Spectroscopic investigation of the reaction mechanism of
CopB-B, the catalytic fragment from an archaeal
thermophilic ATP-driven heavy metal transporter
Christian Vo
¨
llmecke, Carsten Ko
¨
tting, Klaus Gerwert and Mathias Lu
¨
bben
Lehrstuhl fu
¨
r Biophysik, Ruhr-Universita
¨
t Bochum, Germany
Introduction
The biological role of P-type ATPases is ATP-driven
transport of ions against their concentration gradients
along membranes. They form a heterogeneous super-
family, which has been divided into several categories
according to sequence similarity and substrate specific-
ity [1]. Among these, the Ca- and Na ⁄ K-ATPases
belong to the well-studied class II enzymes. Another
large group (class Ib) comprises the so-called CPX-
ATPases, which are responsible for the import or
export of soft metals, such as copper, zinc, silver, lead,
cobalt or cadmium.
CPX-ATPases are evolutionarily related and have a
common architecture, consisting of a hydrophobic part
with a predicted eight transmembrane helices, in which
the central ion binding site resides. Their peripheral

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(Received 14 May 2009, revised 24 July
2009, accepted 21 August 2009)
doi:10.1111/j.1742-4658.2009.07320.x
The mechanism of ATP hydrolysis of a shortened variant of the heavy
metal-translocating P-type ATPase CopB of Sulfolobus solfataricus was
studied. The catalytic fragment, named CopB-B, comprises the nucleotide
binding and phosphorylation domains. We demonstrated stoichiometric
high-affinity binding of one nucleotide to the protein (K
diss
1–20 lm). Mg
is not necessary for nucleotide association but is essential for the phospha-
tase activity. Binding and hydrolysis of ATP released photolytically from
the caged precursor nitrophenylethyl-ATP was measured at 30 °C by infra-
red spectroscopy, demonstrating that phosphate groups are not involved in
nucleotide binding. The hydrolytic kinetics was biphasic, and provides
evidence for at least one reaction intermediate. Modelling of the forward
reaction gave rise to three kinetic states connected by two intrinsic rate
constants. The lower kinetic constant (k
1
= 4.7 · 10
)3
s
)1
at 30 °C) repre-
sents the first and rate-limiting reaction, probably reflecting the transition
between the open and closed conformations of the domain pair. The subse-
quent step has a faster rate (k

which precluded the use of site-directed mutant proteins
or group-specific isotopically labelled proteins for spec-
tral comparisons, which are crucial for assignment of
protein-associated absorbance difference bands.
Bacterial CPX-ATPases consist of a single subunit
and can be readily expressed in the heterologous host
Escherichia coli. Proteins of this subclass are therefore
suited for site-directed mutagenesis, and would be ideal
candidates for the study of molecular reaction mecha-
nisms. However, the 3D structure, which would be
enormously helpful in understanding the molecular
mechanism of CPX-ATPase, is unknown. Previously,
various attempts at comparative modelling have created
a structural model of the holoenzyme [14–16]. Using
‘divide and conquer’ strategies, the partial 3D structures
of various modules have been determined, such as the
HMA domain of the CPX-ATPases of Listeria mono-
cytogenes and Bacillus subtilis, the N ⁄ P and A domains
of Archaeoglobus fulgidus CopA and the N ⁄ P domains
of Sulfolobus solfataricus CopB [17–21]. In order to
study the reaction mechanism of the ATPase, we
explored here whether a truncated variant of CopB could
act as model for the holoenzyme. Therefore, the soluble
catalytic fragment CopB-B, comprising the hydrophilic
N ⁄ P domains of CopB from Sulfolobus solfataricus
(Fig. 1) was probed. The activities of the catalytic
fragment were investigated using enzymological, fluores-
cence [22] and infrared spectroscopy [23] methods.
Results
Nucleotide binding to CopB-B

