Boogerd, F C

Stouthamer, A.H.

van Verseveld, H W

F.C. Boogerd

H.W. van Verseveld

A.H. Stouthamer

Crossref

FEBS Letters

Electron transport to nitrous oxide in<i>Paracoccus denitrificans</i>

Boogerd, F.C.

van Verseveld, H.W.

Elsevier - Publisher Connector 

Volume 113, number 2 FEBS LETTERS May 1980 
ELECTRON TRANSPORT TO NITROUS OXIDE IN PARA COCCUS DENITRIFICANS 
F. C. BOOGERD, H. W. van VERSEVELD and A. H. STOUTHAMER 
Department of Microbiology, Biological Laboratory, Free University, 1007 MC Amsterdam, The Netherlands 
Received 7 February 1980 
Revised version received 10 March 1980 
1. Introduction 
Many bacteria can use nitrogenous oxides, instead 
of oxygen, as terminal electron acceptor during 
respiration [ 11. Puracoccus denitrijkans is capable of 
growing anaerobically in the presence of nitrate, nitrite 
or nitrous oxide. When nitrate is the electron acceptor 
growth is accompanied by the evolution of nitrogen 
and nitrous oxide [2]. Cells grown anaerobically with 
nitrate as electron acceptor contain flavin, UQ-10, b-, 
c-, and o-type cytochromes, a membrane-bound 
nitrate-reductase and a soluble two-haem (c- and d- 
type) nitrite-reductase [3,4]. There are strong indica- 
tions that b-type cytochromes are involved in elec- 
tron transport to nitrate and c-type cytochromes in 
electron transport to nitrite [5,6]. The occurrence of 
obligate intermediates, like nitrous oxide, in the 
reduction of nitrite to nitrogen gas is a point of 
controversy [7-lo]. Therefore we started a more 
profound study of the role of nitrous oxide in deni- 
trification by Pa. denitrificans. The results indicate 
that nitrous oxide could be an obligate intermediate 
in the reduction of nitrite to nitrogen gas and that c- 
type cytochrome(s) are involved in electron transport 
to nitrous oxide. 
2. Materials and methods 
Pa. denitrificans NCIB 8944, formerly Micrococcus 
denittificans [ 111, was the experimental organism. 
Bacteria were grown anaerobically at 37°C in the 
liquid medium described in [ 121 with succinate as 
carbon and energy source, NH4C1 as nitrogen source 
and nitrate as electron acceptor. Anaerobic chemostat 
experiments were performed as described in [ 131 in a 
Bioflo C30 chemostat (New Brunswick Scientific Co., 
ElsevierlNorth-Holland Biomedical Press 
Inc., New Brunswick, NJ, USA). The culture volume 
was -300 ml. The pH was controlled at a value of 
7.5, unless otherwise stated. In succinate-limited 
continuous cultures the concentrations of succinate 
and nitrate were 25 mM and 100 mM, respectively. 
With electron acceptor limitation the concentration 
of nitrate was 40 mM, while succinate was added in a 
concentration of 50 mM. Succinate- and nitrate- 
limited chemostat cultures were grown at dilution 
rates of 0.13 and 0.18/h, respectively. After at least 
six generation times, the chemostat effluent was 
collected overnight in an ice-cooled vessel and cells 
were harvested by centrifugation. After washing twice 
with phosphate-buffer (10 mM K-phosphate, pH 7.0) 
cells were resuspended in the same buffer. 
Bacteria were grown in batch culture on methanol 
(100 mM) and nitrate (100 mM) in a 1-l vessel at 
35’C under stagnant conditions. The medium was 
supplemented with 0.01% yeast-extract (Difco) and 
0.05% NaHCOs. The starting pH was 7.5. Cells were 
harvested when the culture had reached an absorbance 
of 0.25-0.35, measured at 660 nm. 
Cells were grown in batch culture at 35°C on 
succinate (50 mM) in an anaerobic jar, only filed 
with NzO. After 4 to 5 days the cells were harvested. 
All cytochrome studies were performed using an 
Aminco DW-2 UV/Vis spectrophotometer. Cyto- 
chrome oxidation-reduction kinetics were studied in 
dual wavelength experiments under anaerobic con- 
ditions in a Thunberg cuvette at 25°C. Nitrous oxide 
was added by means of injection of a known volume 
of N,O-saturated water at 25°C (nitrous oxide con- 
centration: 24.5 nmol/pl). Known quantities of 
nitrite were introduced by injecting an anaerobic 
NaNOz solution. NaNs and KCN were dissolved in 
10 mM K-phosphate buffer (pH 7.0). Rotenone and 
antimycin A were added as ethanolic solutions. In 
279 
Volume 113, number 2 FEBS LETTERS May 1980 
experiments with Pa. denitrificans cells, grown on 
