Modulation of activity of NADH oxidase from
Thermus thermophilus
through change in flexibility in the enzyme active site induced
by Hofmeister series anions
Gabriel Z
ˇolda
´k
1
, Mathias Sprinzl
2
and Erik Sedla
´k
1
1
Department of Biochemistry, Faculty of Sciences, P. J. S
ˇafa
´rik University, Kos
ˇice, Slovakia;
2
Laboratorium fu
¨r Biochemie,
Universita
¨t Bayreuth, Germany
The conformational dynamics of NADH oxidase from
Thermus thermophilus was modulated by the Hofmeister
series of anions (H
2
PO
4–
, SO
42–
, CH
3
COO
–
, Cl
–
, Br
–
,I
–
,
ClO
4–
, SCN
–
) in the concentration range 0–3
M
. Both cha-
otropic and kosmotropic anions, at high concentration,
inhibit the enzyme by different mechanisms. Chaotropic
anions increase the apparent Michaelis constant and decre-
ase the activation barrier of the reaction. Kosmotropic ani-
ons have the opposite effect. Anions from the middle of the
Hofmeister series do not significantly affect the enzyme acti-
vity even at high concentration. We detected no significant
changes in ellipticity of the aromatic region in the presence
of the anions studied. There is a decreased Stern–Volmer
quenching constant for FAD fluorescence quenching in the
presence of kosmotropic anions and an increased quench-
ing constant in the presence of chaotropic anions. All of
this indicates that active site flexibility is important in the
function of the enzyme. The data demonstrate that both the
high rigidity of the active site in the presence of kosmotropic
anions, and its high flexibility in the presence of chaotropic
anions have a decelerating effect on enzyme activity. The
Hofmeister series of anions proved to be suitable agents for
altering enzyme activity through changes in flexibility of the
polypeptide chain, with potential importance in modulating
extremozyme activity at room temperature.
Keywords: activation; conformational dynamics; flavopro-
teins; NADH oxidase; Thermus thermophilus.
The native conformation of an enzyme is produced by the
complex interaction of van der Waals interactions, hydro-
gen bonds and ionic interactions. These interactions
produce stability of the enzyme under physiological condi-
tions and prevent deleterious conformational changes from
perturbations in the environment that would cause deacti-
vation. These interactions, however, must not result in
protein rigidity because the enzyme active site requires
flexibility for optimal catalytic function. The balance of
these two tendencies is sensitively adjusted for the physio-
logical conditions at which the enzyme works. Examples
of such adjustments are enzymes from hyperthermophiles
and psychrophiles which have optimal activity at high
(> 80 C) and low (< 20 C) temperatures, respectively
[1,2]. Enzymes from thermophiles are almost inactive at
room temperature because of polypeptide and side chain
rigidity induced by higher-order interactions within secon-
dary and tertiary structures. Psychrophilic enzymes are
inactive at room temperature because the high flexibility of
their polypeptide and side chains results in partial/local or
complete unfolding of the tertiary structure. Modulation of
the balance between the rigidity and flexibility of the
polypeptide and side chains can be achieved by changing the
solvent properties. Stabilization of psychrophilic enzymes
without affecting their activity, or activation of thermophilic
enzymes without affecting their stability, is interesting for
both basic and applied protein chemistry.
The use of chaotropic agents (urea, guanidinium
hydrochloride) to activate different enzymes has been
reported in several papers [3–8]. The change in activity
resulted from conformational changes in the tertiary and
secondary structure of the enzymes studied. We have
shown recently that it is possible to activate NADH
oxidase from Thermus thermophilus with urea without
affecting the global stability of the enzyme at room
temperature [8a]. NADH oxidase (EC 1.6.99.3) from
T. thermophilus is a dimeric flavoprotein containing one
molecule of FMN or FAD per 25 kDa monomer, which
catalyzes hydride transfer from NADH to an acceptor
such as FAD, ferricyanide and oxygen [9]. It belongs to the
flavin reductase/nitroreductase family, which has similar
broad substrate specificity, fold and quaternary structure
[10,11]. Localization of the active site of NADH oxidase at
the edge of the dimeric interface (Fig. 1) is in agreement
with the fact that the active sites of enzymes are usually the
most labile part of the enzyme structure [12]. Perturbation
of either the static or dynamic state of the active site may
lead to significantly changed activity. Previous studies in
our laboratory have indicated that activation of NADH
oxidase is not achieved by conformational change but is a
result of the increased dynamics of the polypeptide/side
chain in the enzyme active site. To substantiate these
observations and analyze the role of dynamics in enzyme
Correspondence to E. Sedla
´k, Department of Biochemistry, Faculty
of Sciences, P. J. S
ˇafa
´rik University, Moyzesova 11, 041 54 Kos
ˇice,
Slovakia. Fax: + 421 55 622 21 24, Tel.: + 421 55 622 35 82,
E-mail: sedlak_er@saske.sk
Enzyme: NADH oxidase (EC 1.6.99.3).
