Redox-sensitive loops D and E regulate NADP(H) binding in domain III
and domain I–domain III interactions in proton-translocating
Escherichia coli
transhydrogenase
Carina Johansson
1
, Anders Pedersen
1
,B.Go¨ ran Karlsson
2
and Jan Rydstro¨m
1
1
Department of Biochemistry, Go
¨
teborg University, Sweden;
2
Department of Molecular Biotechnology, Chalmers University of
Technology, Go
¨
teborg, Sweden
Membrane-bound transhydrogenases are conformationally
driven proton-pumps which couple an inward proton
translocation to the reversible reduction of NADP
+
by
NADH (forward reaction). This reaction is stimulated by an
electrochemical proton gradient, Dp, presumably through an
increased release of NADPH. The enzymes have three
domains: domain II spans the membrane, while domain I
and III are hydrophilic and contain the binding sites for

membrane of bacteria. It couples the reduction of NADP
+
by NADH to the electrochemical proton gradient (Dp)
according to the reaction
NADH þ NADP
þ
þ H
þ
ðoutÞ
! NAD
þ
þ NADPH þ H
þ
ðinÞ
ð1Þ
‘Out’ and ‘in’ denote the cytosol and matrix, respectively,
in mitochondria and periplasmic space and cytosol, respect-
ively, in bacteria. Key features of this reaction is that?p
stimulates the rate of reduction of NADP
+
by NADH
some 10-fold and causes a shift in the apparent equilibrium
constant from 1 to approximately 500 [1].
Transhydrogenase from Escherichia coli is composed of
an a subunit of about 54 kDa and a b subunit of about
48 kDa. The active form of the enzyme is a
2
b
2
.Likeall

m, Department of Biochemistry,
Go
¨
teborg University, Box462, 405 30 Go
¨
teborg, Sweden.
Fax: + 46 31 7733910, Tel.: + 46 31 7733921,
E-mail:
Abbreviations: dI, transhydrogenase domain I; dIII, transhydrogenase
domain III; ecI, E. coli dI; ecIII, E. coli dIII; rrI, R. rubrum dI;
rrIII, R. rubrum dIII; cfTH, cysteine-free transhydrogenase;
AcPyAD
+
, oxidized 3-acetylpyridine-NAD
+
MIANS, 2-(4¢-malei-
midylanilino)naphthalene-6-sulfonic acid (sodium salt).
(Received 30 May 2002, revised 12 July 2002,
accepted 26 July 2002)
Eur. J. Biochem. 269, 4505–4515 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03144.x
surrounded by helices and irregular loops. All dIII
structures were solved using dIII with NADP
+
bound in
a nonclassical binding mode of the substrate, i.e. as
compared to other NADP(H)-dependent enzymes,
NADP
+
in dIII is bound in a reversed orientation. The
3D structure of dIII with bound NADPH is still unknown,

2
-dIII com-
plex from R. rubrum [13], loops D and E have previously
been suggested to be important in NADP(H)-binding
and possibly also coupling to proton translocation
[4,13]. The corresponding regions in ecIII are indicated in
an NMR-derived structural model (Fig. 1) and in the
amino-acid sequence with secondary structure elements
(Fig. 2).
The functional importance of loops D and E have so far
not been functionally established. In the present work, the
roles of these regions were investigated in greater detail
using site-directed mutagenesis. The results suggest that
loop D is important in communicating affinity changes in
the NADP(H)-binding site to domain I, and that both loop
D and E regulate the release of NADP(H). In support of the
previous suggestion [4,13] both loops are suggested to play a
key role in the regulation of the enzyme by an electrochem-
ical proton gradient.
MATERIALS AND METHODS
Site-directed mutagenesis
The Quikchange mutagenesis kit (Stratagene) was used to
introduce single cysteine mutations in isolated ecIII and
cfTH. A modified pET8c plasmid used for the prepar-
ation of histidine-tagged ecIII [14] served as a DNA
template in the construction of the seven cysteine
mutants ecIIIA398C, ecIIIS404C, ecIIII406C, ecIIIG408C,
ecIIIM409C, ecIIIV411C and ecIIIY431C. In addition,
four previously produced mutants, i.e. ecIIID392C [15],
and ecIIIT393C, ecIIIG430C and ecIIIA432C [9], were