methanol and N03-, rotenone and antimycin A were 
solubilized in ~~dimethyl form~ide, because 
ethanol is an electron donor for methanol dehydro- 
genase [14,15]. 
3. Results 
A number of steady-state ‘dithionite-reduced minus 
nitrous oxide-oxidized’ difference spectra of cells of 
Pa. denitrificans are shown in fig. 1. From the peaks at 
522-523 nm and 550-552 nm and the shoulders at 
528-529 nm and 558-559 nm in the Q- and &region 
of the spectra, it is clear that nitrous oxide oxidizes 
b- and c-type cytochromes in Pa. denitri’ficans cells, 
grown under succinate- (a) or NO,--limitation (c) in 
the chemostat, with NzO and succinate in batch cul- 
ture (b) and with NOa- and methanol in batch cul- 
ture (d). There are no spectral indications for the 
oxidation of a-type cytochromes. To investigate the 
involvement of cytochromes in the reduction of NzO, 
kinetic experiments with cytochrome c were per- 
formed. The absorption of cytochrome c was measured 
at 55 l-552 nm minus 577 nm. The effects of several 
electron transport inhibitors (KCN, NaN, and anti- 
mycin A) on the nitrous oxide reduction rate and the 
oxidation level of cytochrome c were studied. It 
appeared that NzO-reduction by cells of succinate- 
limited chemostat cultures (pH 6.9) was very sensitive 
to cyanide. A concentration as low as 2 PM KCN in 
the cuvette, containing about 2 mg dry weight cells/ 
ml, resulted in a 50% inhibition of the rate of reduc- 
tion of nitrous oxide by endogenous substrates. 90% 
inhibition was achieved at a KCN-concentration of 
10 PM. The steady-state oxidation level of cytochrome 
c gradually decreased by increasing the KCN-concen- 
tration from 0 to 33 PM. We found essentially the 
same results with cells from a batch culture of Pa. 
denitrifcans, grown on succinate and NzO as electron 
acceptor. 
The reduction of nitrous oxide by cells from a 
Fig.1. Dithionite-reduced minus N,Ooxidized difference spectra (490-700 nm) of Pa. denitrificnns cells, grown (a) under 
succinate-limitation (pH of the chemostat culture was 6.9; 1.4 mg dry weight cells/ml); (b) with N,O and succinate in batch cd- 
true (2.4 mg dry weight cells/ml); (c) under NO,- limitation (2 mg dry weight cells/ml); and (d) with NO,- and methanol in batch 
culture (2 mg dry weight ceBs/ml). 
280 
Volume 113. number 2 FEBS LETTERS May 1980 
su~~~ate-limited continuous culture with nitrate as 
electron acceptor was also rather sensitive td azide. 
The NzO-reduction velocity was 50% inhibited by 
15 PM NaNs, while 90% inhibition was achieved at a 
concentration of 65 PM (about 2 mg dry weight cells/ 
ml). As in the case of KCN, addition of NaNs in 
increasing concentrations (O-200 PM) resulted in a 
gradual decrease of the steady-state N,O-induced oxi- 
dation level of cytochrome c. The same picture was 
obtained for the inhibition of N,O reduction by azide 
with bacteria from a nitrate-limited chemostat cul- 
ture and a batch culture, grown on succinate and N,O. 
Although these effects of KCN and NaN3 are com- 
patible with an interaction of NzO with the respiratory 
chain after cytochrome c, of course it is no con- 
clusive evidence. Therefore we also used antimycin A 
as electron transport inhibitor, which is known to 
i~ibit electron transfer between ~ytochromes b and 
c [ 15,161. After addition of 230 nmol NzO to a 3-ml 
cell suspension of Pa. denitrzficans, grown under 
nitrate limitation, a drop in the absorbance precedes 
the N,O-steady-state (fig2a,b). A possible xplanation 
for this phenomenon will be given in section 4. Fol- 
lowing the addition of 10 (lg antimycin A the oxida- 
tion level of cytochrome c due to NaO is higher than 
the one before addition of this inhibitor and the con- 
sumption of NzO takesmore time (fig.2b). Obviously, 
the reduction of NzO by endogenous substrates i
~bited by ant~yc~ A. This experiment suggests 
that cytochrome c indeed is involved in electron trans- 
port to nitrous oxide. 
,p 
AA 
To obtain more evidence in favour of this view we 
decided to grow Pa. denitrificans on methanol as 
carbon and energy source and nitrate as electron 
acceptor. It has been shown that Pa. denitrificans is 
able to grow under these conditions 1141. Reduction 
equivalents of methanol enter the respiratory chain at 