(Received 26 August 2003, revised 8 October 2003,
accepted 28 October 2003)
Eur. J. Biochem. 271, 48–57 (2004) FEBS 2003 doi:10.1046/j.1432-1033.2003.03900.x
activity, we have investigated the effect of the Hofmeister
series of anions on the activity of NADH oxidase from
T. thermophilus.
The crystal structure provides information about the
flexibility of a given structure by comparison of temperature
B factors. Temperature B factors are atomic mean square
displacements obtained from the intensity of the diffractive
spots [13]. The absolute value of the B factor is dependent
on the refinement method and the conditions of crystalliza-
tion [14]. It is therefore only correct to compare B factors
within a particular structure, although such data must also
be handled with caution. Data from the crystals are
averaged over crystal space and time, therefore they reflect
crystal defects, static disorders and other parameters [15].
NADH oxidase has an overall low temperature factor for
the whole structure (23 A
˚
2
) [9] that is in accordance with
the high stability of the protein conformation (Fig. 1). The
flavin moiety, with a low B factor, indicates rigidity and
strong binding to the protein matrix. Trp47, the only
tryptophan residue located in close proximity to the flavin
cofactor (within 10 A
˚), is almost parallel to the isoalloxazine
ring, but the elevated temperature factor indicates it has
high flexibility. The indole ring is stabilized through
hydrophobic interactions (side chains of Ala46, Leu49,
Phe120, Ala121, Ala122, Met123) from helix F. The crystal
structure of a homologous nitroreductase in various states
shows that binding of the substrate (nicotinic acid) is
accompanied by the induced fit of helix F and helix E [10].
Rearrangement of helix F during the binding event results in
a change in the torsional angle of several residues.
Remarkably, the residues that are involved in substrate
binding through changes in their dihedral angles are those
with the highest temperature factor, and are mostly from
helix F. Similarly, the high B factors of helix F indicate that
it is highly flexible in NADH oxidase (Fig. 1). Stabilization
or destabilization of this helix would affect interactions with
Trp47 and thus the opening of the active site, which is
necessary for activity (unpublished observation). This would
indicate a mechanism of interaction of NADH substrate
with the enzyme common to this flavoenzyme family.
The Hofmeister series of anions were chosen as suitable
candidates for stabilization/destabilization of this part of
NADH oxidase. There are numerous reports on the effect
of the Hofmeister series of salts on folding and stability of
proteins [16–18] and enzyme activity in both aqueous
solutions [19–26] and organic solvents [27]. It is generally
accepted that the effect of these salts on protein results from
interactions of the salt with the polypeptide chain (enthalpic
contribution) and, indirectly, through effects on the water
structure (entropic contribution) [28–36]. For our study, we
chose the Hofmeister series of anions: H
2
PO
4–
, SO
42–
,
CH
3
COO
–
, Cl
–
, Br
–
,I
–
, ClO
4–
, SCN
–
(ordered from
kosmotropic to chaotropic). Anions are more efficient than
cations in affecting the properties of polypeptide chains. The
anion–water interaction is stronger than the cation–water
interaction, thus anions have a greater effect on water
ordering. The explanation for this is the asymmetry of the
charge in a water molecule, with the negative end of the
molecule’s dipole being nearer the center than the positive
end [34,36].