phate region, and should not be interpreted as assign-
ing possible protein interaction sites to functional
groups of the substrate [21].
The binding interaction of CopB-B with various
adenine nucleotides under stoichiometric conditions
was qualitatively verified by gel filtration of the nucleo-
tide ⁄ protein complex and subsequent analysis of the
nucleotides of the collected fractions using high-perfor-
mance liquid chromatography on a reverse-phase
column (see Appendix S1). Equilibrium binding of
nucleotides was quantitatively investigated using the
fluorescent analogue 3¢-N-methylanthraniloyl-ATP
(mant-ATP) (Fig. 2). Binding to the protein at saturat-
ing nucleotide concentrations resulted in a 4.5-fold
increase of emission intensity, demonstrating that the
fluorophore becomes positioned in a location that is
less exposed to quenching molecules. In addition, the
emission peak shifts from 444 to 434 nm, indicating
that, upon binding, the fluorescent substituent moves
from the hydrophilic solvent into the more hydropho-
bic protein environment (Fig. 2A). To assess the speci-
ficity of binding, we displaced the bound mant-ATP
by addition of excess ATP. The kinetic dissociation of
the mant-ATP ⁄ protein complex appears to be rela-
tively rapid, as the process could not be resolved
within the manual mixing time. This reversible ligand
competition shows that the nucleotide portion of the
analogue is responsible for the specific interaction with
the protein.
A titration of the nucleotide binding site under stoi-

units; [E
t
] = total concentration of CopB-B. (C) Determination of
ligand dissociation constants from competitive titrations of 0.5 l
M
CopB-B with mant-ATP in the presence of the indicated total con-
centrations ([L
0
]) of ATP (squares), ADP (circles) and AMP (trian-
gles) for determination of the apparent K
app
diss
. Data were analyzed
according to Eqn (4). The bars indicate K
app
diss
errors from individual
fits of titration curves obtained at fixed competitor concentrations.
Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo
¨
llmecke et al.
6174 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS
curve under experimental condition 1 described in the
Experimental procedures (mant-ATP held constant) is
shown in Fig. 2B. A non-linear regression fit of the
measured data results in a binding constant of 1 lm
according to Eqn (1). The same results were obtained
when titrations were performed under experimental
condition 2 (protein held constant). Nucleotide binding
was highly sensitive to the salt concentration, with the

nucleotide. Based on a series of fluorescence titrations
of mant-ATP to CopB-B in the presence of various
competitor concentrations [L
0
], the binding constant of
the nucleotide can be determined from the slope of
the linear plot of the apparent binding constants K
app
diss
and [L
0
]. With the ligand ATP, a binding constant
K
lig
diss
of 10 lm was obtained (Fig. 2C). The non-hydro-
lysable analogue adenosine 5¢(b,c-imido)triphosphate
(AMPPNP) had binding properties comparable to
those of ATP (Table 1). Structural modification of the
purine moiety had no significant effect, as ATP and
GTP showed affinities in the same order of magnitude.
On the other hand, ADP, the product of the ATPase
reaction, bound to CopB-B with approximately half of
the affinity of ATP. AMP had a comparable K
lig
diss
of
approximately 30 lm (Table 1), which indicates that
the b- and c-phosphate groups are less important for
the binding process than the base ⁄ sugar part. A

constant K
lig
diss
of ATP (Fig. 3A). Nevertheless, these
relationships are consistent because high substrate con-
centrations are needed to overcome the high-affinity
binding of the product ADP (Table 1) under kinetic
steady-state conditions. No production of inorganic
phosphate was observed in the absence of Mg
2+
, which
indicates that Mg-ATP is the substrate of CopB-B.
Furthermore, the ATPase activity increased in the
temperature interval between 20–70 °C (Fig. 3B). At
higher incubation temperature, the thermophilic protein
starts to denature. The protein is an active hydrolase
under single turnover conditions at room temperature
as demonstrated for stoichiometric loading with
Mg-ATP by HPLC analysis (data not shown). Notably,
the catalytic fragment is still active at a temperature of
30 °C, which is important with regard to our approach
to investigate the molecular reaction mechanism using
time-resolved FTIR spectroscopy (see below).
Table 1. Binding of nucleotides to the catalytic fragments of CPX-
ATPase CopB. The interaction is quantified from apparent binding
constants obtained by competitive binding titration of mant-ATP in
the presence of various concentrations of nucleotides. Unless
indicated otherwise, Mg
2+
was omitted to prevent phosphatase

FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6175
Molecular interaction of ATP with CopB-B
Transient reactions were routinely observed using
rapid mixing techniques. However, these are difficult
to perform in the case of time-resolved FTIR spectros-
copy. The use of cuvettes with an optical path length
of less than 10 lm is imperative due to the high absor-
bance of water in the infrared region. Under these cir-
cumstances, the reaction mechanism of the ATPase
can best be studied by release of ATP from the caged
precursor compound cgATP by photochemical activa-
tion according to the following reaction scheme:
where k
ph
represents the kinetic constant describing
the fast photolytic cleavage of the caged compound. It
is clear from equilibrium binding of cgATP (Table 1)
that the CopB-BÆcgATP complex has already formed
before photolysis. To this end, samples were prepared
in special FTIR cuvettes with high concentrations of
CopB-B and the Mg
2+
complex of cgATP. The com-
ponents were present at a 1 : 1 ratio in order to pre-
vent more than a single catalytic turnover. Upon light
activation for an integrated duration of 0.12 s, the
genuine substrate is released.
In order to clearly differentiate the post-flash IR
absorbance signals into the photochemical processes of
ATP release [25] and the subsequent hydrolytic protein

resolved by global fit analysis (see below) were assigned
using substrate isotopologues [26]. The IR difference
spectrum recorded directly after photo-release indicates
the binding state of the pre-existing CopB-BÆATP com-
plex before the start of hydrolysis (Fig. 4B). Negative
difference bands at 1525 and 1347 cm
)1
refer to the
symmetric and anti-symmetric vibrations of the NO
2
group in cgATP identified previously [25]. For compari-
son and further band assignment, spectra were run
under identical conditions with ATP isotopically
labelled at specific positions, i.e. by chemical substitu-
tion of
16
O for
18
O in the phosphate groups. The
increase in weight results in higher reduced masses of
the molecular oscillators and therefore lowering of the
A
B
Fig. 3. Catalytic properties of CopB-B. (A) Substrate kinetics of
10 l
M CopB-B with Mg-ATP at 70 °C. (B) Temperature dependence
of 10 l
M CopB-B at an Mg-ATP concentration of 5 mM. The pH of
the Na ⁄ Mes incubation medium at various temperatures was kept
constant between 5.9 and 6.2.

)]. Minor deviations of the observed band
frequencies from tabulated values could relate to the
pH dependence of phosphate resonances and their
shifts induced by formation of Mg complexes [25,27].
Further band assignments are summarized in
Table 2 (corresponding spectra not shown). It is worth
noting that, in the CopB-B-bound state, the phosphate
vibrations are coupled, as seen for example in the
absorbance band at 1123 cm
)1
, which is shifted to
1101 cm
)1
irrespective of placement of the
18
O label in
the b or a group. Strong phosphate coupling is other-
wise known only for nucleotides in free aqueous solu-
tion [26]. In sharp contrast to CopB-B, phosphate
coupling is abolished in the case of the GTP-binding
protein Ras, in which phosphate absorbances are
significantly shifted with respect to the non-bound
state [26] and coupling between the a and b groups is
removed. The close similarity of IR difference spectra
of nucleotides in the presence and absence of CopB-B
leads to the conclusion that the phosphate groups of
ATP apparently do not contribute significantly to the
formation of the nucleotide–protein complex; instead
they are positioned in a hydrophilic environment or
even remain solvent-exposed.