1 L culture, the supernatant was loaded onto a 15-mL
Q-Sepharose HP column (Pharmacia) equilibrated with
20 m
M
Tris/HCl and 10 m
M
(NH
4
)
2
SO
4
(pH 8.0). Protein
was eluted with about 60 mL of the same buffer, after which
(NH
4
)
2
SO
4
was added to a final concentration of 1.6
M
.
After 10 h of incubation the sample was centrifuged for 1 h
at 18 000 r.p.m. in a Beckman JA20 rotor, and the
supernatant loaded onto a 15-mL Butyl Toyopearl column
(Tosohas). Protein was eluted with a gradient (300 mL) of
1.6 to 0
M
(NH

was determined by absorbance spectroscopy at 339 nm,
using e
NADPH
¼ 6100
M
)1
Æcm
)1
, whereas the content of
NADP
+
was determined by fluorescence using a modified
Klingenberg procedure as described previously [14].
Activity assays
Unless stated otherwise, transhydrogenation reactions cata-
lyzed by mutant and wild-type ecIII were assayed as
described [19] in buffer A [20 m
M
each of Mes, Mops, Ches,
and Tris, 50 m
M
NaCl (pH 7.0)], using rrI. Protein-protein
titrations were performed in which the ecIII concentration
was kept constant and the rrI concentration varied until a
maximal rate was reached.
The forward and reverse reactions catalyzed by the cfTH
and cfTH mutant enzymes were measured as described [20]
in buffer B [20 m
M
each of Mes, Ches, Tris and Hepes,

section was used and the excitation and emission slits were
both 2.5 nm.
Determination of the NADPH release rate from ecIII
by fluorescence
The release rate of NADPH from ecIII was determined
from the exponential decrease in fluorescence as bound
NADPH was released from ecIII and oxidized by glutathi-
one and glutathione reductase using excitation and emission
wavelengths of 340 and 460 nm, respectively. Oxidized
glutathione (2 m
M
) was added to the cuvette containing
1.5–2 l
M
of mutant ecIII enzyme and 1–4 U of glutathione
reductase; the fluorescence was monitored for up to 20 min.
In the case of the ecIIIV431C mutant, it was preincubated
with an equimolar concentration (about 1 l
M
)ofNADPH
for 5 min prior to the assay. The measurements were carried
out in a buffer composed of 20 m
M
Mops and 5 m
M
MgCl
2
(pH 7.0).
RESULTS
Characterization of single-cysteine mutations in ecIII

m and B. G. Karlsson,
unpublished results).
Content of bound NADP(H) in dIII
The increased affinity of dIII for NADP(H) is reflected in
the content of tightly bound NADP(H) in almost 100% of
the molecules of separately expressed ecIII [10,19]. The
presence of NADP(H) changes the absorbance maximum,
k
max
, from 278 nm for the apo-protein to 268 nm for the
NADP(H)-containing ecIII. Consequently, k
max
is an
indication of the fraction of apo-protein. An additional
Ó FEBS 2002 Redox-sensitive loops D and E in transhydrogenase (Eur. J. Biochem. 269) 4507
typical property of isolated ecIII is the percentage of bound
NADP(H), which is highly reproducible for different
preparations. In order to assess the effects on substrate-
binding site occupancy, the proportions of bound NADP
+
and NADPH were determined for the mutants generated in
this investigation i.e. ecIIIA398C, ecIIIS404C, ecIIII406C,
ecIIIG408C, ecIIIM409C, ecIIIV411C and ecIIIY431C
(Table 1).
Fig. 3. NADP(H) binding region of dIII viewed
perpendicular to (A) and parallell with the
b sheet (B). The structure was modelled using
MOLMOL
[26] based on the crystal structure of
the bovine dIII with bound NADP