the level of cytochrome c in aerobic [ 151 and anaerobic 
cells [ 141 of Pa. denitrificans. Consequently, when 
cytochrome c is involved in NzO reduction, it must 
be possible to couple oxidation of methanol to 
formaldehyde with the reduction of NaO. A way to 
demonstrate his is to block electron transport 
between endogenous substrates and cytochrome c
and observe whether subsequent addition of methanol 
to the cell suspension results in a stimulation of NaO 
reduction. A typical experiment is shown in fig3. 
Endogenous substrates reduced the injected NzO 
within 1 min (fig.3a). After addition of 10 &g AA and 
subsequent addition of NaO to the suspension, the 
oxidation level of cytochrome c is increased and 
much more time is needed for reduction of the added 
NzO (fig.3b). When thereafter methanol (1.8 mmol) 
is added to the cuvette the N~O-educed oxidation 
level of cytochrome c is lowered and equal to that 
before the addition of antimycin A and the time 
required for N,O consumption isstrikingly reduced 
(fig.3c). Because oxidation of methanol to formalde- 
hyde can be followed by oxidation of formaldehyde 
to formate by the No-linked form~dehyde-dehy- 
drogenase and of formate to CO* by the NAD-linked 
formate-dehydrogenase, it is possible, although not 
I aA : 0 005 c 
bf 
1 mtn 
Fig.2. Absorbance at 551 nm minus 576 nm (cyt. c) of cells of Pa. denifrificcns grown under succinate-limitation (2 mg dry 
weight cells/ml) after successive addition of (a) nitrous oxide (230 nmol), antimycin A (AA; 3.3 pg/ml); and (b) nitrous oxide. 
281 
Volume 113. number 2 FEBS LETTERS May 1980 
-1” AA me thonol 
a) - 
r I nA,O 01 
Fig.3. Absorbance at 549 nm minus 576 nm (cyt. c) of Pa. denitrificans cells grown in batch culture with methanol and NO,- 
(1.5 mg dry weight cells/ml) after successive addition of (a) nitrous oxide (230 nmol), antimycin A (3.3 pg/ml); (b) N,O, 
methanol (0.6 G),; and (c) N,O. 
probable, that the antimycin A blockade is overrun 
by the extra NADH formed in this way. To exclude 
this possibility two experiments were done. Firstly, 
the tricarboxylic acid cycle intermediate L-malate 
was used as a potential source of NADH. Addition of 
excess L-malate before and after addition of antimycin 
A did not result in a stimulation of the nitrous oxide 
reduction, on the other hand, methanol stimulated 
the consumption of NzO in the presence of antimycin 
A and L-malate (results not shown). Secondly 
rotenone, an electron transport inhibitor acting on or 
in a very close proximity of iron-sulfur centre 2 of 
the NADH-ubiquinone segment of the respiratory 
chain [17], was used next to antimycin A. 300 PM 
rotenone alone inhibited the N,O-reduction by about 
60%. Addition of 1.8 mmol methanol resulted again 
in an increased nitrous oxide consumption rate, 
approximately equal to the one before addition of 
rotenone. Moreover methanol also increased the N,O- 
reduction which was inhibited by the simultaneous 
presence of 6.5 pg antimycin A/ml and 300 PM 
rotenone, while L-malate did not. The results of these 
experiments strengthen the view that cytochrome c is 
involved in electron transport to nitrous oxide. 
The reduction of nitrite by Pa. denitrificans has 
also been studied with the aid of dual wavelength 
spectroscopy. The oxidation level of cytochrome c 
(552-557 nm) is followed with time. As can be seen 
in fig.4 the back-reduction of cytochrome c after a 
NO*--puIse is biphasic (fig.4b). The first steady-state 
must result from reduction of nitrite, while the second 
one is most probably caused by reduction of nitrous 
oxide, because a N,O-pulse results in an almost iden- 
282 
tical steady-state oxidation level of cytochrome c 
(fig.4a). The addition of azide up to a concentration 
of 155 I.~M has no significant influence on the rate of 
NO,--reduction and on the NO*--induced oxidation 
level of cytochrome c. However, azide has a pro- 
nounced effect on the second steady-state (fig.4c, d 
and e). Increasing concentrations of NaN, result in a 
decreasing second oxidation level of cytochrome c 
and increasing length of the second steady-state. Addi- 
tion of NzO in the presence of 155 PM NaNa brings 
about inhibition of the reduction of nitrous oxide 
and the same oxidation level as from the NzO pro- 