(continuous line) was obtained with unlabelled ATP. The absor-
bance difference band (hatched upwards) is downshifted to another
position (hatched downwards) in the spectrum obtained using
c-
18
O
4
-labelled ATP under otherwise identical conditions.
C. Vo
¨
llmecke et al. Hydrolytic mechanism of the catalytic CPx-ATPase domain
FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6177
extremely fast initial photolytic phase (Fig. 4A), the
relatively slow hydrolytic reaction rates were kinetical-
ly analysed by global fitting. We were able to simulate
the spectral absorbance changes by multi-exponential
regression analysis with two rate constants k
1
app
and
k
2
app
. Thus, to describe the overall hydrolysis reaction,
we derived a tentative working model displayed in
Scheme 2, consisting of the pre-hydrolytic initial state
(CopB-BÆATP), an intermediate (I) and a final state
(CopB-BÆADP):
In addition to the quickly formed so-called photoly-
sis spectrum ‘CopB-BÆATP–CopB-BÆcgATP’ (Fig. 4B),

)1
(Fig. 5A, bottom).
Kinetic modelling of CopB-B’s ATPase reaction
If the apparent rate constants k
1
app
and k
2
app
derived
from the global fitting differ only by a factor of four,
as in our case (Table 3), analysis of the spectral com-
ponents of the amplitude spectra )a
1
and )a
2
(Fig. 5A) becomes complicated due to mixing of states.
In such a case, apparent and intrinsic rate constants
often deviate drastically from each other. For deter-
mination of intrinsic rate constants for the ATP hydro-
lysis, we applied the kinetic modelling program
KinTek Global Kinetic ExplorerÔ [28] using the fol-
lowing model (Scheme 3) with intrinsic rate constants
k
1
, k
)1
, k
2
and k

) = 1 and c
¥
(CopB-BÆATP) = c
0
(P
i
) = 0. Due to
the unknown absorption coefficient of the intermediate
I, we arbitrarily averaged both normalization factors for
CopB-BÆATP and P
i
to obtain a reference for its relative
concentration. Based on these assumptions, we consid-
ered models 1 and 2 described below.
Model 1 is a simulation based on free parameter
optimisation of the program, and yields k
1
= 4.7 ·
10
)3
s
)1
, k
)1
= 3.0 · 10
)4
s
)1
, k
2

O
2
-a (cm
)1
)
Photolysis 1123 v
s
a-b-ATP
a
1101 1101 u
1137 v
as
c-ATP 1089 u
1213 v
as
b-a-ATP 1206 u
1250 v
as
a-b-ATP sp.
b
u
)a
1
c
1108 ATP ⁄ ADP sp. d
)a
2
1078 v
s
(PO

Scheme 3.
Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo
¨
llmecke et al.
6178 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS
k
)2
=1.0 · 10
)4
s
)1
(Table 3). The corresponding con-
centration profiles of the three components (Fig. 6A)
agree well with our normalized data (squares), indicat-
ing reasonable selection of scaling factors. The main fea-
tures of this kinetic model are that k
2
> k
1
(k
2
$ k
1
app
;
k
1
$ k
2
app

and k
)1
= k
)2
= 0. Given these assump-
tions, Fig. 6B shows that the measured normalized
absorbance at 1255 cm
)1
, indicative of the time course
of educt concentration, clearly deviates from its calcu-
lated concentration profile. Moreover, this simulation
yields notably higher concentrations of the intermedi-
ate than the former model.
To further check the rationality and stability of our
model assumptions, we varied the extinction coefficient
of the intermediate I for both models 1 and 2 (see Dis-
cussion and Fig. S3). In neither case did the simulated
curves give better fits to the measured data than the
ones displayed in Fig. 6A. Of even greater significance
than the extinction coefficient of the intermediate I are
the concentration profiles of educt and product, which
both match optimally with curve fit 1. In summary, fit
1, based on program-chosen intrinsic constants, maps
the time course of the reactant concentrations much
better than fit 2, based on fixed constants; fit 1 therefore
supports a credible model. The data from model 1 were
thus used to calculate the relative contributions of the
states to the amplitude spectra )a
1
and )a

app
()a
1
, top)
and the rate k
2
app
()a
2
, bottom). (B) Band assignment verifying
phosphate production in the k
2
app
transition by comparison of ampli-
tude spectra recorded with
16
O (continuous line) and
18
O (dotted
line) ATP isotopologues (top) and after double difference calculation
(
16
O–
18
O difference spectra) (bottom). The hatched zones indicate
the loss of c-ATP in the precursor state and the formation of
inorganic phosphate at the final stage of the phosphatase reaction.
Table 3. Kinetic constants obtained by various theoretical methods
of examination.
Kinetic step