with an increased content of NADPH. This effect was
most pronounced in the case of the ecIIIS404C,
ecIIIM409C and ecIIIY431C mutants, with 33%, 27%
and 28% bound NADPH, respectively, and a relatively
constant amount of apoprotein. EcIIIV411C and
ecIIIY431C did not follow the same pattern, but
contained approximately 54% and 29% apoprotein,
respectively (Table 1). Thus, it is clear that the mutations
in the redox-sensitive loops D and E strongly affected the
binding site.
In all mutants in loop D, except ecIIIV411C, the
nucleotide content was at least 89%, with a 3–5 fold
increase in the percentage of bound NADPH. (Table 1).
Clearly, the small fraction of apo-protein in these mutants
reflected the fact that this region of the protein is not directly
involved in substrate binding.
Forward reaction catalyzed by rrI + ecIII mutant mixtures
The forward reaction (reduction of thio-NADP
+
by
NADH) catalyzed by rrI and ecIII mutant mixtures was
examined by protein-protein titrations in which the ecIII
concentration was kept constant and the rrI concentration
varied until a maximal rate was reached. In this and other
dI-dependent assays in this investigation, rrI rather than ecI
was used due to the generally higher activities obtained with
this dI preparation (cf. Fig. 2). The [rrI]/[ecIII] ratio at half-
saturation is a measure of the affinity of the complex formed
for rrI and ecIII [10] and may be used to gain information
about the role of a particular amino-acid residue in the

position caused by the mutation. An obvious candidate
responsible for this effect is the conserved bD392 residue,
which is located within 6 A
˚
from bI406 in the NADP
+
-
complexed dIII crystal structure [6,7].
The [rrI]/[ecIII] at half-saturation was increased for all
cysteine mutants, but appeared to be correlated to the
maximal rate displayed by the mutants (Table 2).
Reverse reaction catalyzed by rrI + ecIII mutant mixtures
Analyses of the reverse reaction catalyzed by rrI + ecIII
mutant mixtures were performed by protein-protein titra-
tions in the same way as for the forward reaction. Like the
forward reaction, the reverse reaction is limited at pH 7.0 by
the slow release of the product bound to dIII [16], but is
several-fold faster. The maximal rate of the reverse reaction
is thus an excellent tool for examining if a mutation has
altered the rate of dissociation of NADP
+
. The [rrI]/[ecIII]
necessary for half V
max
is an indication of the dissociation
constant for the rrI + ecIII complex but, like the forward
reaction, this ratio is also dependent on how fast the product
is released from ecIII. An elevated release rate needs more dI
to saturate the reaction.
As shown in Table 3 the maximal rates obtained for both

(n
M
)
[rrI]
(n
M
)
[rrI]/
[ecIII]
V
max
(mol thio-NADPH)Æ
(mol ecIII)
)1
Æmin
)1
%
ecIII 5000 5 0.001 0.04 100
ecIIIA398C 2500 3 0.001 0.12 300
ecIIIS404C 5000 4 0.0008 0.08 200
ecIIII406C 2500 20 0.08 1.4 3500
ecIIIG408C 2500 5 0.002 0.13 325
ecIIIM409C 5000 15 0.003 0.13 325
ecIIIV411C 5000 10 0.002 0.22 550
ecIIIG430C 2500 400 0.16 11 27500
ecIIIY431C 2500 140 0.06 4 10000
ecIIIA432C 2500 10 0.004 0.13 325
Ó FEBS 2002 Redox-sensitive loops D and E in transhydrogenase (Eur. J. Biochem. 269) 4509
was kept constant and the rrI concentration varied until the
reaction rate reached a maximum. NADP(H) remains

