© Copyright 2004 by Humana Press Inc.

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1085-9195/04/41:357–365/$25.00

ORIGINAL ARTICLE

Hydrogenase 3 But Not Hydrogenase 4 is Major

in Hydrogen Gas Production by Escherichia coli
Formate Hydrogenlyase at Acidic pH
and in the Presence of External Formate
Nelli Mnatsakanyan, Karine Bagramyan, and Armen Trchounian*
Department of Biophysics of the Biological Faculty, Yerevan State University,
1 Alex Manougian Str., 375025 Yerevan, Armenia

Abstract
Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H2) when
grown on glucose. H2 formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4),
those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-asso-
ciated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H2 was found
to be dependent on Hyd-4 and the F0F1-adenosine triphosphate (ATPase), whereas external for-
mate increased the activity of Hyd-3. In this study with cells grown without and with external for-
mate, H2 production dependent on pH was investigated. In both types of cells, H2 production was
increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to
N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H
and Hyd-3 but not Hyd-4 and the F0F1-ATPase. The results indicate that Hyd-3 has a major role in
H2 production at acidic pH independently on the F0F1-ATPase.

Index Entries: Formate hydrogenlyase; pH; Escherichia coli.

INTRODUCTION of acid-regulated pathways, particularly fer-

Escherichia coli has a remarkable flexibility in
adaptation to anaerobic environments with a
wide range of pH, probably by carrying out a
mixed-acid fermentation of sugars (glucose).
Anaerobic enhancement is found in a number
*Author to whom all correspondence and reprint
requests should be addressed. E-mail: Trchounian@ysu.am
mentation components such as formate hydro-
genlyase (FHL) (1). Formate produced from the
anaerobic cleavage in the absence of exogenous
electron acceptors is converted to carbon diox-
ide (CO2) and molecular hydrogen (H2) by
FHL. As the production of fermentation acids
increases, there is a concomitant decrease in
pH of the medium.
E. coli maintains cytoplasmic pH ([pH]in)
within a narrow range, approx pH 7.5 (2,3). To

Cell Biochemistry and Biophysics 357 Volume 41, 2004

358 Mnatsakanyan et al.

compensate for the pH drop, formate is taken and (2) to become resistant to N,N′-dicyclo-
up by the formate channel protein, FocA, via a hexylcarbodiimide (DCCD) or to osmotic
proton-symport mechanism. After reaching a shock and be largely dependent on Hyd-3 but
critical intracellular concentration, formate not Hyd-4 activity and not on F0F1.
activates the regulator protein, FhlA, which
then induces the expression of the fdhF gene
(encoding formate dehydrogenase H, Fdh-H) MATERIALS AND METHODS
and the hyc operon (encoding electron carries
and hydrogenase 3, Hyd-3) (4,5). Direct trans- Bacterial Strains and Growth
fer of formate to FhlA protein would also min- and Preparation of Bacteria
imize formate accumulation in the cytoplasm
The E. coli strains used in this study are
(6). This FHL pathway, constituted by the com-
detailed in Table 1. Bacteria were grown under
plex of Fdh-H and Hyd-3 and called FHL-1,
anaerobic conditions at 37°C in peptone (2%
thus provides a means of maintaining [pH]in
peptone, 0.5% NaCl, and 0.2% K2HPO4) growth
homeostasis. It has been proposed that hydro-
medium with 0.2% glucose at different pH. The
genase 4 (Hyd-4) together with Fdh-H forms a
pH of the medium was adjusted to 7.5 or 5.5 by
second FHL pathway (FHL-2) (7).
the addition of HCl and NaOH. The pH of the
The activity of FHL-1 and FHL-2 both seems
medium was decreased to 7.0 or 5.2 at the late
to be dependent on external pH and formate
logarithmic growth phase, respectively. When
concentration when the latter is included into
studied, 30 mM sodium formate was added into
the growth medium. At alkaline pH FHL activ-
the growth medium (9). Bacterial growth was
ity has recently been shown to be largely
monitored by measuring the absorbance of the
dependent on Fdh-H, Hyd-4, and the F0F1-
culture at 600 nm using a spectrophotometer.
adenosine triphosphate (ATPase) (8), and for-
Growth rate was determined over the interval at
mate added into the growth medium increases
which the logarithm of absorbance increased
the activity of Hyd-3 but not Hyd-4 (9).
linearly with time and expressed as 0.693/dou-
Although Fdh-H activity is maximal at alkaline
bling time. Fermentation under the growth con-
pH (10), FHL activity is stimulated after lower-
ditions used was described previously (18).
ing pH (1). At slightly acidic pH, H2 produc-
For DCCD inhibition studies, cells were incu-
tion became mostly dependent on Fdh-H and
bated with DCCD at 0.02 mM for 10 min at
Hyd-3 (8), but distinction between Hyd-3 and
37°C. After transfer into a medium of higher
Hyd-4 in H2 production at acidic pH has not
osmolarity, cells were subjected to an osmotic
yet been shown.
upshock, whereas a transfer into a medium of
It should be noted that although H2 can be
lower osmolarity was of downshock; medium
reoxidized by hydrogenase 1 (Hyd-1) or hydro-
osmolarity was estimated by calculations. All
genase 2 (Hyd-2) in E. coli; neither Hyd-1 nor
data were averaged from duplicate or triplicate
Hyd-2 are responsible for increase in hydroge-
independent measurements.
nase activity when cells were grown in the
presence of external formate (11).
The studies of relevant genes and of forma-
H2 Production Assay
tion of the FHL complexes are making good Hydrogen gas production by E. coli cells was
progress (7,12–15); however, their activity and measured using a pair of the oxidation-reduc-
regulation are still poorly understood. tion, platinum, and titanium-silicate electrodes
In the present article, hydrogen gas produc- (State Enterprise of Electrometer Equipment,
tion by FHL in fermenting E. coli was studied at Gomel, Belarus), as described previously (19).
alkaline and acidic pH: H2 production is In contrast to titanium-silicate electrode, a plat-
shown (1) to be enhanced after medium acidi- inum electrode is sensitive to H2 (19), allowing
fication that is dependent on external formate detection of H2 production. The rate of this