1.0 · 10
)4
a
The steps are defined according to Schemes 2 or 3.
b
Rate con-
stants were calculated by data approximation via global fit [apparent
rate constants (k
i
app
)] or via kinetic modelling (model 1; k
i
).
C. Vo
¨
llmecke et al. Hydrolytic mechanism of the catalytic CPx-ATPase domain
FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6179
the bands facing downwards derive from the final ADP
state. The intensities are 38% compared to pure states.
The bands facing downwards in )a
2
derive from both
the intermediate state (38%) and the initial ATP state,
and the bands facing upwards in )a
2
derive from the
final ADP state.
The intermediate state during ATP hydrolysis
As the absorbances of the intermediate are facing
upwards in )a

and )a
2
amplitude
spectra (Fig. 5A). Signals in this region point to the
prevalence of protonated aspartic or glutamic acid side
chains either undergoing protonation ⁄ deprotonation
reactions or conformational reorganizations.
End product state of CopB-B-catalysed ATP
hydrolysis
As mentioned above, the bands of the end product are
the bands facing upwards in )a
2
(Fig. 5A, bottom).
The shift of the positive band from 1078 to 1043 cm
)1
upon c-
18
O-ATP labelling clearly demonstrates the for-
mation of free inorganic phosphate in the product
state, which becomes obvious in the absorbance differ-
ence, and especially in the double difference spectrum
(Fig. 5B). Further product bands are found at 1220
and 1098 cm
)1
, which are assigned to the a and b
vibrations of the hydrolysis product ADP (Table 2).
Isotopic labelling at the c-
18
O-ATP position shifts the
negative m

the normalized measured absorbances of educt at 1255 cm
)1
(CopB-BÆATP), of reaction intermediate at 1338 cm
)1
(unidentified
protein functional group) and of product at 1078 cm
)1
(inorganic
phosphate P
i
) are plotted (squares). Simulations were performed
under the two conditions: fit 1, for which intrinsic rate constants
k
1
, k
2
, k
)1
and k
)2
were optimized using the program KinTek Global
Kinetic ExplorerÔ (continuous lines) (A), and fit 2, for which fixed
rate constants k
1
= k
1
app
and k
2
= k

bond in Ca-ATPase [30]. At 30 °C, the ATP hydrolysis
rate of CopB-B is fairly low, but still allows observa-
tion of the reaction with substrate produced from
cgATP under single turnover conditions with a half-life
of approximately 3 min.
Nucleotide binding to CopB-B
In order to precisely define the reaction conditions of
the spectroscopically observed CopB-B reaction with
ATP, the interaction of nucleotides with CopB-B was
explored by direct equilibrium binding or competition
assays using the fluorescent nucleotide mant-ATP. As
has also been observed with other purine nucleotides,
cgATP has high affinity for CopB-B, which proves
that, within the applied concentration range of the
FTIR experiments ([cgATP]
0
>> K
diss
lig
(cgATP)), a
complex between the components has already formed
before photolysis. After laser flash photolysis of
cgATP, the substrate ATP is released at the position
of its binding site, so this aspect of complex associa-
tion can be ignored for the kinetic interpretation of
our data.
The nucleotide binding spectrum of CopB-B
obtained immediately after photolysis (Fig. 4B) shows
a striking similarity to the spectrum of free ATP,
which is in sharp contrast to observations made with