–dIII complex [13], these results show that both
loops D and E directly or indirectly make crucial contacts
with dI. Mutations in the I406–V411 region affected the
hydride transfer efficiency and only 24–31% of the
maximal rate of the cyclic reaction could be reached,
despite saturating concentrations of rrI. Interestingly, the
ecIIIS404C and ecIIIA398C mutants were still able to
catalyze the hydride transfer at a wild-type rate, even though
the affinity for rrI had been substantially lowered.
Release rate of NADPH measured by fluorescence
The fact that NADPH, but not NADP
+
, fluoresces at
460 nm when using excitation at 340 nm, was utilized in
order to determine the rate of release of NADPH from
ecIII. By this method bound NADPH was oxidized by
glutathione reductase and glutathione. The reaction is
limited by the rate of release of NADPH and the resulting
decrease in fluorescence could consequently be used to
calculate the K
offNADPH
[22]. Fig. 4 shows the oxidation by
glutathione and glutathione reductase of NADPH bound to
ecIIIG432C and ecIIIG430C. The rate obtained with
ecIIIA432C is representative of the wild-type rate. In
contrast, the ecIIIG430C mutant showed a dramatic 110-
fold increase in oxidation rate. Based on similar oxidation
traces, K
offNADPH
values for several ecIII mutants were

M
)
[rrI]
(n
M
)
[rrI]/
[ecIII]
V
max
(mol AcPyADH)Æ
(mol ecIII)
)1
Æmin
)1
%
ecIII 4900 20 0.004 4 100
ecIIIA398C 2500 30 0.012 4 100
ecIIIS404C 4000 30 0.008 3 75
ecIIII406C 2500 250 0.100 18 450
ecIIIG408C 2500 40 0.016 4 100
ecIIIM409C 5000 150 0.030 3 75
ecIIIV411C 2500 110 0.044 5 125
ecIIIY431C 2500 240 0.096 18 450
Table 4. Properties of the cyclic reaction catalyzed by wild-type and mutant ecIII in the presence of rrI. The values are estimations from protein-
protein titration curves in which the ecIII concentration was fixed and the rrI concentration varied (not shown). The assays were performed as
described in Materials and Methods. The [ecIII] values refer to the fixed enzyme concentration used in the titrations. The [rrI] values correspond to
the concentration of rrI at
1
/

ecIII 40 70 50 4900 100
ecIIIA398C 12.5 300 294 5000 102
ecIIIS404C 12.5 95 89 4900 100
ecIIII406C 40 250 230 1400 29
ecIIIG408C 40 200 180 1400 29
ecIIIM409C 40 450 430 1200 24
ecIIIV411C 40 150 130 1500 31
ecIIIY431C 40 470 450 800 16
4510 C. Johansson et al.(Eur. J. Biochem. 269) Ó FEBS 2002
resulting mutant protein was characterized with respect to
catalytic activities. The kinetic properties of the various
transhydrogenation activities catalyzed by the cfTHG430C
mutant are summarized in Table 6. The severe effect of
mutating this conserved glycine into a cysteine was clearly
reflected in the resulting maximal rates of the reverse,
forward and cyclic reactions which were all between 7 and
10% of the corresponding wild-type cfTH activities. The K
m
for NADPH in the reverse reaction was 40 times higher
than that for wild-type cfTH whereas the K
m
for thio-
NADP
+
in the forward reaction, remained essentially
unchanged. Consequently, the cfTHG430C mutation resul-
ted in a substantial loss of affinity for NADPH, while the
affinity for NADP
+
was unaffected (Table 6).

ecIIIM409C 0.004 0.8
ecIIIG430C 0.560 110
ecIIIY431C 0.025 5
ecIIIA432C 0.011 2
Table 6. Kinetic parameters of purified cfTH and cfTHG430C enzymes. The K
thioÀNADPþ
m
, K
NADPH
m
and V
max
values were derived from Eadie-Hofstee plots. The fixed concentration of AcPyAD
+
used in the
reverse reaction was 400 l
M
for cfTH and 1500 l
M
for cfTHG430C. The fixed concentration of NADH used in the forward reaction was 400 l
M
for cfTH and 800 l
M
for cfTHG430C. For the cyclic reaction
the following concentrations were used for cfTH; 200 l
M
NADP
+
,200l
M