duced from NOz- (figAt). Consequently the second 
steady-state, which appears after addition of NO*-, 
most probably can be attributed to the production of 
NzO from NOz-. 
4. Discussion 
The inhibition of the nitrous oxide reduction by 
antimycin A and the stimulation of the N,O-reduc- 
tion by methanol in the presence of rotenone and 
antimycin A clearly indicate the involvement of c- 
type cytochrome(s) in electron transport to nitrous 
oxide. Because we have no evidence for the presence 
of a-type cytochromes in Pa. denitrijkans cells grown 
under anaerobic conditions, it is very likely that the 
N,O-reductase is attached to the respiratory chain in 
the region of the c-type cytochromes. 
The observation that KCN and NaN,, in concen- 
trations which inhibit the N,O-reduction by endog- 
enous substrates, have no significant effect on the 
Volume 113, number 2 
_j2O 
155,UM N3- 
fiJ 9 
FEBS LETTERS 
6,uM Nj 
& 
NO3 
4 
May 1980 
Fig.4. Absorbance at 552 nm minus 577 nm (cyt. c) of Pa. denitrijkans cells grown under succinate-limitation (2 mg dry weight 
cells/ml) after successive addition of (a) N,O (230 nmol); (b) NO,- (200 nmol), azide (6 PM); cc> NO,- 9 azide (30 PM); W NO,-, 
azide (155 PM); (e) NO,-; and (0 $0. 
j NO; - -_--+N20---+N~ 
? ? 
I 
FeS Mo 
rotenone 
NAOH jFp*IFeS)l_4 +UQ-10 
T succrnoteBFp 
Fig.5. The proposed scheme of the anaerobic respiratory chain of Pa. denittificans. The concentrations of KCN and NaN, at 
which 90% inhibition of respiration occurs are given. Abbreviations: Fp, flavoprotein; FeS, iron-sulfur center; UQ-10, ubiquinone- 
10; cyt, cytochrome; AA, antimycin A; X, methoxatin [ 191; MO, molybdenum. 
283 
Volume 113, number 2 FEBS LETTERS May 1980 
NOz--reduction probably means that these inhibitors 
exert their inhibiting effect after cytochrome c, where 
the N,O-reductase itself is the most likely candidate. 
Therefore we propose that the anaerobic electron 
transport chain of Pa. denitrificans has the composi- 
tion described in fig.5 (an extended version of the 
one presented in [IS]). 
We believe that the often observed change in 
absorbance immediately after injection of N,O is 
caused by a relative slow establishment of the NzO- 
steady-state (fig.2). Besides this, sometimes a short 
O,-steady-state, preceding the N,O-steady-state, 
could be observed (for example in fig.3). This con- 
tamination with oxygen did not significantly influence 
the results reported here. 
It has been reported that azide inhibits nitrogen 
production from nitrous oxide in Pa. denittificans at 
low concentrations, bus has little effect on the pro- 
duction of nitrogen from nitrite [8]. Sidransky et al. 
[20] have suggested that an explanation of this 
phenomenon might be that nitrite can counteract the 
inhibition by azide of nitrous oxide reductase activity. 
However in fig.4 we have shown that azide has little 
or no influence on the NOz- reduction but a clearly 
inhibiting effect on the reduction of NzO produced 
from NOz- in the same order as in the case of the 
reduction of NzO itself. This result is opposite to the 
findings of Sidransky et al. [20] with Pseudomonas 
aemginosa but in agreement with those of Matsubara 
and Mori [21], who claimed that only N,O was pro- 
duced from nitrite in Pseudomonas denitrificans in 
the presence of an adequate concentration of azide. 
Concerning the role of NzO in the denitrification 
of NO*- the results presented in fig.4 do suggest hat 
NzO is an obligate intermediate in the conversion of 
NO?- to N,. A more penetrating study is necessary 
for a clear-cut conclusion about the obligate occur- 
rence of N20. 
Acknowledgements 
This investigation was supported by the Nether- 
lands Foundation for Chemical Research (S. 0. N.) 
with financial aid from the Netherlands Organization 
for the Advancement of Pure Research (Z. W. 0.). 
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284 


Electron transport to nitrous oxide in Paracoccus denitrificans 

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Electron transport to nitrous oxide in Paracoccus denitrificans

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Electron transport to nitrous oxide in Paracoccus denitrificans

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