Cell Biochemistry and Biophysics Volume 41, 2004

H2 Production at Acidic pH and Formate
Escherichia coli Strains Used in the Experiments
Strain Genotype
FRAG90 lacZ gal kdpABC5 trkD1
FRAG115 FRAG90 ∆(atpB-D)
FM911 MC4100 ∆fdhFrecA56
HD700 MC4100 ∆(hycA-H)
HD705 MC4100 ∆hycE
JRG3621 MC4100 ∆(hyfB-R)::spc
JRG3933 MC4100 ∆hycE ∆(hyfB-R)::spc
MC4100 araD139 ∆(argF-lac)U169 ptsF
relA1 fibB5301 rpsL150
process was determined as the difference
between the initial rates of decreasing oxida-
tion-reduction potentials (ORP) for these elec-
trodes (mV ORP/min/mg dry weight) (8). Dry
weight was determined as described previ-
ously (17). H2 production was detected after
the addition of glucose or formate into the
assay medium. The pH of assay medium was
the same as the initial pH of growth medium
because the change in H2 production rate was
absent with decreasing pH of assay medium of
even one unit of change of pH (8). Km for for-
mate was determined using Lineweaver-Berk
plot. Hydrogen gas production data were veri-
fied by using the chemical assay (19) and the
Durham tube method (8) for bacterial cultures
grown to stationary phase.
RESULTS AND DISCUSSION
H2 Production at Alkaline and Acidic pH
and External Formate
Alkaline and acidic pH have effects on the
FHL activity in E. coli (1,20). Fermenting E. coli
Cell Biochemistry and Biophysics
359
Table 1
Source and reference
W. Epstein (Department of Molecular Genetics and
Cell Biology, The University of Chicago, Chicago Il
60637) (16)
W. Epstein (17)
A. Böck via K. Bagramyan (8)
A. Böck (Laboratory of Microbiology, Munich University,
D 80638 Munich 19, Germany) (5)
A. Böck (5)
S. C. Andrews (Division of Microbiology,
School of Animal and Microbial Sciences, The
University of Reading, Reading RG6 6AJ, UK) (7)
S. C. Andrews (8)
A. Böck (5)
MC4100, wild-type strain, grown on glucose
at alkaline or acidic pH (see Materials and
Methods) evolved hydrogen gas. The latter
was observed after osmotic upshock (Fig. 1).
H2 production rate at pH 7.5 was 5.2 mV
ORP/min/mg dry weight. This value is con-
sistent with previous work from our labora-
tory with either whole cells or protoplasts (8).
As external pH decreased, this seriously
affected H2 production rate: it was approx 1.3-
fold higher at pH 5.5 than that at pH 7.5 (see
Fig. 1). Similar increase in H2 production was
observed when cells were grown in the pres-
ence of external formate (see Fig. 1). This con-
firms previous data showing an increase in H2
production rate after a shift from alkaline to
slightly acidic pH (from 7.5 to 6.5) (8) and
indicating almost similar H2 production by E.
coli grown in the presence of formate at alka-
line pH (9).
In contrast, the growth rate of E. coli fermen-
ting glucose under anaerobic conditions
decreased significantly with lowering pH: it
was approximately threefold less at pH 5.5
than that at pH 7.5 (Table 2). 30 mM formate
added in the growth medium inhibited this
Volume 41, 2004
360 Mnatsakanyan et al.