(l
M
)(lmol AcPyADH)Æ(mg cfTH)
)1
Æmin
)1
%
K
NADPH
m
(l
M
)(lmol AcPyADH)Æ(mg cfTH)
)1
Æmin
)1
%
cfTH 0.5 100 67 3.6 100 4 13.4 100
cfTHG430C 0.05 10 47 0.3 8 175 0.9 7
Ó FEBS 2002 Redox-sensitive loops D and E in transhydrogenase (Eur. J. Biochem. 269) 4511
were all analyzed in buffer C. The maximal rates listed in
Table 7 were obtained from the pH optima of the
respective mixtures of domains or enzymes. For the
reconstituted system, rrI + ecIII, there was a pronounced
difference between the rates of the forward and reverse
reaction, the reverse rate being 150 times higher. For the
reconstituted rrI + ecIIIG430C mutant, this difference in
rates had largely disappeared, the reverse reaction being
only 4.5 times faster (Table 7). Thus, the ratio between the
forward and the reverse reaction rates was approximately

. The distance between
I406 and the nicotinamide ring is about 6 A
˚
[13]. Based on
this structural information, it was proposed that loop D and
E(theÔlidÕ)wereinvolvedintheinteractionwithdIand
binding of NADP
+
, respectively, especially the change from
the ÔoccludedÕ state to the open state and vice versa [2,4,13].
These conclusions were supported by NMR studies where
NADP
+
hadbeenreplacedbyNADPHintheR. rubrum
dIII [11]. Studies of ecIII by NMR [10], especially chemical
shifts caused by the presence of ecI and/or NADP(H),
showed that the ecIII itself and the ecI–ecIII interface were
altered upon a change in the redox-state of the bound
NADP(H).
Despite the large amount of structural information
available for loops D and E, their functional roles have
not been systematically examined by site-directed mutage-
genesis. Residues subjected to mutagenesis were therefore
chosen based on the magnitude of their chemical shift
perturbations in the NMR experiments [10], i.e. especially
G389-I406 and G430-V434, and surrounding residues, and
on their conservation among 62 transhydrogenase gene
sequences. Some of these mutants, i.e. ecIIIK424C,
ecIIIH345C, ecIIIA348C, ecIIIR350C [15], and ecIIIT393C,
ecIIIR425C, ecIIIG430C and ecIIIA432C [19], were partly

pH
rrI + ecIII 0.04 7.0 6 7.0 2800 7.0
rrI + ecIIIG430C 7 7.0 32 7.0 850 7.0
cfTH 48 7.0 370 7.0 1370 6.0
cfTHG430C 5 7.0 31 5.0 87 6.0
4512 C. Johansson et al.(Eur. J. Biochem. 269) Ó FEBS 2002
The role of loop D
Mutations were introduced in loop D in ecIII, a region that
was suggested by NMR experiments to be involved in
redox-regulation of the interactions of ecIII with ecI [10]. In
addition to ecIIIA398C, ecIIIS404C and ecIIII406C, muta-
tions were also made in the adjacent G408-M409-P410-V/
I411 region, i.e. ecIIIG408C, ecIIIM409C and ecIIIV411C.
Earler made mutants in this region include ecIIID392C [15]
and ecIIIT393 [10]. All of these mutants show a varying
content of bound NADP(H), the most conspicuous being
ecIIIR425C [10] and ecIIID392C [15] which are isolated as
100% apo-form, and ecIIIV411C which has 54% apo-form
(Table 1).
Introduction of a cysteine in the S404 position of ecIII
had little or no effect on the substrate-binding properties
and affinity for dI. This ecIII mutant behaved wild-type like
in all experiments. It should be noted, however, that the
replacement of serine with cysteine is a rather mild
substitution. When the ecIIIS404C mutant was reacted
with MIANS and NEM (A. Pedersen, C. Johansson and
J. Rydstro
¨
m, unpublished results), the reverse reaction was
stimulated by a factor of 1.6 and 2.0, respectively, indicating