The modulation of the conformational dynamics of the
enzyme by the Hofmeister anions enabled us to show that
both stabilization and destabilization of the active site of
NADH oxidase by kosmotropic and chaotropic anions,
respectively, inhibits its activity. Application of the Hof-
meister series of anions may be a suitable approach to
modifying properties of enzymes from extremophiles. The
work presented is the result of a continuation of our interest
in understanding the role of flexibility for catalytic efficiency
of enzymes. NADH oxidase from T. thermophilus is a good
candidate for such a study because the flexibility of its
polypeptide chain is adjusted to the harsh conditions of
thermophilic bacteria. Therefore, the addition of chaotropic
agents at room temperature will not significantly perturb
the enzyme’s global structure [8a] but will modulate the
flexibility of most of its labile parts, i.e. the part of the
protein structure where the active sites are usually located
[9].
Experimental procedures
Analytical-grade biochemicals were obtained from Merck
(Germany). Urea (high purity grade) was purchased from
Sigma. Urea concentrations were determined from refract-
ive index measurements using an Abbe Refractometer AR3-
AR6. The pH values of the solutions were measured with a
Sensorex glass electrode before and after measurement at
room temperature. Only the measurements at which the pH
change was less than 0.2 pH unit were used.
Protein expression and purification
The NADH oxidase from T. thermophilus was overpro-
duced in Escherichia coli JM 108. The purification proce-
dure for the overproduced NADH oxidase was described
Fig. 1. Homodimeric structure of NADH oxidase from T. thermophilus
colored according to temperature B factor. Low B factor (< 15 A
˚
2
rigid structure) is shown by a dark blue color, intermediate B factor
(30–45 A
˚
2
) by green/yellow, and high B factor (> 60 A
˚
2
) by red. Flavin
cofactor and the closest tryptophan, Trp47, are shown. The thin line
indicates dimeric interface. The isoalloxasine ring of flavin is localized
in the rigid part of the homodimer, and Trp47 is localized on the most
flexible a-helix of the protein structure, helix F (shown within elliptical
traces).
FEBS 2003 Anion effect on activity of NADH oxidase (Eur. J. Biochem. 271)49
previously [37]. The final product provided a single band on
a SDS/polyacrylamide gel stained with Coomassie Brilliant
Blue. Before use, the protein was dialyzed against 5 m
M
phosphate buffer, pH 7.0, in the absence of FAD. The
specific activity of NADH oxidase is 1.9 UÆmg
)1
in 5 m
M
phosphate buffer, pH 7.0.
Steady-state kinetics
All kinetic measurements were performed on a Shimadzu
UV3000 spectrophotometer. The kinetic parameters were
determined from the initial decrease in NADH absorbance
at 340 nm (e
340nm
¼6220
M
)1
Æcm
)1
), at 20 C. Measure-
ments were performed after incubation (12 h) in 120 n
M
NADH oxidase, 5 m
M
phosphate, pH 7.0, containing
0.12 m
M
FAD and different concentrations of salts. The
reaction was started by the addition of 180 l
M
NADH. To
determine the K
m
value the concentration of NADH was
varied in the range 5–200 l
M
. It is not possible to use
NADH at higher concentrations because of its large
absorbance. The data were fitted to the Michaelis equation:
m¼Vmax
½NADH
½NADHþKNADH
m;app
ð1Þ
where, K
m,app
is the apparent Michaelis constant and the
apparent V
max
is the maximum velocity for the catalytic
reaction. The experimental data were also plotted according
to the Lineweaver–Burk equation and analyzed by linear
regression. Similar results were obtained using both meth-
ods. The apparent k
cat
was determined as V
max
/[E]
0
, where
[E]
0
is the total concentration of NADH oxidase in solution.
Determination of the Michaelis–Menten parameters has
not been possible in the presence of some concentrations of
iodine anions because of a spectral overlap of iodine
(product of the peroxide and iodide) and NADH. At high
concentrations of rhodanide, perchlorate, sulfate and phos-
phate, the activity of NADH oxidase is very low and
determination of the Michaelis–Menten constants has large
errors.
Temperature dependence of enzyme activity
Enzyme activity was determined in 5 m
M
phosphate buffer
containing 0.12 m
M
FAD and 120 n
M
NADH oxidase.
Reactions were started by the addition of NADH to achieve
a final concentration of 0.18 m
M
NADH. Initial velocities
were measured in the range 20–40 C. Temperature during
measurements was kept constant by temperature controlled
water circulation around the cuvette. Temperature depend-
encies were analyzed with a simple Arrhenius equation
lnkcat ¼Ea
RT þC1ð2Þ
where, Ris the gas constant (8.314 JÆK
)1
Æmol
)1
), E
a
is the
activation energy for the observed reaction, and C
1
is a
temperature-independent constant. At least five values were
plotted as ln (k
cat
)vs.T
)1
and analyzed by linear regression.