Fig. 1. H2 production rate by E. coli MC4100 grown under fermentative conditions at slightly
alkaline or acidic pH. Bacteria were grown in glucose without (A) or in the presence of 30 mM
sodium formate (B). Cells were harvested at the mid-to-late logarithmic phase of growth by cen-
trifugation, washed with distilled water or 0.8 M sucrose, and transferred into 100 mM trisphos-
phate buffer to subject bacteria to an upshock or downshock, respectively. H2 production by
unshocked cells (washing in the buffer) was almost similar to that of downshocked ones. Data are
averaged from duplicate and triplicate measurements.

Table 2
Escherichia coli MC4100 Growth Rate Under Fermentation Conditions
on Glucose at Alkaline and Acidic pH
Growth conditions* µ (h–1) Growth conditions µ (h–1)
pH 7.5 0.70** pH 5.5 0.23
pH 7.5 + formate 0.40 pH 7.5 + formate 0.12
* When indicated, 30 mM formate was introduced into the growth medium.
**Average data from double measurements are represented.

growth rate either at alkaline or acidic pH
(Table 2). These data might be explained by
that weak acids can inhibit bacterial growth in
a pH- and concentration-dependent manner
(21,22). This is also likely because at acidic con-
ditions fermentation acids remain in a nondis-
sociated form (23).
Thus in cells grown both without and with
external formate, H2 production was increased
with lowering pH. The results are correlated
with data reported by Rossmann with cowork-
ers (20) that Fdh-H and Hyd-3 activity was
stimulated with lowering pH. But our results
seem to contradict with data of Bock and
coworkers (1,20) showing that FHL pathway is
significantly induced by formate. pH and for-
mate might induce an expression of genes
encoding FHL components (4,5); however,
these factors are not adequate in the activity of
the FHL complex.
Thus in cells grown both without and with
external formate H2 after formate was added
into the assay medium (formate was added in
the same concentration as in the growth

Cell Biochemistry and Biophysics Volume 41, 2004

H2 Production at Acidic pH and Formate
Fig. 2. Increase in H2 evolution rate with for-
mate in E. coli MC4100. The cells were grown
and assayed at pH 7.5 in the presence of 30 mM
sodium formate. The assay mixture contained
100 mM trisphosphate buffer, pH 7.5; formate
was added. Error bars represent the standard
deviations from duplicate or triplicate cultures.
medium). This production of H2 was depen-
dent on formate concentration (Fig. 2) and had
Km of 13 mM, whereas insignificant depen-
dence was observed with the cells grown with-
out external formate (data not shown). Such a
difference in H2 production was also detected
at acidic pH (data not shown). Formate-depen-
dent H2 production by whole cells is of impor-
tance, suggesting that during the growth or the
assays at alkaline and acidic pH external for-
mate can pass into the cell and stimulate FHL
pathway or initiate Fdh-H activity. If so, a way
to transport formate is proposed. Osmotic
effect of formate added either in growth or
assay medium could be ignored based on data
obtained (unpublished data).
Effects of DCCD and Osmotic Shock
on H2 Production
DCCD inhibiting the F0F1-ATPase and the
other proton-translocating membrane proteins
in bacteria (24) was used to establish the nature
and mechanism of H2 production in certain
conditions. Although H2 production by E. coli
Cell Biochemistry and Biophysics
361
Fig. 3. Inhibition of H2 evolution by DCCD
in E. coli MC4100 grown without external for-
mate at alkaline pH. For details, see Materials
and Methods; other conditions and designa-
tions are as in Fig. 2 legend.
(19,25) and Salmonella typhimurium (26) has
been shown to be inhibited by DCCD suggest-
ing a requirement in F0F1, a specific study will
be done. It is likely that FHL activity is driven
by a proton gradient established by F0F1 (7,25);
however, a direct involvement of this ATPase
in H2 production should be also considered
(8,26). Moreover, to understand H2 production
mechanisms at different pH, effect of DCCD at
acidic pH should be also determined because
DCCD inhibition was demonstrated at alkaline
and slightly acidic pH (8,19,25).
Indeed, H2 production by E. coli MC4100
grown without external formate at alkaline pH
was almost completely inhibited by 0.02 mM
DCCD (Fig. 3), confirming a requirement in
F0F1. However, at acidic pH, this inhibition dis-
appeared (see Fig. 3). It was possible to suggest
that at acidic pH F0F1 was not required or
DCCD was unable to interact with F0F1. The
latter seems to be likely because F0F1 can itself
be changed at acidic pH (27), elucidating
inhibitory effect of DCCD. But increased H2
production by an atpB-D mutant deleted for
F0F1 (FRAG115) at pH 5.5 (Fig. 4) (compare
with only residual H2 production at pH 7.5)
ruled out a requirement in F0F1.
H2 production by E. coli MC4100 grown at
alkaline pH was observed in the previously
Volume 41, 2004
362 Mnatsakanyan et al.

