spectroscopic investigation of the reaction …gerwert/125.pdfspectroscopic investigation of the...

Spectroscopic investigation of the reaction mechanism ofCopB-B, the catalytic fragment from an archaealthermophilic ATP-driven heavy metal transporterChristian Vollmecke, Carsten Kotting, Klaus Gerwert and Mathias Lubben

Lehrstuhl fur Biophysik, Ruhr-Universitat 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

part is extensively hydrophilic and contains several

structural and functional modules, such as nucleotide

binding (N), phosphorylation (P), actuator (A) and

heavy metal binding (HMA) domains.

During the catalytic cycle, P-type ATPases, also

called E1E2-ATPases, undergo ordered large-scale

domain movements, in which ion translocation is

coupled to the energy released from ATP hydrolysis.

Starting from the E1 state, with high binding affinity

for the substrates (ions and nucleotides) on one side

of the membrane, the terminal c-phosphate group of

ATP is transiently transferred to a conserved aspartic

acid, forming a covalently bound aspartyl-phosphate

Keywords

fluorescence spectroscopy;

Fourier-transform infrared spectroscopy;

heavy metal translocation; P-type ATPase;

reaction mechanism

Correspondence

M. Lubben, Lehrstuhl fur Biophysik,

Ruhr-Universitat Bochum, Universitatsstr.

150, D-44780 Bochum, Germany

Fax: +49 234 32 14626

Tel: +49 234 32 24465

E-mail: [email protected]

(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 (Kdiss 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 (k1 = 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 (k2 = 17 · 10)3 s)1 at 30 �C), leading to prod-

uct formation. Although the latter appears to be a single step, it probably

comprises several reactions with presently unresolved intermediates. Based

on these data, we suggest a model of the hydrolytic mechanism.

Abbreviations

cgATP, caged ATP; mant-ATP, 3¢-N-methylanthraniloyl-ATP; AMPPNP, adenosine 5’(b,c-imido)triphosphate.

6172 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS

intermediate. The phosphorylated E1 state switches

to the phosphorylated E2 state with low affinity for

the substrate ion, which is released to the other side

of the membrane after hydrolysis of the phosphoryl

bond. Extensive information about the catalytic

mechanism has been obtained from investigations of

various P-type ATPases [2–5]. Many details on the

molecular function and structural models of ground

state and various intermediate states have been

obtained for Ca-ATPase [6], which is regarded as

virtually paradigmatic for the P-type ATPases.

Ca-ATPase and Na ⁄K-ATPase have been extensively

investigated by time-resolved FTIR absorbance differ-

ence spectroscopy using various nucleotides and nucleo-

tide analogues [7–13]. These studies have suffered from

the fact that the described mammalian proteins could

only be purified from native tissue material. The holo-

proteins were difficult to express in Escherichia coli,

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

The catalytic fragment N ⁄P, also called CopB-B, con-

sists of the nucleotide binding and phosphorylation

domains of the thermophilic CPX-ATPase CopB from

S. solfataricus. It was expressed in E. coli, crystallized

in a nucleotide-free state, and its structure was deter-

mined [21] (see Fig. 1). The domains are connected by

hinge peptides, which allow substantial flexibility of

both domains relative to each other. The domains

appear to be in a so-called closed orientation, into

which the substrate nucleotide, ATP, has been

modelled by superposition on the nucleotide-bound

structure of Ca-ATPase (Fig. 1). The purine moiety fits

into a cleft of the nucleotide-binding domain, whereas

Fig. 1. 3D structural model of the catalytic fragment CopB-B of the

heavy metal-translocating CPX-ATPase CopB from Sulfolobus solfa-

taricus (PDB code 2IYE). The protein is displayed in half-transparent

molecular surface representation, and the conserved phosphoryl-

atable Asp416 is shown. The adenine nucleotide shown was

modelled after structural superposition with the ADP ⁄ AlF3-bound

structure of Ca-ATPase (PDB code 1WPE).