Coefficients of linearity were typically higher than 0.98.
From comparison of the Arrhenius equation and the
transition state theory, the enthalpy (DH*) and entropy
(DS*) of activation were calculated
DH¼EaRTð3Þ
Tln kcat
T

¼TDS
RþC2ð4Þ
C
2
is the temperature-independent constant.
Thefreeenergyofactivation(DG*) was calculated from
the equation:
DG¼DHTDSð5Þ
Fluorescence emission spectroscopy
The fluorescence steady-state measurements were per-
formed on a Shimadzu RF5000 spectrofluorophotometer.
The fluorescence spectrum of tryptophan residues was
obtained on excitation at 295 nm. The cuvette contained
5m
M
sodium phosphate, pH 7.0, with various concentra-
tions of salts and 2.4 l
M
dimeric protein in a total volume of
2.5 mL. Fluorescence measurements were performed at
20 C. Temperature was kept constant (± 0.3 C) by
temperature controlled water circulation.
Quenching of FAD fluorescence
The fluorophores in NADH oxidase make it possible to
perform fluorescence quenching experiments to investigate
the dynamics of the environment near the fluorophore and
the accessibility of the fluorophores to solvent. Tryptophan
moieties are widely used in quenching experiments. NADH
oxidase contains four tryptophans at different positions,
which complicates a detailed analysis. The flavin cofactor is
another fluorophore that could be used as an intrinsic probe
quenched by externally added quenchers, e.g. iodide and
rhodanide anions. The commonly used noncharged quen-
cher acrylamide is not an efficient quencher of FAD
fluorescence. The FAD fluorescence is not affected even at
relatively high (0.2
M
) concentrations of acrylamide. Fluor-
escence quenching of the FAD was performed using iodide
anions (KI). Stock solution (4
M
KI in 5 m
M
phosphate
buffer, pH 7.0) was freshly prepared to avoid oxidation of
iodide [38]. Sodium dithionite could not be used in the stock
solution of KI (inhibition of iodine formation) because of
concomitant changes in the redox state of the flavin. As it is
a single population of the FAD, it is possible to use a simple
Stern–Volmer equation:
F0
F¼1þKsm½KIð6Þ
where, K
SV
is the Stern–Volmer quenching constant.
Comparison of the values of K
SV
allows us to assess the
accessibility of the FAD cofactor and, indirectly, the
dynamics of its environment. Fluorescence was monitored
at 525 nm after excitation by 450 nm in the absence (F
0
)and
presence of various concentrations of KI (F). The linearity
of the experimental data (coefficient of linearity r0.99)
confirms the validity of the simple model (Eqn 6).
CD measurements
CD measurements were performed on a Jasco J-810
spectropolarimeter (Jasco, Tokyo, Japan) at 20 Cwith
50 G. Z
ˇolda
´ket al.(Eur. J. Biochem. 271)FEBS 2003
27.4 l
M
NADH oxidase in 50 m
M
sodium phosphate,
pH 7.0, and at different concentrations of salt. A 1 cm path-
length cuvette was used for the aromatic region. Each
spectrum was an accumulation of 10 consecutive scans.