strated earlier for Ca-ATPase [12], could not be
resolved in our samples. Details on the as yet unre-
solved catalytic steps may be disclosed after careful
adjustment of reaction conditions by either freezing
otherwise invisible intermediates or investigating
site-specific mutants.
Kinetic process of ATP hydrolysis
Kinetic modelling requires theoretical values for cata-
lytic events as an input, but delivers a more detailed
interpretation of measured data than global fitting.
Obvious deviations from recorded absorbance data
occur, as in fit 2 (Fig. 6B), in which the intrinsic rate
constants were arbitrarily chosen as equal to the
apparent constants. In contrast, concentration profiles
closely matched the absorbance time courses in the
case where the intrinsic constants were adjusted (fit 1,
Fig. 6A). The educt decrease (CopBÆATP) takes place
with the slower intrinsic rate k
1
, and the product
increase (P
i
) proceeds with the faster rate constant k
2
.
Therefore, a relatively low concentration of intermedi-
ate is seen, as the decay rate k
2
of intermediate I is
faster than its production rate k

large change in the amide I band upon intermediate
formation, and the subsequent reversal of this change
during the product formation. Once this catalytically
active conformation is formed, the subsequent
processes are fast. The reaction intermediate could
be CopB-BÆATP in a short-lived ‘closed conformation’
or another rapidly forming and decaying, and so far
unresolved, state.
A model of CopB-B-catalysed ATP hydrolysis
Our data on equilibrium binding and kinetic features
of CopB-B can be combined to form a consistent
hypothetical model of the ATP hydrolysis reaction
sequence (Fig. 7). CopB-B in its ‘open conformation’,
with distant nucleotide-binding and phosphorylation
domains, binds nucleotides such as mant-ATP, ATP,
ADP and cgATP with relatively high affinity even in
the absence of Mg
2+
, as represented by species 1. A
pre-photolytic state complex of CopB-B loaded with
cgATP (species 1a) could easily be transformed to the
ATP-bound state (species 1) by UV irradiation. For
electrostatic reasons, the open conformation may be
even more favoured in the nucleotide-bound state,
because a number of negatively charged amino acid
side chains are located close to the phosphorylatable
Asp416 [21], and may reject the strongly charged
triphosphate. Substrates could be attached by their
purine ⁄ ribose moieties to the binding cleft of the nucle-
otide-binding domain, and the phosphates project into

) to the closed
conformation (species 2) is followed by
rapid transition to the phosphorylated form
(species 3) and nucleotide ⁄ phosphate-bound
form (species 4). Species 2–4 cannot be
resolved spectroscopically and are grouped
as intermediate I. The rapidly produced
ADP-bound species 5 (at k
2
) is the end
product under single turnover conditions
adjusted for infrared spectroscopic measure-
ments whereas during steady-state catalysis
the bound ADP is displaced by ATP to initi-
ate another round of substrate hydrolysis.
See text for further details.
Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo
¨
llmecke et al.
6182 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS
[21]. However, under steady-state turnover conditions,
the tightly bound ADP could become readily displaced
by the substrate ATP, which is present in vast excess,
offering an explanation of why the K
M
value of the
ATPase reaction is relatively high compared with the
fairly low equilibrium binding constant K
diss
lig

sis of cgATP (see Fig. 4A for structural formula) and its
isotopologues, which photo-release ATP labelled with
18
O
at certain phosphate positions, was performed for a-[
18
O
2
]-
cgATP as described previously [38,39], and for b-[
18
O
3
]-
cgATP and [b,c-
18
O,c-
18
O
3
]-cgATP (here termed [c-
18
O
4
])
using a procedure analogous to that described by Du et al.
[40]. Coupling of the 2-nitrophenylethyl caged group to the
terminal phosphate was performed as described previously
[41]. The concentration of cgATP was determined spectro-
photometrically using an extinction coefficient (e

performed in two ways. In the first method, the concentra-
tion of mant-ATP or mant-ADP (0.5 lm) was kept
constant, and small amounts of CopB-B (between 0 and
15 lm) were added from a 200 lm stock solution. The
binding data were calculated as described previously [42]
using the formula
F ¼ F
0
þ
EA½
A
t
½