C. Vollmecke 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 6173

the phosphate groups are located in the vicinity of the

phosphorylation domain. It should be taken into

account that our model of the nucleotide-bound state

of CopB-B is relatively crude with respect to the phos-

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-

chiometric conditions (i.e. when the molar concentra-

tions of mant-ATP and protein have values much

greater than Kdiss) resulted in a linear increase of fluo-

rescence with ligand addition up to the saturation

point, and above it in constant fluorescence (data not

shown). Extrapolating the lines to their intercept gave a

binding stoichiometry of one nucleotide per CopB-B

fragment.

For determination of the binding constant Kdiss, the

conditions were adjusted such that the concentrations

of mant-ATP and protein were of the same order as

the expected Kdiss. The hyperbolically shaped titration

A

B

C

Fig. 2. Equilibrium binding of CopB-B with nucleotides. (A) Fluores-

cence spectra of 0.5 lM mant-ATP in 5 mM Na ⁄ Mes buffer, pH

6.2, at room temperature in the absence (dashed lines) or presence

(continuous lines) of CopB-B in large stoichiometric excess (15 lM).

(B) Fluorescence titration of 0.5 lM mant-ATP with CopB-B. The

fluorescence at emission wavelength 434 nm is given in arbitrary

units; [Et] = total concentration of CopB-B. (C) Determination of

ligand dissociation constants from competitive titrations of 0.5 lM

CopB-B with mant-ATP in the presence of the indicated total con-

centrations ([L0]) of ATP (squares), ADP (circles) and AMP (trian-

gles) for determination of the apparent Kappdiss . Data were analyzed

according to Eqn (4). The bars indicate Kappdiss errors from individual

fits of titration curves obtained at fixed competitor concentrations.

Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vollmecke et al.


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

Kdiss increasing to 40 lm at 100 mm NaCl or

(NH4)2SO4. Notably, binding does not require Mg2+;

the affinity is reduced by a factor of 10 in the presence

of 1 mm MgCl2 (Table 1).

The binding specificity of the protein to mant-ATP

can be demonstrated by its displacement by other

nucleotides that are added in slight excess to the

complex. It is clear from the displacement of bound

mant-ATP by ATP and related compounds that these

nucleotides interact with the same protein binding

site. Ligand competition could thus be exploited for

determination of binding constants of non-fluorescent

nucleotides. According to Eqn (4), the apparent affin-

ity Kappdiss of CopB-B for mant-ATP is significantly

increased with higher concentrations of competitor

nucleotide. Based on a series of fluorescence titrations

of mant-ATP to CopB-B in the presence of various

competitor concentrations [L0], the binding constant of

the nucleotide can be determined from the slope of

the linear plot of the apparent binding constants Kappdiss

and [L0]. With the ligand ATP, a binding constant

K ligdiss 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 ligdiss 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

remarkable observation is the binding of caged ATP

(cgATP) with an affinity similar to that of ATP

(Table 1), which was verified independently by HPLC

(see Fig. S1).

Catalytic activity

During catalytic activity, the c-phosphate of ATP is

transiently transferred onto the strictly conserved

aspartic acid located in the phosphorylation domain,

which is Asp416 in CopB-B [21]. In the P-type ATPase

holoprotein, the A domain comes into contact with the

N ⁄P domain pair, promoting the hydrolysis reaction

by release of inorganic phosphate from the phosphory-

lated intermediate state [5]. Formation of the phos-

phorylated intermediate of CopB-B with the substrate

ATP has been shown previously [24], as well as its

hydrolytic activity with the artificial substrate p-nitro-

phenyl phosphate, even though the A domain is absent

in this construct. This is probably due to thermal

activation of the phosphatase reaction. The catalytic

activity using the native substrate Mg-ATP

gave approximately five times higher rates, amounting

to 50–70 nmol (mgÆmin))1. Variation of substrate

concentration revealed a simple hyperbolic Michaelis–

Menten-type dependence and a KM of 1 mm, which

reflects relatively poor kinetic substrate affinity

compared with the thermodynamic ligand association

constant K ligdiss 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 Mg2+, 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, Mg2+ was omitted to prevent phosphatase

activity.