Results
The parameters characterizing the activity of NADH
oxidase, i.e. the apparent rate constant, k
cat
, and the apparent
Michaelis constant, K
m
, strongly depend on the ionic
strength of the solution. Increasing the ionic strength from
5m
M
to 50 m
M
potassium phosphate results in a sixfold
increase in the k
cat
value, from 1.1 to 6.6 s
)1
, and a slight
decrease in the K
m
value, from 8.5 to 5.2 l
M
. In Table 1, it
can be seen that NADH oxidase is nonspecifically activated
by increased ionic strength, as all the salts studied at 0.5
M
induced an increase in the k
cat
value of the enzyme. However,
a further increase in ionic strength enabled us to distinguish
the effects of the different anions. Anions from the middle
part of the Hofmeister series, Br
–
, Cl
–
, CH
3
COO
–
, without
significant chaotropic or kosmotropic properties did not
affect the value of k
cat
even at high concentrations. On the
other hand, both chaotropic and kosmotropic anions caused
adecreaseinthek
cat
value with increased concentration. As
confirmed by parallel experiments with KCl and NaCl that
provided identical results within the margin of error, cations
do not have an effect on the kinetic parameters of NADH
oxidase. Figure 2 shows the relative activity of the enzyme in
the presence of 1
M
and 2
M
salt concentrations. Whereas the
apparent k
cat
decreased in the presence of both chaotropic
and kosmotropic anions, the apparent K
m
significantly
increased in the presence of chaotropic anions and decreased
in the presence of kosmotropic anions (Table 1). It should be
noted that the real k
cat
(k
cat real
) is underestimated when the
substrate concentration is lower than 10 ·K
m
.Fromthe
Michaelis–Menten equation (Eqn 1), we know that in the
presence of [S] ¼10 ·K
m
the apparent k
cat
is related to
the real catalytic rate, as k
cat real
¼11/10 ·k
cat
.Inthe
presence of high concentrations (> 0.5
M
) of chaotropic salt,
the substrate (NADH) concentration [S] is related to K
m
as
[S] ffi3K
m
(Table 1). In this case, k
cat real
is related to k
cat
as
k
cat real
ffi4/3k
cat
. However, at high concentrations of
chaotropic salt, the absolute value of k
cat
is 7 times lower
than in the presence of neutral salt. Thus k
cat real
in chaotropic
salts is related to k
cat real
in neutral salts as: k
cat real chaotrop
/
k
cat real neutral
¼(4/3) ·(1/7), i.e. significantly less than 1.
Therefore, even if K
m
increases by 2–3-fold, the bell shape
of k
cat real
(the relative values of k
cat real chaotrop
/k
cat real neutral
)
will not be significantly affected.
As the result of decreased conformational dynamics,
enzymes from thermophiles have very low activity at low
temperatures [39]. The protein dynamics and thermal
stability are inversely related to each other [40,41]. The
dependence of the enzyme activity on temperature in
the presence of the salts was investigated to assess how the
conformational dynamics of the active site is dependent on
the type of salt present (Fig. 2). Figure 3 shows the
temperature dependence of the relative rate constant k
cat
in the presence of 2
M
salt. For each salt, k
cat
at 20 Cwas
Table 1. Apparent rate constant (k
cat
), Michaelis constants (K
m
) and their ratio rat various concentrations of the salts. Assays were performed using
120 n
M
enzyme and 0.12 m
M
FAD in 5 m
M
potassium phosphate buffer and the given concentration of salts, pH 7.0 at 20 C. The reaction was
started by the addition of 0.18 m
M
NADH in the absence of salts. Apparent k
cat
¼1.10 ± 0.11 s
)1
,K
m
¼8.5 ± 0.9 l
M
and catalytic efficiency
k
cat
/K
m
¼r¼1.30 ·10
5
M
)1
Æs
)1
. Errors in determination of k
cat
and K
m
are within 10%. This value was calculated from several (2–5) independent
measurements. ND, Not determined.
Anion
0.5
M
1.0
M
1.5
M
2.0
M
k
cat
(s
)1
)
K
m
(l
M
)
r·10
)5
(
M
)1
Æs
)1
)
k
cat
(s
)1
)
K
m
(l
M
)
r·10
)5
(
M
)1
Æs
)1
)
k
cat
(s
)1
)
K
m
(l
M
)
r·10
)5
(
M
)1
Æs
)1
)
k
cat
(s
)1
)
K
m
(l
M
)
r·10
)5
(
M
)1
Æs
)1
)
SCN
–
5.36 15.60 3.43 1.00 30.55 0.33 0.78 ND ND 0.49 ND ND
ClO
4–
5.10 15.65 3.26 0.81 44.22 0.18 0.54 ND ND 0.27 ND ND
I
–
7.18 29.34 2.45 6.97 ND ND 3.86 ND ND 1.29 ND ND
Br
–
7.61 20.90 3.64 6.75 21.71 3.11 6.22 22.51 2.76 5.36 28.14 1.91
Cl
–
5.01 13.25 3.78 7.50 13.67 5.55 6.64 13.72 4.84 6.43 13.40 4.80
CH
3
COO
–
4.82 12.86 3.75 4.82 10.05 5.80 5.36 18.94 2.83 4.95 28.12 1.76
SO 2
42.68 11.25 2.40 2.19 7.23 3.03 1.07 ND ND 0.86 ND ND
H
2
PO
4–
3.32 13.67 2.43 2.14 10.45 2.05 1.18 8.94 1.32 0.38 ND ND
Fig. 2. Relative activity of NADH oxidase from T. thermophilus in the
presence of 1
M
(gray histogram) and 2
M
(black) sodium or potassium
salts of the designated anions, in 5 m
M
phosphate buffer, pH 7.0, at
20 °C. Activity was initiated by the addition of 0.2 m
M
NADH.