Fig. 4. Effect of pH on H2 production rate in E. coli wild-type, atp, hyc, and hyf mutants. Cells were
grown on glucose without (A) or in the presence (B) of 30 mM sodium formate. The assay pH was
the same as the initial value of the pH of the growth medium. For mutants, see Table 1; other des-
ignations are as in Fig. 2 legend.

described conditions after an osmotic upshock to operate in an osmotically regulated mode

(see Fig. 1). An osmotic downshock suppressed
H2 production (see Fig. 1). This may indicate
that FHL activity is sensitive to medium osmo-
larity independently on the expression level of
Hyd-4 (15). Although external osmolarity may
moderately affect fermentation enzymes such
as Fdh-H and hydrogenase (28), osmotic sensi-
tivity of Hyd-4 has not yet been studied.
Alternatively, F0F1-ATPase has been proposed
(18). Because F0F1 is required for FHL activity
at alkaline pH (8), such an osmotic effect on
FHL activity may be mediated through F0F1.
Interestingly, external formate recovered H2
production after osmotic upshock and down-
shock both at alkaline and acidic pH. This may
indicate a way in FHL activity can be in accor-
dance with the proposal that formate increases
an activity of Hyd-3 but not Hyd-4 (9).

Cell Biochemistry and Biophysics Volume 41, 2004

H2 Production at Acidic pH and Formate
H2 PRODUCTION BY HYD-3 BUT NOT
HYD-4 AT ACIDIC PH
To understand effect of formate on the FHL
activity at acidic pH, the E. coli strains deleted
for Fdh-H, Hyd-3, and Hyd-4 were studied. H2
production was only residual (0.5 to 1.0 mV
ORP/min/mg dry weight) in fdh (FM911) or
hyc (HD700, HD705) strains (see Fig. 4).
However, there was H2 production by hyf
(JRG3615) but not hyc and hyf double strain
(JRG3933) (see Fig. 4) showing that under
acidic, pH Fdh-H- and Hyd-3-constituted path-
way is responsible for H2 formation. These
results seem to contradict data of Skibinski et
al. (15) as a result of different growth condi-
tions used and assays.
Interestingly, H2 production in fdh or hyc
strains could not be recovered by 30 mM for-
mate added in the growth medium at acidic
pH (see Fig. 4). However, it was increased in hyf
strain by formate at alkaline pH (see Fig. 1),
confirming the previous finding (9). Therefore,
one can see that formate is able to induce Hyd-
3 but not Hyd-4 activity only at alkaline pH.
Such an effect is not observed with wild-type
cells, however. It is likely that total activity of
multiple transport systems and membrane-
associated enzymes may be not a sum of each
mechanism alone (16,29).
In any case, absence of H2 production in hyc
and hyf double strain (JRG3933) (seeFig. 4) con-
firms the results of Sawers with coworkers (11)
that neither Hyd-1 nor Hyd-2 are responsible
for FHL activity observed.
CONCLUSION
The results reported here indicate that H2
production by E. coli formate hydrogenlyase can
be regulated by pH and formate. Such a regula-
tion might be due to Hyd-3 but not Hyd-4 activ-
ity. Bock with coworkers (20) suggested that the
lack of hyc operon expression during respiratory
conditions was due to a lack of sufficient level of
formate to interact with FhlA. This allows us to
distinguish the significance of two FHL path-
Cell Biochemistry and Biophysics
363
ways. At alkaline pH, Hyd-4 in conjunction
with Fdh-H constituting FHL-2 (7) is responsi-
ble for H2 production (8), but Hyd-3-dependent
activity induced by formate may become essen-
tial under defined conditions (see Fig. 2,4). Hyd-
3 activity has a major role at acidic pH.
Bock and Sawers (1) propose that FHL path-
ways are a means of maintaining [pH]in.
Therefore, a requirement of the F0F1-ATPase
for FHL activity composed with Hyd-4 and
maintaining [pH]in at alkaline pH seems to be
quite reasonable. Although the operon encod-
ing Hyd-4 is suggested to have genes for pro-
ton-translocating components, no proton
pumping is detected yet. However at acidic
pH, [pH]in might be maintained in spite of
F0F1-ATPase because of FHL complex com-
posed with Hyd-3 operating to pump protons
(30) or in the other order.
ACKNOWLEDGMENTS
We thank S. C. Andrews, A. Bock, and W.
Epstein for supplying E. coli strains, valuable
advice, and discussion. The study was sup-
ported by a grant no. 0461 from the Ministry of
Education and Science of Armenia and in part
by award no. AB1–2307-YE02 of the US Civilian
Research and Development Foundation.
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