Nucleotide Binding constant Kligdiss (lM)a

mant-ATPb 0.8

mant-ATPb ⁄ 1 mM MgCl2 10.0

ATP 10.0

ADP 18.9

AMP 29.8

AMPPNP 3.5

cgATP 9.5

GTP 12.6

a According to Eqn (4). b For mant-ATP in the absence of competi-

tor, the value for Kdiss is given.



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 kph 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 Mg2+ 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

reactions, the photochemical non-enzymatic process,

which is strongly dependent on temperature and the

pH of the medium, must be the fastest reaction step.

The rapid appearance of positive absorbance changes

at 1123 cm)1 generated from free cgATP (Fig. 4A,

continuous line) and from cgATP in the presence of

CopB-B (Fig. 4A, dotted line) within the phosphate

region of the infrared spectrum is indicative of product

formation. This band was assigned to the symmetric

stretching vibration of the c-PO32) group of ATP [25],

thus providing information on the photochemical

release rate of ATP from its caged precursor molecule.

The time course of the difference band corresponds to

rates of 4 and 7 s)1 in the presence or absence of

CopB-B, respectively, which demonstrates that the

release of ATP is much faster than all subsequent

partial reactions (see below), and, furthermore, gives a

constant reference line for the pre-photolytic state of

CopB-BÆATP after less than 2 s (Fig. 4A).

Static photolysis spectrum and phosphate band

assignment

The absorbance difference bands that are directly visi-

ble in the spectra after photolysis of cgATP and those

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 NO2

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 16O for 18O 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 lM CopB-B with Mg-ATP at 70 �C. (B) Temperature dependence

of 10 lM 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.

Scheme 1.



vibrational frequencies. As a typical example, Fig. 4C

shows the photolysis spectrum of CopB-B with ATP

and c-18O4-ATP, respectively. The positive band at

1137 cm)1 observed in the 16O compound is down-

shifted to 1089 cm)1 in the c-18O4-labelled ATP, and

this band can therefore be assigned to the anti-symmet-

ric stretching vibration of the c-phosphate group [ma(c-PO3

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 18O 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.

Dynamic interaction of ATP with CopB-B:

time-resolved hydrolysis spectra revealing a

reaction intermediate

After rapid release of the substrate ATP, its hydrolysis

was observed to occur at comparatively low rates. As

a control, the time course of the absorbance changes

after photo-release was recorded in the spectral range

from 1000–1800 cm)1 in the absence of protein, which

demonstrates insignificant spectral contributions from

cgATP and its photolysis alone (for details, see

Fig. S2). Upon elimination of the data related to the

A

B

C

Fig. 4. Investigation of the ATPase reaction by FTIR spectroscopy.

(A) Time course of ATP photo-release from cgATP. The absorbance

changes of the symmetric coupled a,b-phosphate band of ATP at

1123 cm)1 (cgATP photolysed in presence of CopB-B, continuous

line; cgATP photolysed alone, dotted line) were recorded by rapid-

scan FTIR spectroscopy. (B) Photolysis spectra of cgATP in the

presence (continuous line) and absence of CopB-B (dotted line).

The difference spectrum was obtained after 2 s, when ATP was

fully released. (C) Principle of band assignment of phosphate absor-

bance difference bands in the photolysis spectrum by means of18O-labelled phosphates (dotted line). The reference spectrum

(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-18O4-labelled ATP under otherwise identical conditions.



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 k1app and

k2app. 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),

the consecutive reaction of the three protein states

connected by the two apparent rate constants is repre-

sented by two amplitude difference spectra )a1 and

)a2 for the two rate constants k1app (Fig. 5A, top) and

k2app (Fig. 5A, bottom). Under the applied reaction

conditions, the first amplitude spectrum (k1app) could

be resolved with a rate constant of 1.9 · 10)2 s)1

(Fig. 5A, top) and the second with a rate constant

(k2app) of 5 · 10)3 s)1 (Fig. 5A, bottom).