FEBS 2003 Anion effect on activity of NADH oxidase (Eur. J. Biochem. 271)51
taken as the reference value. Figure 3 shows that the slope
of the observed dependencies increases according to the
position of the anions in the Hofmeister series, in the order
from chaotropic to kosmotropic anions. This indicates that,
in the presence of chaotropic anions (SCN
–
,ClO
4–
)the
activation energy is temperature independent, whereas in
the presence of kosmotropic anions (SO
42–
,H
2
PO
4–
)itis
strongly temperature dependent.
To determine how activation parameters are affected in
the presence of various concentrations of different salts, the
temperature dependencies of the rate constants were
measured at 20–40 C (Supplementary material). Figure 4
shows a dependence of DG*, at 20 C, on the concentration
of perchlorate, chloride and sulfate anions. In the range
0.5–1.0
M
salt, there is a minimum of this dependence for all
anions studied. Whereas in the presence of chloride (neutral)
anions, the dependence achieves a local minimum, in the
case of both sulfate (kosmotropic) and perchlorate (chao-
tropic) anions, the observed minimum is global. It should be
noted that, although the observed minima are not pro-
nounced, a similar tendency of DG* is observed for all
anions, indicating that the observed dependencies are real.
A double minimum or wide minimum, in the range 0.5–
2.0
M
salt, of DG* vs. concentration is also observed for
bromide, iodide and acetate anions, i.e. anions from the
middle part of the Hofmeister series. The wide minimum in
the case of these anions also supports the relative independ-
ence of k
cat
on the salt concentration (Table 1). Only one
minimum and one relatively sharp maximum activity of
NADH oxidase is observed for both chaotropic and
kosmotropic anions. The DG* and k
cat
dependencies
correlate in this sense that the minimum of DG* is located
at a similar (same) concentration range as the maximum of
k
cat
for each given anion.
To demonstrate that the observed changes in enzyme
activity are related to conformational changes in the active
site, we analyzed the CD spectra of the peptide (data not
shown) and aromatic regions (Fig. 5). The CD spectrum of
NADH oxidase in the aromatic region consists of a positive
Fig. 3. Dependence of relative activity of NADH oxidase from
T. thermophilus on temperature in the presence of 2
M
sodium or
potassium salts of the following anions: H
2
PO
4–
(j), SO
42–
(
~
), Cl
–
(d),
Br
–
(.), I
–
(e), CH
3
COO
–
(h), ClO
4–
(,), SCN
–
(r)in5mM
phosphate buffer, pH 7.0, at 20 °C.
Fig. 4. Dependence of activation free energy (DG*) of the reaction
catalyzedbyNADHoxidasefromT. thermophilus at 20 °Cinthe
presence of 2
M
NaCl (d), NaClO
4
(,), or Na
2
SO
4
(
~
), in 5 m
M
phosphate buffer, pH 7.0.
Fig. 5. CD spectra of NADH oxidase from T. thermophilus in the
aromatic region in the presence of 2
M
NaCl (dashed line), NaClO
4
(dotted line), or Na
2
SO
4
(thick solid line) and in the absence of salts (thin
solid line), in 5 m
M
phosphate buffer, pH 7.0, at 20 °C. Inset: Nor-
malized tryptophan fluorescence (excitation wavelength 295 nm) of
NADH oxidase from T. thermophilus in the aromatic region in the
presence of 2
M
NaCl (dashed line), NaClO
4
(dotted line), ans Na
2
SO
4
(thick solid line) and in the absence of salts (thin solid line), in 5 m
M
phosphate buffer, pH 7.0, at 20 C.
52 G. Z
ˇolda
´ket al.(Eur. J. Biochem. 271)FEBS 2003