the H + / O ratios were obtained from the acidification phases shown in Fig.
1 using the extrapolation procedure of Scholes and
Mitchell [13]. By this method values for H + / O of
2.4, 1.8 and 1.6 were obtained for experiments in
which 10 ffl (record 1), 20/~1 (record 2) and 20 #1
(record 3) air-saturated buffer were added. The
results with a 30 #1 (record 3) addition of buffer
have not been calculated owing to uncertainty as
to whether the base line had been re-established.
The H + : O ratios obtained from Fig. 1 are consistent with electron flow from isoascorbate to
oxygen being linked to proton translocation by t h e
respiratory chain. This view is supported by the
loss of the acidification phase when a protonophore was present (Fig. 1). The overall reaction of
isoascorbate oxidation by oxygen results in an
uptake of 1H ÷ per pair of electrons transferred
[12]. Hence the overall disappearance of H +
observed after addition of air-saturated solution in
the presence of a protonophore is to be expected.
However, in this type of experiment we were not
able to obtain proportionality between the quan-
G
Q
-I 20nmolH*
®
I
--~ 6 atin ~,Fig. 1. Proton translocation linked to oxidation of isoascorbate
plus TMPD by cells of N. winogradskyi. The cells were taken
from a culture that was grown for 10 days before, and for 7
days after, feeding with NaNO2 (see MATERIALS AND
METHODS). The reaction chamber contained an anaerobic
suspension of cells (4.6 mg protein-ml-l) (see MATERIALS
AND METHODS for medium) together with 5 mM isoascorbate plus 50 #M TMPD. After 30 min incubation, 10 p~l,2 x 20
#1 and 30 #1 volumes of an air-saturated solution of 150 mM
KCI were added as shown. 13 /~M TTFB was added as indicated and caused the displacement of the electrode response
shown. Further additions of air-saturated solution were made
as shown in the presence of TTFB.
tity of oxygen added and the extent of proton
uptake (Fig. 1). A complicating factor is that hydrolysis of dehydro- isoascorbate can subsequently
release an additional proton [10].
Introduction of oxygen in small pulses to
anaerobic suspensions o[ cells in the presence of
potassium ferrocyanide caused an acidification of
the external medium except when the protonophore carbonylcyanide p-trifluoro-methoxyphenylhydrazone (FCCP) was present (Fig. 2). Since
90
20 nmol H +
QI
,-~ 3 min l.*-
Fig. 2. Proton translocation associated with oxidation of ferrocyanide by cells of N. winogradskyi. The cells were taken from
a culture that was grown for 10 days before, and for 5 days
after, feeding with N a N O 2 (see M A T E R I A L S A N D M E T H ODS). The reaction chamber contained an anaerobic suspension of cells (1.2 mg p r o t e i n / m l ) in the medium described in
M A T E R I A L S A N D METHODS. After 30 min incubation
potassium ferrocyanide was added to a final concentration of 1
mM. 20 /tl volumes of an air-saturated solution of 150 m M
KCI were added as shown. Record 3 is an experiment in which
2.7 # M FCCP was present.
oxidation of ferrocyanide by cytochrome c does
not involve release of a proton, the interpretation
of the experiments shown in Fig. 2 is that the
acidification is a consequence of a proton pumping activity of cytochrome oxidase. The data shown
in Fig. 2 indicate that 1.5 (record 1) and 1.7
(record 2) H ÷ were detected per oxygen atom
reduced.
There are undoubtedly technical problems concerning the measurement of proton translocation
associated with electron transport in Nitrobacter.
First, we found that satisfactory results were obtained only if freshly harvested cells from a growing culture were used and preincubated for 30 min
in the reaction chamber. Second, 5-1 culture provided sufficient cells for only a very small number
of experiments. Third, the rate of oxidation of
isoascorbate plus T M P D was approx. 20% of the
nitrite oxidation rate (which was 160-200 ng atom.
O . min -1. mg -1 protein) and the rate of ferrocyanide oxidation even lower at 5% of this value.
This might account for the rather slow rate of
proton extrusion seen in the experiments with
ferrocyanide (Fig. 2) as well as the failure to
observe an overall alkalinisation that is expected
for the oxidation of ferrocyanide by oxygen in the
presence of FCCP. Experiments designed to detect
the proton pumping activity of cytochrome aa 3 in
either mitochondria or bacteria have been criticised on the basis that the observed proton translocation might arise from the activity of other
segments of the electron transfer chain [14]. This
objection can probably be discounted for Nitrobacter, at least with isoascorbate plus T M P D as
substrate. Although we were not able to inhibit the
endogenous respiration of this organism, the rate
of endogenous respiration was between 10% and
25% of the rate of respiration with ascorbate plus
T M P D and therefore the proton translocation observed with this substrate (Fig. 1) can be attributed
to the activity of the cytochrome aa 3 segment of
the electron transfer chain.