Kinetic modelling of CopB-B’s ATPase reaction

If the apparent rate constants k1app and k2

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 )a1 and )a2(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 Explorer� [28] using the fol-

lowing model (Scheme 3) with intrinsic rate constants

k1, k)1, k2 and k)2:

In order to determine the intrinsic rate constants, we

assumed that the concentration changes of CopB-

BÆATP, the intermediate I and Pi are proportional to the

absorption changes at 1255 cm)1 (vas a-b-ATP band),

1338 cm)1 (unidentified protein side chain band) and

1078 cm)1 (inorganic phosphate band), respectively. In

addition, we normalized both the starting reactant

(educt) absorbance at 1255 cm)1 and the product absor-

bance at 1078 cm)1, so that c0 (CopB-BÆATP) = c¥

(Pi) = 1 and c¥ (CopB-BÆATP) = c0 (Pi) = 0. Due to

the unknown absorption coefficient of the intermediate

I, we arbitrarily averaged both normalization factors for

CopB-BÆATP and Pi 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 k1 = 4.7 ·10)3 s)1, k)1 = 3.0 · 10)4 s)1, k2 = 1.7 · 10)2 s)1 and

Table 2. Assignment of phosphate vibration detected in the Mg-adenine nucleotide complexes of CopB-B by means of 18O-labelled ATP iso-

topologues.

Spectrum according

to global fit v (cm)1) Band assignment

Band position after shift upon addition of isotopologDeflection of the

difference bandd18O4-c (cm)1) 18O3-b (cm)1) 18O2-a (cm)1)

Photolysis 1123 vs a-b-ATPa 1101 1101 u

1137 vas c-ATP 1089 u

1213 vas b-a-ATP 1206 u

1250 vas a-b-ATP sp.b u

)a1c 1108 ATP ⁄ ADP sp. d

)a2 1078 vs (PO2)) phosphate 1043 u

1098 v b-ADP sp. u

1136 vas c-ATP sp. d

1220 vas a-ADP sp. u

1255 vas a-b-ATP sp. sp. d

a Assignment to more than one phosphate group indicates strong vibrational coupling [27]. b sp., superposed. Absorbance difference bands

disappear upon isotopic labelling, but shifts are not observed due to complex band superposition. c Amplitude spectra corresponding to the

apparent rate constants k1app and k2

app due to global fitting. d u = upward, d = downward.

Scheme 2.

Scheme 3.



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 k2 > k1 (k2� k1app;

k1 � k2app), and that back reactions are negligible. The

faster decline of the intermediate compared to its forma-

tion leads to only small concentrations of intermediate I

during the reaction. The maximum concentration of I is

approximately one-eighth of that of c0 (CopB-BÆATP).

This is similar to the relatively small absorbance change

at 1338 cm)1 compared to 1078 or 1255 cm)1, and thus

in line with our measurements.

In model 2, parameters were fixed as suggested by

global fitting, namely k1 > k2 and k1 = k1app, and

k2 = k2app 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 )a1 and )a2 of the

global fit as detailed in Appendix S1. The result of this

calculation is that the bands facing upwards in )a1(Fig. 5A, top) derive from the intermediate state, and

A

B

Fig. 5. FTIR spectroscopic measurement of the ATPase reaction as

performed by CopB-B, initiated by flash-initiated substrate liberation

of ATP from cgATP. Rapid scan spectra recorded with a repetition

time of 185 ms (using double-sided forward–backward mode) fitted

to two rate constants by global fit analysis, k1app = 1.9 · 10)2 s)1

and k2app = 5 · 10)3 s)1, starting from 2 s after the flash. The band

labelled X is an artefact that also occurs in the sample without pro-

tein. (A) Amplitude spectra corresponding to the rate k1app ()a1, top)

and the rate k2app ()a2, bottom). (B) Band assignment verifying

phosphate production in the k2app transition by comparison of ampli-

tude spectra recorded with 16O (continuous line) and 18O (dotted

line) ATP isotopologues (top) and after double difference calculation

(16O–18O 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 stepa Rate constantb (s)1)

First k1app 1.9 · 10)2

k1 4.7 · 10)3

k)1 3.0 · 10)4

Second k2app 5.0 · 10)3

k2 1.7 · 10)2

k)2 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 (kiapp)] or via kinetic modelling (model 1; ki).



the bands facing downwards derive from the final ADP

state. The intensities are 38% compared to pure states.