¨
m, unpublished,
results). This difference might reflect a movement of the
I406C side-chain, and probably the entire loop D, that is
coupled to events in the NADP(H)-binding site.
Mutations in the G408-M409-P410-V/I411 region did
not influence the substrate-binding characteristics of ecIII,
but caused a substantial decrease in the affinity for domain
I. The K
d
for the rrI + ecIIIM409C complex was ninefold
higher than that for wild-type ecIII and the maximal rate of
the cyclic reaction was only about 24%, indicating that the
complex was distorted by this mutation.
The role of loop E
Mutations in loop E in isolated ecIII had dramatic
consequences on its interactions with both dI and
NADP(H). The most remarkable property of these mutants
was the high dissociation rates of NADP(H), suggested by
both high forward (Table 2) and reverse (Table 3) reaction
rates, particularly the fast release of NADPH in fluores-
cence measurements. The ecIIIG430C and ecIIIA432C
mutants were earlier shown to be catalyze 850- and 150-fold
increased reverse rates, respectively [10]. In the presence of
rrI, the forward reaction catalyzed by the ecIIIG430C and
ecIIIY431C mutants was 275 and 100 times faster, respect-
ively, than that of wild-type ecIII. As this reaction catalyzed
by the rrI + ecIII complex normally is limited by the slow
release of the NADPH, these high rates indicate high
dissociation rates of NADPH. Indeed, measured directly, a

respectively, higher than that for the rrI + ecIII complex.
As expected, the rate of the cyclic reaction was inversely
proportionate to the K
d
value (Table 4 and [10]), whereas
the rate of the reverse reaction catalyzed by the
ecIIIG430C [10], ecIII/431C (Table 3) and ecIIIA432C
[10] mutants was proportionate to the [rrI]/[ecIII] ratio at
1/2 V
max
, respectively.
The inherent tight binding of NADP(H) in isolated
domain III has previously been explained by the hypo-
thesis that separately expressed dIII mimics the ‘occluded’
conformation of domain III in the intact enzyme [2,4,16].
This occluded conformation is assumed to correspond to a
state in which the hydride transfer step takes place [2].
Consequently, the activities of the reverse and forward
reactions catalyzed by rrI + ecIII, in which release of
NADP(H) is limiting, were very low as compared to that
of intact cfTH (Table 7). The comparison of the rates of
the various transhydrogenation reactions catalyzed by
wild-type and mutants of rrI + ecIII complexes and
intact transhydrogenases allowed an important conclusion
regarding the differences between isolated ecIII and dIII
as it functions in intact transhydrogenase. The ratio of the
rates of the reverse and forward reactions catalyzed by
rrI + ecIIIG430C was similar to that for cfTHG430C, i.e
approximately 7 (Table 6). Normally, this ratio is the
same for cfTH, but 150 for rrI + ecIII (Table 6).

and release of NADP(H) [2,4], loop E may play a major role
in the coupling mechanism of transhydrogenase. In addi-
tion, the changes in affinity for NADP(H) could be
communicated to domain I as loop E forms part of the
region that confers a redox regulation of the ecI + ecIII
complex interface.
In conclusion, the present results suggest that loop D is
involved in the interactions with domain I and that the I406
residue is a potential candidate for the regulation of the
accessibility of the side-chain of the D392 residue that is
essential for proton-pumping. Moreover, the results support
the notion that loop E functions as a mobile lid [4,7,13],
regulating the release of NADPH, a step that probably is of
central importance in the coupling mechanism of transhy-
drogenase. It is proposed that movements of these two loops
work in concert to regulate the affinity of NADP(H),
protonation events in their surroundings and to communi-
cate these changes to domain I.
ACKNOWLEDGEMENTS
This work was supported by the Swedish Natural Science Research
Council. AP acknowledges a grant from the Sven and Lilly Lawski
Foundation.
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Ó FEBS 2002 Redox-sensitive loops D and E in transhydrogenase (Eur. J. Biochem. 269) 4515