5. DISCUSSION
The data presented in the present paper give
evidence for a proton pumping capacity of the
cytochrome aa 3 of N. winogradskyi, as expected
on the basis of a scheme for energy conservation
in N i t r o b a c t e r put forward by Ferguson [3]. The
conclusion is supported by a re-examination of
data obtained some years ago by Ingledew [15],
who found that introduction of pulses of oxygen
to anaerobic suspensions of N. w i n o g r a d s k y i in the
presence of valinomycin and ascorbate plus T M P D
resulted in the appearance of protons external to
the cells at a stoichiometry of 2H ÷ :O. It was
assumed that all the protons released originated
from ascorbate, but as explained above the chemical reaction of ascorbate oxidation by the oxidised
form of T M P D (Wurster's blue) should yield only
one proton per oxygen atom reduced. Thus reinterpretation of Ingledew's data [15] also supports a
role for cytochrome aa 3 as a proton pump. This
91
conclusion does not agree with the results obtained
with a purified cytochrome aa 3 from Nitrobacter
agilis [16]. This protein has been incorporated in
phospholipid vesicles and tests made to determine
whether it has a proton-pumping function. The
results have been negative [16]. The discrepancy
between the latter work and the findings both of
the present work and of Ingledew [15] could have
several causes. Amongst these is the possibility
that the proton pumping activity of the purified
enzyme is low and difficult to detect. When the
cytochrome aa 3 from Paracoccus denitrificans was
first purified only a very low proton pumping
activity could be detected [17], contrary to the
indications from experiments from whole cells [10],
but subsequent work has demonstrated proton
pumping with the purified cytochrome, provided
certain reaction conditions are used [18].
In recent work, Hollocher et al., [4] have concluded that protonmotive force in Nitrobacter is
not established by a proton-translocating respiratory chain. This has led to consideration of schemes
for ATP synthesis via a mechanism independent of
a protonmotive force [5]. A protonmotive force
would be generated subsequently by a proton
translocating ATPase. This conclusion was reached on the basis of experiments in which evidence for proton translocation was sought by introducing pulses of oxygen into an anaerobic suspension of Nitrobacter supplied with the physiological substrate nitrite, but in the presence of
reagents to collapse the membrane potential. These
conditions are necessary for detection of proton
translocation with this type of experiment [13].
However, a difficulty is that it is well known that
nitrite oxidation by Nitrobacter is dependent on
the presence of a sizeable membrane potential
[1-3]. Thus, attempts to detect respiratory chainlinked proton translocation with this physiological
substrate could be doomed to failure, because
during short pulses of respiration the membrane
potential is probably not generated, and thus nitrite
oxidation will be inhibited. In the steady state the
membrane potential may not be completely dissipated and thus nitrite respiration, albeit at a
reduced rate [4], can proceed in the presence of
permeant ions. Furthermore, we have observed
that when an anaerobic suspension of Nitrobacter
is added to an aerobic solution, the subsequent
rate of nitrite oxidation shows a lag phase before
the maximum rate is reached unless a membrane
potential is first generated by several minutes of
slow respiration using endogenous substrates. This
observation also indicates that addition of an
oxygen pulse to an anaerobic suspension of
Nitrobacter containing nitrite is unlikely to permit
detection of proton translocation associated with
electron flow from nitrite to oxygen. The data in
the present paper give good evidence that the
respiratory chain of Nitrobacter does translocate
protons, most probably at the level of cytochrome
aa3, although technical difficulties and low cell
growth yields prevent characterisation of this proton translocation in the detail possible with either
mitochondria or bacteria that can be grown more
readily.
ACKNOWLEDGEMENTS
H.G.W. was the recipient of a Royal Society
European Science Exchange Fellowship under an
agreement with the Deutsche Forschungsgemeinschaft.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Cobley, J.G. (1976) Biochem. J. 156, 481-491.
Cobley, J.G. (1976) Biochem. J. 156, 493-498.
Ferguson, S.J. (1982) FEBS Lett. 146, 239-243.
Hollocher, T.C., Kumar, S. and Nicholas, D.J.D. (1982) J.
Bacteriol. 149, 1013-1021.
Hollocher, T.C. (1984) Arch. Biochem. Biophys. 233,
721-727.
Wikstr6m, M. and Saraste, M. (1984) in Comprehensive
Biochemistry, Bioenergetics (Ernster, L., Ed.) pp. 49-94,
Elsevier, Amsterdam.
Sundermeyer, H. and Bock, E. (1981) Arch. Microbiol.
130, 250-254.
Hungate, R.E. (1969) in Methods in Microbiology (Norris,
J.R. and Ribbons, D.W., Eds.) Vol. 3B. pp. 117-132.
Academic Press, New York.
Madeira, V. (1975) Biochem. Biophys. Res. Commun. 64,
870-876.
Van Verseveld, H.W., Krab, K. and Stouthamer, A.H.
(1981) Biochim. Biophys. Acta 635, 525-534.
Lowry, O.H., Roseborough, N.J., Farr, A.L. and Randall,
R.J. (1951) J. Biol. Chem. 193, 265-275.
92
[12] Wikstr6m, M. and Krab, K. (1979) Biochim. Biophys.
Acta 549, 177-222.
[13] Scholes, P. and Mitchell, P. (1970) J. Bioenerg. 1,309-323.
[14] Moyle, J. and Mitchell. P. (1978) FEBS Lett. 88, 268-272.
[15] Ingledew, W.J, (1974) Ph.D. Thesis, University of Bristol,
Bristol, U.K.
[16] Sone, N., Yanagita, Y., Hon-Nami, K., Fukumori, Y. and
Yamanaka, T. (1983) FEBS Lett. 155, 150-154.
[17] Ludwig, B. (1980) Biochim. Biophys. Acta 594, 177-189.
[18] Solioz, M., Carafoli, E. and Ludwig, B. (1982) J. Biol.
Chem. 257, 1579-1582.