The bands facing downwards in )a2 derive from both

the intermediate state (38%) and the initial ATP state,

and the bands facing upwards in )a2 derive from the

final ADP state.

The intermediate state during ATP hydrolysis

As the absorbances of the intermediate are facing

upwards in )a1 and downwards in )a2 (Fig. 5A), the

appearance and disappearance of a band at 1338 cm)1

may be regarded as a marker of this intermediate. It

represents an unknown absorbing group of the protein,

because absorbances in this region are clearly distinct

from the phosphate vibrations. Furthermore, the

amplitude spectra displayed in Fig. 5A indicate signifi-

cant changes in the broad amide I band centered at

approximately 1650 cm)1, and especially pronounced

at 1676 cm)1, and in the amide II band position at

1546 cm)1. This is not unexpected, as it is known that

P-type ATPases undergo remarkable structural

changes during catalysis. Another interesting feature is

the reproducible occurrence of small positive and nega-

tive absorbance difference signals in the carbonyl

region of the IR spectra in the region of 1720–

1740 cm)1, seen in both the )a1 and )a2 amplitude

spectra (Fig. 5A). Signals in this region point to the

prevalence of protonated aspartic or glutamic acid side

chains either undergoing protonation ⁄deprotonationreactions 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 )a2 (Fig. 5A, bottom).

The shift of the positive band from 1078 to 1043 cm)1

upon c-18O-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 bvibrations of the hydrolysis product ADP (Table 2).

Isotopic labelling at the c-18O-ATP position shifts the

negative ms c-ATP band from 1136 to 1108 cm)1

(Fig. 5B, curved arrow). As expected, the negative

bands at 1255 and 1136 cm)1 (Fig. 5A, bottom) corre-

spond well with the positive bands in the photolysis

spectrum (Fig. 4B) from a-, b- and c-coupled ATP

vibrations (Table 2).

Discussion

CopB-B is a suitable model to study ATP

hydrolysis of the P-type ATPase CopB

We have measured significant basal ATPase activity of

CopB in absence of the heavy metals (M. Zoltner &

M. Lubben, unpublished observations). Similarly,

metal-independent hydrolytic activity has also been

observed with the CPX-ATPase CopA of Thermo-

toga maritima [29]. CopB-B can mimic the effects of

A

B

Fig. 6. Time course of computed reactant concentrations after

kinetic modelling of the reaction between CopB-B and ATP. The

normalized concentrations of reactants were plotted as fractions of

1 over time (educt CopB-BÆATP, red line; reaction intermediate I,

black line; product inorganic phosphate Pi, blue line). In addition,

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 Pi) are plotted (squares). Simulations were performed

under the two conditions: fit 1, for which intrinsic rate constants

k1, k2, k)1 and k)2 were optimized using the program KinTek Global

Kinetic Explorer� (continuous lines) (A), and fit 2, for which fixed

rate constants k1 = k1app and k2 = k2

app, k)1 = k)2 = 0 were chosen

(B).



CopB-ATPase, which are entirely independent of the

translocated heavy metals, as the fragment naturally

carries out ‘uncoupled’ hydrolytic activity. Our efforts

demonstrate that spectroscopic methods can be used to

study the substrate binding and catalytic activity of the

hyperthermophilic Sulfolobus enzyme CopB, because it

is easily handled at room temperature. The catalytic

fragment CopB-B, consisting of nucleotide-binding

and phosphorylation domains, is the natively folded

‘business end’ of the holoenzyme CopB. It is expected

that this fragment, whose 3D structure is known,

behaves similarly to the holoenzyme with respect to

ATP hydrolysis and thus serves as a model of it. The

protein is capable of forming an intermediate with

covalently bound inorganic phosphate [24], and has

considerable ATPase activity despite the absence of the

actuator domain (A domain), which is considered to

promote rapid cleavage of the aspartyl phosphate

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 >> Kdisslig (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

several GTP-binding proteins such as Ras, Ran, Rab,

Rap and Rho, which exhibit vibrational uncoupling of

the phosphate resonances and significant shifts of the

a, b and c absorbance bands, resulting from strong

interactions of phosphate groups with amino acid side

chains lining the nucleotide binding site of the protein

[26,31–34]. It is concluded that, in CopB-B, the phos-

phates stay in contact with the solvent, and the tightly

bound ATP becomes immobilized by other molecular

parts of the nucleotide, presumably the purine moiety,

which apparently protrudes into a binding pocket

formed by CopB-B as seen in Fig. 1.

CopB-B interacts with ATP in a multi-step

process

ATP hydrolysis of CopB-B apparently includes two

phases. These are kinetically resolved by global fit

analysis and reflect the formation and decay of a single

observable reaction intermediate. Given the many

intermediates that have been recognized during the

reaction mechanism of P-type ATPases [2,35], more

than one intermediary state would also be expected to

occur during observation of hydrolysis with FTIR

spectroscopy. For example, there is spectral evidence

for protonated carboxyl groups, of which one is

expected as a potential phosphate acceptor in P-type

ATPases [13], within the absorbance region of 1720–

1740 cm)1 (Fig. 5A,B). Spectroscopic signatures of a

transiently phosphorylated aspartic acid, as demon-

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 k1, and the product

increase (Pi) proceeds with the faster rate constant k2.

Therefore, a relatively low concentration of intermedi-

ate is seen, as the decay rate k2 of intermediate I is

faster than its production rate k1. The slower rate k1should be associated to the first process after release of

ATP, i.e. the conformational change of CopB-B

leading to the ‘closed conformation’. In this step, the

hydrophilic environment of the phosphate groups of

ATP is substituted by a specific catalytic environment

within a binding pocket of the protein. This should

induce dramatic absorption changes within the phos-



phate region [26]. The absence of more intense phos-

phate absorbance difference bands in our measure-

ments further supports the kinetic model in which

k2 > k1, because the concentration of the reaction

intermediate is low, giving rise to only weak absorp-

tion changes. Thus, we conclude that the rate-deter-

mining step of our reaction is the slow ‘snapping‘

process of the domains to the intermediate I form with

rate constant k1. The conformational rearrangements

involved in this ‘snapping’ are shown by the relatively

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 Mg2+, 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

the solution. No hydrolysis occurs in the absence of

Mg2+, but, after its addition, the readily formed

Mg-ATP complex partially attenuates the electrostatic

repulsion, allowing by approximation of both

domains with k1 the slow adoption of a closed confor-

mation (species 2). The ‘snapping’ of domains would

initiate a rapid succession of catalytic steps (species 3

and 4 in Fig. 7), of which the existence of aspartyl-

phosphate and non-covalently bound phosphate states

has been proven in earlier experiments [21,24]. Because

these states have not yet been time-resolved by IR

spectroscopy, they are tentatively combined as interme-

diate I (Fig. 7, species 2–4). Species 5 is the final state

after a single substrate turnover; it represents CopB-B

with ADP bound with high affinity but dissociated

inorganic phosphate due to its low affinity for CopB-B

Fig. 7. Schematic model of the interaction

of ATP and cgATP with CopB-B during catal-

ysis. The nucleotide-binding and phosphory-

lation domains are shown as indented oval

symbols; they are connected by a short

linker and can adopt ‘open’ or ‘closed’

orientations. Various nucleotides (species 1

and 1a) bind to the open conformation (spe-

cies 0). Slow transition (at k1) 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 k2) 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.



[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 KM value of the

ATPase reaction is relatively high compared with the

fairly low equilibrium binding constant Kdisslig of ATP.

Conclusions

Partial reactions of the CPX-ATPase holoenzyme

CopB can be investigated using the catalytic fragment

CopB-B. Despite the fact that no information on the

fate of the translocated heavy metal ion can be

obtained, the nucleotide-binding ⁄phosphorylation dou-

ble domain of CopB-B alone exhibits high-affinity

ATP binding, protein phosphorylation and ATPase

activity. After binding of ATP to the nucleotide-bind-

ing domain, the first rate-limiting step consists of asso-

ciation of nucleotide and phosphorylation domains to

allow the subsequent second step involving rapid phos-

phoryl transfer and phosphoenzyme hydrolysis, which

are not yet resolved. As such, the fragment CopB-B

can be regarded as a valuable simple tool to facilitate

simulation of the partial reactions at a less-complex

level than the highly demanding holoenzyme.

Experimental procedures

Purification of CopB-B

Heterologous expression of the N ⁄P catalytic fragment

(CopB-B) of Sulfolobus solfataricus in E. coli and its purifi-

cation were performed as described previously [21]. After

the last concentration step, the protein was shock frozen in

small aliquots. Samples were thawed immediately prior to

the experiments. Protein content was determined by the

bicinchoninic acid method [36].

Synthesis of nucleotide analogues

mant-ATP (see Fig. 2A for structural formula) was synthe-

sized using the procedure described previously [37]. Synthe-

sis of cgATP (see Fig. 4A for structural formula) and its

isotopologues, which photo-release ATP labelled with 18O

at certain phosphate positions, was performed for a-[18O2]-

cgATP as described previously [38,39], and for b-[18O3]-

cgATP and [b,c-18O,c-18O3]-cgATP (here termed [c-18O4])

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 (e260) of

26 600 m)1Æcm)1.

Fluorescence spectroscopy and equilibrium

binding of nucleotides

Fluorescence measurements were performed using a Jasco-

6500 instrument (Jasco, Gross-Umstadt, Germany) oper-

ated at room temperature. By excitation at 356 nm, emis-

sion spectra were recorded between 400 and 500 nm, at a

scanning speed of 200 nmÆmin)1 and bandwidths of 3 nm, a

response time of 0.5 s, and with a constant photomultiplier

voltage of 600 V.

Equilibrium binding of mant nucleotides

Binding studies were performed at room temperature in

1 mL Na ⁄Mes buffer, pH 6.2, with and excitation wave-

length of 356 nm and an emission wavelength of 434 nm.

Stoichiometric titrations were performed by adding up to

50 lm of CopB-B in small intervals to a constant concen-

tration of mant-ATP (25 lm) (for details, see Results and

Discussion). Data were obtained 30 s after addition of the

ligand. Read-outs were corrected for dilution due to the

added volumes. Titrations for determination of Kdiss were

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 F0 þEA½ �At½ �

� �F1 ð1Þ

where F0 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. [At] represents the total

concentration of ligand (independent variable), and [EA]

is the actual concentration of the mant-ATP–CopB-B

complex, which is given by

EA ¼ ðð Et½ � þ Kdiss þ ½At�Þ � ðð½Et� þ Kdiss þ ½At�Þ2

� 4 Et½ � At½ �Þ0:5Þ=2 ð2Þ

where [Et] indicates the total concentration of the added

CopB-B protein. The measured data were fitted to the bind-

ing equation by means of a non-linear regression algorithm

implemented within the computer program ORIGIN

(OriginLab Corporation, Northamptonm, MA, USA).

In the second method, the conditions were as described

above except that the concentration of CopB-B was kept

constant ([Et] = 0.5 lm) and small amounts of mant

nucleotide ([At], 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



mant-ATP were performed in parallel experiments. In this

case, the binding data are calculated by the formula

F ¼ F0 þEA½ �Et½ �

� �F1 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

[43] were performed as described above for the second

method, but with the additional presence of 0, 5, 10, 20 or

25 lm of the competitor nucleotide. In this case, the binding

constant Kdiss of mant-ATP (in the absence of competitor)

increases to the apparent binding constant Kappdiss (in the pres-

ence of competitor). Under the assumption that the free

concentration of competitor ligand [L] is negligible

compared to its total concentration [L0], the apparent

binding constant for mant-ATP is expressed as

Kappdiss ¼ Kdiss 1þ L0½ �

K ligdiss

!ð4Þ

in which Kdiss represents the binding constant of the

mant-ATP complex in the absence of competitor, and

K ligdissrepresents the binding constant of the competitor

ligand. K ligdiss may be read from the slope of the linear plot

of the apparent binding constant Kappdiss versus the total

concentrations of competitor ligand [L0].

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 lL in

a medium containing 60 mm Na ⁄Mes, pH 6.2, 10 mm MgCl2,

5 mm ATP, for 20 min at 70 �C. The reaction was stopped by

addition of 400 lL molybdate reagent, which contained

0.5% w ⁄ v (NH4)2Mo7O24, 2% w ⁄ v H2SO4 and 0.5% w ⁄ vSDS, and colour formation was induced using 20 lL of 10%

w ⁄ v ascorbic acid. To correct for non-enzymatic phosphate

release, samples without protein were run in parallel. After

shaking for 2 min at room temperature, the absorbance was

read at 750 nm using an Ultrospec-3000 spectrophotometer

(GE Healthcare, Munich, Germany). The concentrations of

liberated phosphate were determined from a linear standard

curve by averaging the readings of five samples.

Sample preparation for FTIR spectroscopy

A notched CaF2 window of 2 mm thickness and 20 mm

width was greased using Apiezon (M&I Materials Ltd.,

Manchester, UK) and covered with a 3 lm Mylar spacer

(DuPont de Nemours, Bad Homburg, Germany) at its

outer circumference. A mixture of 300 lg (10.7 nmol)

CopB-B in 5 mm Na ⁄Mes, pH 6.2, and 10.7 nmol cgATP

in 10 lL of a buffer containing 25 mm Na ⁄Mes, pH 6.2,

2 mm MgCl2, 10 mm 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 MgCl2, 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

an instrumental scanner speed of 100 kHz. Before photoly-

sis, a reference spectrum was taken by averaging 50 scans.

After light activation, interferograms were averaged in the

following order to cover approximately the time ranges

0.2–2 s (1–10; one scan), 2–60 s (11–40; 10 scans), 60–600 s

(41–70; 100 scans) and 600–1000 s (71–72; 1000 scans). The

averaged interferograms were manipulated by zero filling

using a factor of 2, and Fourier-transformed using Mertz

phase correction and the Blackman–Harris three-term

apodization function. Absorbance spectra and absorbance

time courses are displayed as differences between the light

intensity I(t) and the reference intensity I0 of the sample

before photolysis at t = 0, namely DA(t) = )log (I(t) ⁄ I0).The kinetics of spectra evolution between 950 and

1800 cm)1 were approximated to multiple exponentials in

the period of 2–1000 s by global fit analysis [45], in which

the absorbance change DA(m,t) is fitted with a sum of n

exponential terms, by calculating the amplitudes ai (m) at

specific wavenumbers m:

DAðm; tÞ ¼Xn

i¼1

aiðmÞe�kappi

t �Xn

i¼1

aiðmÞ þ aphðmÞ ð5Þ

where aph (m) represents the amplitude of the initial state

after photolysis and kiapp the apparent rate constants. For

convenience, the physical quantity of )ai (m) is displayed in

the amplitude spectra.



Kinetic modelling

Simulation of concentration profiles of educts, products

and reaction intermediates by means of the intrinsic rate

constants k1 and k2 as well as k)1 and k)2 was performed

using the program KinTek Global Kinetic Explorer�(KinTek Corporation, Austin, TX, USA) [28] using a

model according to Scheme 3 (see above).

Acknowledgements

We thank Dr Yan Suveyzdis for chemical synthesis of

the isotopologues of cgATP and Ingo Rekittke for the

preparation of mant nucleotides. This work was

supported by grants LU405 ⁄3-1 from the Deutsche

Forschungsgemeinschaft and I ⁄ 78128 from the Volks-

wagenStiftung to M.L.

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Supporting information

The following supplementary material is available:

Appendix S1. Additional nucleotide binding data;

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.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers,

this journal provides supporting information supplied

by the authors. Such materials are peer-reviewed and

may be re-organized for online delivery, but are not

copy-edited or typeset. Technical support issues arising

from supporting information (other than missing files)

should be addressed to the authors.



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