F
1
ð1Þ
where F
0
indicates the initial fluorescence intensity in the
absence of protein, F indicates the measured fluorescence
intensity at a given concentration of CopB-B (dependent
variable), and F
¥
indicates the fluorescence intensity at sat-
urating concentration of protein. [A
t
] represents the total
concentration of ligand (independent variable), and [EA]
is the actual concentration of the mant-ATP–CopB-B

above except that the concentration of CopB-B was kept
constant ([E
t
] = 0.5 lm) and small amounts of mant
nucleotide ([A
t
], varying between 0 and 14 lm) from a
stock solution of 200 lm mant-ATP or mant-ADP were
added. Corrections for the fluorescence increase of free
C. Vo
¨
llmecke et al. Hydrolytic mechanism of the catalytic CPx-ATPase domain
FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6183
mant-ATP were performed in parallel experiments. In this
case, the binding data are calculated by the formula
F ¼ F
0
þ
EA½
E
t
½

F
1
ð3Þ
Equilibrium binding of other nucleotides in the
presence of mant-ATP
For determination of binding constants of non-fluorescent
nucleotides, a series of competition titration experiments

in which K
diss
represents the binding constant of the
mant-ATP complex in the absence of competitor, and
K
lig
diss
represents the binding constant of the competitor
ligand. K
lig
diss
may be read from the slope of the linear plot
of the apparent binding constant K
app
diss
versus the total
concentrations of competitor ligand [L
0
].
ATPase activity assay
The hydrolytic activity of CopB-B was measured by a modifi-
cation of the procedure described previously [44], quantifying
the liberated inorganic phosphate. Samples of 10 lm CopB-B
(final concentration) were incubated in a volume of 100 lLin
a medium containing 60 mm Na ⁄ Mes, pH 6.2, 10 mm MgCl
2
,
5mm ATP, for 20 min at 70 °C. The reaction was stopped by
addition of 400 lL molybdate reagent, which contained
0.5% w ⁄ v (NH

2mm MgCl
2
,10mm DTT, was deposited on top in a cen-
tral position, gently evaporated to dryness under a nitro-
gen stream, and subsequently rehydrated by addition of
0.7 lL of water. The resulting final concentrations were
357 mm Na ⁄ Mes, pH 6.2, 28.6 mm MgCl
2
, 143 mm DTT,
15.3 mm cgATP and 15.3 mm CopB-B.
Measurement of FTIR spectra and mathematical
data conversion
After sample equilibration for 5 h at 30 °C, spectra were
recorded using a IFS66VS vacuum instrument (Bruker
Optik, Ettlingen, Germany) equipped with a liquid nitro-
gen-cooled mercury cadmium telluride detector. An excimer
laser (Lambda Physics, Dieburg, Germany) operated with a
pulse energy of 130–140 mJ (output read from an internal
power meter) at 308 nm was used to photoactivate cgATP
by 60 flashes with pulse durations of 20 ns at a repetition
rate of 500 Hz. The total irradiation duration (120 ms) was
sufficient to release 90% of ATP from cgATP. Interfero-
grams at a nominal resolution of 4 cm
)1
were recorded
under rapid scan conditions with low-pass filter cutting at
1950 cm
)1
and an aperture width of 3.5–4 mm using the
double-sided, forward–backward data acquisition mode and

i¼1
a
i
ðmÞe
Àk
app
i
t
À
X
n
i¼1
a
i
ðmÞþa
ph
ðmÞð5Þ
where a
ph
(m) represents the amplitude of the initial state
after photolysis and k
i
app
the apparent rate constants. For
convenience, the physical quantity of )a
i
(m) is displayed in
the amplitude spectra.
Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo
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mathematical derivations; alternate kinetic fits.
Fig. S1. Binding of cgATP to CopB-B, analysed by
HPLC after centrifuged column separation of non-
bound nucleotide.
Fig. S2. Time courses of reactions of CopB-B with
photo-released ATP, observed at various wavenumbers.
Fig. S3. Alternative fitting conditions.
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Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo
¨
llmecke et al.
6186 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS