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Structural and exchange properties of “Co(III)-phenanthroline-ATP”: a labeling reagent for the active site of ATPases
BIOh’J0RGAN.K
CHEMLSTR Y 9,81-90
81
(I 978)
Structural and Exchange Properties of “Co(III)-PhenanthrolineATP”:
A Labeling Reagent for the Active Site of ATPases
J. GRANOT and M. M. WERBER*
Structural Chemis?q~ and Po&mer Deparhnenrs. The I~‘eizmann Institure of Science. Rehovot, Israel A. DANCHIN Dkpartement de Biolo,oiee-Pllol~cuiaire, Insritut Pasreurand Insrirur de Bisiogie Physico-chimique, Paris, France
ABSTRACT
This paper reports
on 1H and 3lP NMR as well as EPR measurements of This complex is reagent of ATPase sites. “Co(III)_(phen)_ATP~” found to be paramagnetic. as deduced both from its EPR spectrum and from the significant broadening, though almost unshifted. proton and phosphorus resonances. This paramagnetism is a result of the incorporation of the superoxide free-radical anion in the coordination sphere of the trivalent cobalt ion. Evidence for the presence of superoxide in the complex is based on competition experiments with cyanide, which is able to displace the superoxide anion. The latter was identified by its inducing effect on the photoreactivity of luminol. The displacement of superoxide by cyanide was accompanied by the abolition of the paramagnetism of the complex. The relative distances between the protons and phosphorus atoms of ATP and the superoxide anion in the complex were caiculated using the NhiR line-broadening data. Structural models compatrbie with the experimental results are proposed. Under conditions of excess of adenine nucieotides or phenanthroline, the coordinated ATP molecule becomes exchangeable_ This phenomenon is attributed to the labihzation of the cobaltic ion ligands induced by the superoxide anion.
the labeling
INTRODUCTION The potentiality of the use of transition-metal ions in the d3 or d6 low-spin state for the affinity labeling of Mg” or Mg*+ -substrate binding sites in proteins and nucleic acids, has previously been described previously [l-5] _ Recently, several Co(IJI) complexes of nucleotides have been studied in detail as labeling reagents in several different systems:‘glycogen phosphorylase [6] , rabbit-muscle l Present address: Washington 99164.
@ Elsevier North-Holland,
Biochemistry/Biophysics.
Inc.. 1978
Washington
State
University,
Pullman,
J. GRANOT, M. M. WERBER and A. DANCHlN
82
myosin 17, 81, coupling factor 1 ATPase [8] and (Na+ + K+) ATPase [9] . Thus, this method has been shown to be useful for the labeling of a variety of specific sites, especially nucleotide binding sites [lo] , such as allosteric regulatory sites or Gatalytic sites. Cobalt(II1) complexes are usually inert to ligand substitution for either S,! or SN2 reactions. However, when a Co(Ill) complex having the same stereoconfiguration as that of the Mg2+ complex is allowed to bind at a specific site on a protein, both the entropy and energy of activation for a substitution reaction may be considerably lowered. This in turn will favor a ligand exchange, causing a former ligand of the complex to exchange for a new ligand from the protein, and thus a stable substrate-metal-protein complex will be formed. Since the Co(III) analog of a substrate-magnesium complex might be a “blueprint” of the site to which it can rigidly bind, we were prompted to investigate the structural features of the complex *‘Co(lH)-(phen)-ATP” that has been used for the labeling of ATPases. Magnetic cesonace techniques have proved to be excellent tools for the elucidation of geometrical (e.g., interatomic distance) and exchange properties of paramagnetic complexes. In the present study it was found that the complex “Co(III)(phen)-ATP” is paramagnetic, due to the presence of the free-radical 0; anion as a ligand. The characterization of the complex has thus been carried out by means of EPR as well as lH and 31P NMR measurements. EXPERIMENTAL Materials The BDH analytical reagents used were CoC12*6H20 and +phenanthroline (phen). Sodium salts of ATP and ADP were obtained from Sigma. Hydrogen peroxide (30%) was obtained from Merck. Deuterium oxide (99.710) was purchased from Carl Roth KG, Germany. All other reagents were of analytical grade. Double distilled water was used throughout. Preparation of Complex The “Co(III)-(phen)-ATP” was prepared by H,Oa or 02 oxidation ofCo(l the presence of phen and ATP at pH 10. as previously described [ 101 . Its molecular weight, as determined by chromatography on Sephadex G-10, was found to be 850 2 40, which is compatible with a 1 : 1 : I ratio of the initial components. It was lyophilized and redissolved in DzO, pH 10.2 ? 0.1 (rcfcrring to the pH-meter reading without correction for isotope effect). Complex c‘onc‘entrations were determined by atomic absorption of the cobalt, and nucleotidc concentrdtions were obtained from their 259~nm absorption.
STRUCTURAL
AND
I’XCltANGE:
Superoxide-Induced
PROPERTIES
Photoreactivity
OF “Co(II1)-(phen)-ATP”
83
of Luminol
The procedure was csscntially as given in Michelson [ 1 11 To 2 ml ofO.1 M potassium phosphate hul‘t’cr at various pH values (7.5-10) 30 t_tl of 10 mM luminol and 0.5 ml of 1 rt1.11 o< comples. followed or prececicd by 0.5 ml of 10 mM of KCN, were added. The enzyme superoxide dismutase from bovine blood [ 121 (a gift from Dr. A. M. Michelson) was added (10 I.cg/ml final) in either native or heat-denatured form before the addition of cyanide. The light emission was monitored as described by Michelson et al. [ 13) . Nuclear
Paramagnetic
Resonance
Spectra
The ‘H NMR spectra were recorded on a Bruker HFX-IO spectrometer operating at 00 Mf11. The experimental solutions contained a trace of dioxane, which served as an internal reference for shift measurments. Due to the low scjluhility of phenltnthrolinc. titration experiments with it had to be performed in the Fourier transform mode employing a Niculet 1080 computer. The ,jlP NMR spectra were recorded un a Bruker WH-270 spectrometer. equipped with a Nicoiet I IX0 computer. operating at 109.3 MHz in the Fourier transform mode. ShiI‘ts were measured relative to orthophosphvric acid as external reference. All the measurements \vere performed at tin ambient probe temperature of ‘7 ?I 1”. Electron
RESULTS
Paramagnetic
AND
Characterization
Resonanw
Spectra
DISCUSSION of
the “Co( I II )-( phen)-ATP”
complex
C!nlike many other (‘o( III) complcses. such as those with amines. pyrophosphates. ~rtpolypl~c)sph~tes. and adenine nuclcotides [h. 141 . the “Co(lll)-(phenjATP” complex was found to be paramagnetic. Both the ‘11 (Fig. 1 ) and 31P (FIf_. 2) NhiK spectra 01’ the i~~nples in quc~~us soiutions rrvruled strongiy I‘he phcn~ntlirc,linc ~t’\crn;tnc’es \vrre actualI: hr~~~denCd hro:rdcnrd rcsonanccs beyond detcclion. lniply~ng the‘ proUmi1y 01’ this molrculr tcl the paramagneiic center. The more sipnificant broadcnmg 01‘ the H8 lint of ATP as compared to the t 1, ,dnd HI’ lines indicates that Ha is closer to the param~gnetic center.
J. GRANOT,
84
-6
-5
.d PPM
M. M. WERBER
-3
and A. DANCHIN
-2
FIG. I. Proton spectra at pH 10.2 of: (a) “Co(III)_(phen)-ATP” complex (45 mM); (b) complex (45mM) + ATP (80 mhi); (c) complex (45 mM) + ATP (207 mM); (d) complex (4.5 mM) + CN- (188 m,M); (e) free ATP (44 mhi). Chemical shifts are relative to dioxane. This is consistent with the preferred conformation of ATP (i.e., anti) about the glycosidic bond [ 15]_ Comparison of the spectra of the complex with those
of free ATP at the same pH and concentration revealed only small proton shifts (e.g.. 36 Hz and 11 Hz. upheld, for the Ha and HI’ resonances, respectively) and no phosphorus shifts (within experimental error). This suggests that the paramagnetic properties of the complex are not due to the presence of Co(R) impurities, since the direct binding of Co(II) to the phosphate moiety of ATP allows the transfer of unpaired spin density from the metal ion to the
phosphorus nuclei. consequently inducing large contact shifts [16] _ Similarly, the ATP protons in the Co(I1) complex are also shifted considerably, the shifts being partiahy due to contact, and to a larger extent to pseudocontact hyperfine interactions [I7, 18]_ The absence of these shifts in the “Co(lII)rphen)-ATP” complex implies that its paramagnetism arises from a hgand coordinated to the
metal ion. This conclusion is substantiated by the EPR spectrum of the complex (Fig. 3) A broad line (line width 275 G) centered around g = 2 is observed_ The va!ue is very different from those obtained in Co(H) complexes (e.g.. gi = 2.3, gr = 2.0 [Isa] or g = 4.3 [19b] _ However, the splitting of the
STRUCTURAL
AND EXCHANGE
PROPERTIES
.?
OF “Co(iiI)-(phen)-ATP”
85
_.= 27
-
e _.~L.__-‘w_L-___.
_-
L.._ : -
;-
-__-..-_r.C.
._,..
_-
-_.L_.___-._
I_
:i___
_-..
.._
_.:
--- . _.._
_
c.‘--i.-,_
..\
J-
FIG. 2. Phosphorus-3 1 spectra at pH IO.2 of: (a) “Co(III)-(phen)-ATP” complex (13 mM); (b) complex (6.5 mhi) + phenanthroline (10 mM): (c) complex (13 ?nM) + ATP (65 mM); (d) complex (13 mM) + CN(42 mhi); (e) complex (13 nhl) + CN- (I 50 mM): (f) free ATP (15 mM).
resonance to eight/lines, which corresponds to hyperfine interaction via spin transfer, is consistent with the hypothesis that 3 paramagnetic ligand whose spin is f is directly linked to the central Co(II1) ion (nuclear spin $)_ The paramagnetic ligand of the “Co(III)(phen)-ATP” complex thus possesses 3 single unpaired electron partiahy deIocaIized over the metal ion, but apparently not over the nuclei of the other ligands coordinated to Co(II1). It has been previously noted that the formation of the “Co(Il1)-(phen)-ATP” complex requires the presence of O2 [7] or Hz02 18) and pH values higher than 9.5. These ObSerVdtiOnS, in conjunction with the magnetic resonate data (see
also Koda et al. [ZO) ) led to the conclusion
that
the paramagnetic
ligand
in
J. GRANOT.
86
M. M. WERBEK and A. DANCHIN
of the “Co(lll)FIG. 3. Electron paramagnetic resonance powder spectrum 2 was determined (phen)-ATP” complex (SO mg) in X band. The position of g = _ -___ using DPPH as an external marker. Arrows indicate sites that might revcal hyperfine coupling between delocalized unpaired electron and nucleus of 59C’o central atom.
the complex can be identified as the superoxide anion. Oz, whose formation and decomposition are strongly pH-dependent [ Eq. (I)] , as is often the case for OS-con:alning complexes [I I]. alkaline
phen
Co(il)
pH
ATP-
l
phcn
Co(lll)--ATI’
ptf
acidic
0 2
(1)
d 2
Under the experimental conditions (pH - IO), the right-hand side of Eq. (I ) would predominate. The above results are in somplete agreement with this assertion. Further evidence for the presence of the superoxide anion in the campIes is given below. The magnetic resonance results imply that the line broadening (Jv~~~) of both the proton and the phosphorus resonances of ATP can be attributed solely to the dipolar interaction between the magnetic mome;Its of the nucleic and the paramagnetic center. This broadening, which is directly related to the spin-spin relaxation of the nuclei, can be used to calculate the distance (R) between the paramagnetic center and the nucleus under observation by means of the relation [ 2 1] Av
where
K is a constant
containing
2M
nuclear
=
KU--= parameters.
(‘1 In the absence
of a precise
STRUCTURAL
AND EXCHANGE
PROPERTIES TABLE
R,,
Y’
c.fil’
R,,,‘R,,,tHs
F
relative
I’ Distances
mrasurcd
C Model distances
HI3
H2
4’
I
1.4
5.2 1
to H8. calculated
a Distances;
and Various
cr-P
c?-P
y-P
1.6
0.71
0.71
0.72
7.6
8.0
4.’
4.0
4.0
1.5
1.5
0.81
0.77
0.77
according
13~ means of a space-filling
rclativc
87
I
and Model Distances Between Superoxide Anion Nuclei of ATP in “Co(III)-(phen)-ATP” Complex
Experimental
R,:RJH,
OF “Co(lIl).(pi~en)_ATP“
to Eq. (2 1. atomic
model.
IO H,.
knowledge of the constant K, it is possible, under the reasonable assumption that the relasation of ~111the nuclei in thef the rumples. a spacefilling atomic tnodel was used. Two structures were Found to be most compatible with the experimental results. In both structures the hesa-coordinated Co(lI1) is bound to the two nitrogens of phen and to the 0, anion. The other three coordination sires are occupied either by the 0-P and y-P phosphates and NT of ATP [S] or by the three phosphates without direct association between the metal ion and the adenine ring. In the second structure the adenine moiety of ATP can be stahilizcd by ;Ln interaction with the phenanthroline molecule. Indeed, by monitoring the proton ~heunical shifts of .4TP and phen ;IS a function of their it was observed that all the resorelative concentrations in qucous solution. n;mccs \verc shifted upfield. This indicates tl~t ass~lctation via vertic‘ai stacking rings ~11‘the I\v~~ mole~ulcs. On the basis ot takes place between tht aromatic the proposed structures. the dtstances betwrcn the superoxide anion and the various nuclei of the ATP molecule were rncasured by means ot’ the atomic with the model. The average valtres. given in l‘ablc I. are in good agreement NMR esperimental results. change c.o~Id be detected by It is important to note that no observable
J. GRANOT,
88
M. M. WERBER and A. DANCHIN
comparing the solvent (HDO) signal in pure water with that in the complex solutions. It is thus conchided that no water molecules are bound in the inner coordination sphere of the complex. This is aIso consistent with the stability of the complex on lyophilization to complete dryness. Effect of Cyanide Ion
To confirm the presence of 0; in the Co(III) complex, competition studies with CN- were undertaken_ Under conditions of several-fold excess. the stable CN- donor is able to displace 0, from the complex: phen-Go3+-ATP
+ CK- -
b,
3+-ATP phen-::
+ 0,
(3
CN-
If the pH is not sufficiently alkaline, the main pathway is internal electron transfer to form 02 and Co*+ [Eq. (I)]. In any case, the spontaneous dismutation of O,, which considerably reduces its lifetime, has to be taken into account_ The superoxide ions displaced by CN- were monitored by making use of their inducing effect on the photoreactivity of luminol. The specificity of this reaction is limited by spontaneous photoemission, which occurs at pH vaIces higher than !?.O [ 1 I] _ To avoid this effect and since lower pH values would have favored internal electron transfer and hence destruction of 0, [Eq. (I)] _ the experiments had to be performed at pH 8.5. It was found that the time course of 0, liberation in the medium was of biphasic nature; 3 flash of emission was first observed, followed by a continuous slow emission (FigA). This can tentatively be interpreted to represent two kinds of displacement mechanisms by cyanide. The fast and slow emission phases may be the result of different positions of cyanide attack. To ascertain that the effect on luminol is really caused by O,, experiments were also performed in the presence of either native or heat-denatured superoxide dismutase. In addition, to ensure that CN- acts as a displacing agent. experiments were performed with injection of CN- followed by complex and with pre-injection of complex followed by CN-. The results of such experiments (Fig. 4) support the assumption that 0; is a ligand of the cobaltic ion and that it can be displaced by a stable donor, such a CN--. Further evidence is provided by the changes observed in the proton and phosphorous spectra of the complex on addition of CN-. The resulting sharp lines (cf. Figs. 1 and 2) demonstrate the displacement of the paramagnetic ligand by CN-. Effect of Nucleo tides
Addition of ATP to an aqueous solution of both the proton and the phosphorus narrowing was found to be proportional
of the complex caused the narrowing resonances of ATP. The e.xtent of to the ATP concentration. A single
STRUCTURAL
AND EXCHANGE
PROPERTIES
OF “Co(II1)-(phen)-ATP“
89
iiME
FIG. 4. Liberation of 0; on addition of excess cyanide to “Co(III)-(phen)0, was monitored by photoemission of ATP” comolex. The presence of luminol at pH 8.5 as described by Michelson [ 11 I _ Either CN- or the complex were added first: only the presence of both components induces photoemission. Denatured superoxide dismutase (10 pg/ml) does not affect the emission, whereas the native enzyme (10 pg/ml ) reduced the photoemission by a factor larger than 100. signal was observed for each resonance. indicating an exchange. which is fast on the NMR time scale, between the free and the bound ATP molecules. The lability of the ATP molecule in this complex is in contrast with the observations that Co(II1) complexes are usually substitution inert. It is suggested that the presence of a free radical as a ligand in the complex causes the labilization of other ligands. The smaller effect of added ATP on the line widths of the phosphorus as compared to those of the proton resonances may be attributed to different exchange rates of the phosphate and adenine moieties of the coordinated ATP. Whereas the binding to the cobaltic ion of all the ATP molecules involves coordination through phosphate groups, it may be that only part of the molecules are linked through the adenine ring. A similar finding was reported for the
J GRANOT.
90
TABLE
hl. M. WERBER 2
Line fiaoadening of ATP Resonances as Function Total ATP Concentration Ratio
(.4TPlb/‘[ATPI,“ 1 .oo
and A. DANCIIIN
of Bound:
H8
H2
HI’
Cr-P
S60*
90
43
540
580
540
47
‘7
0.50
0-P
--
Y-P
0.36
160
33
17
520
560
520
0.25
110
18
8
480
5’0
490
410
430
410
0.20
X0
12
4
0.17
f)O
8
3
D Ratio of bound
to total
ATP concentrations.
* Average value calculated by means resonance of ATP hound in the complex
of proton line-broadening data. The Ha was hroadencd hcyond detection.
manganese(ll)-ATP complex (221, in which the metal ion was ftlund to associate directly with the phosphate groups and N;7 of the adenine ring. Addition of ADP tv solutions of the complex res:llted in similar. though weaker. effects as compared to those induced by ATP. l’vcn LLlarge csccss CI! ADP could not displace a11 the ATP from the comples. indILating that AlIP cannot compete as effectively as ATP tar the metal ion. The Lompetition studies with ADP supports our suggestion that Co(lll) may be chelated either by two or three phosphate groups. However. the effect of ATP indicates that the species that involve ring c*oordination are less abundant than those involving chelation by all three phosphate groups. Effect of Phenanthroline
Addition of phenanthroline to an aque~~us solution of the CompIcs resulted in considerable narrowing of the proton and the phosphorus (Fig 2) rcsonances. In addition. the signals cjf the added phcnanthrolinc ~c~l~~eti wilh those of the bound molecules. it appears that excess of rt~cnantllrc,lill~ ATP
from
the complex.
It has. however. IO be noted that narrowng
resonances may also be due phenanthroline.
Noting
to the displacement
that addition
of either
the signals of the coordinated phenanthrolinc, be rcgardcd as substitution inert.
of the supcroside
d~splaccs of the ATP anion by
or ADP has no cl‘t’ect on it is wncluckd that this ligand can ATP
STRUCTURAL CONCLUDING
AND EXCHANGE PROPERTIES
OF “Co(lII)-(phen)-ATP”
91
REMARKS
The complex “Co(,fll)-(phen)-ATP” is an analog of the M.;-ATP subtrate of ATPase and can act as a competitive inhibitor without being cleaved by the enzymes [S] . The present study has demonstrated that the metal is directly linked to three ligands in the complex: (1) o-phenanthroline, (2) the superoxide anion, and (3) ATP. With regard to the coordination of ATP, two species may be formed; one involving binding through the three phosphate groups only and the other through two phosphate groups and N7 of the adenine ring. It has been suggested that in Mg-ATP the metal ion is coordinated to the p- and y-phosphates [ 231 and possibly to the adenine ring [ 181 . It was found that the “Co(llI)-(phen)-ATP” complex is labile with regard to ATP. and ADP, in ATP substitution by chelators, such as phenanthroiine, decreasing order of efficiency. However, it should be noted that exchange of ATP takes place only in the presence of excess of chelating agents. The observation that water does not enter the complex provides evidence in favor of the inertness of “Co(lll)-(phen)-ATP” in aqueous solutions in the absence of chelators. The labilization effect is attributed to the presence o1’superoxide anion in the complex. Cobaltic complexes with amine and nucleotide ligands similar to those of the present complex bu: lacking the superoxide anion are known to be substitution inert [4. 141. A&hough the detailed mechanism of this labilization process is not known. it must be noted that the superoxide anion can impart to the metal ran a partial Co(ll) character. which might change its substitution properties. As for the role of the phenanthroline moiety, we would like to suggest that it stabihzes the superoxide anion in the complex by acting as an “zleztron sink’ ]24], which enables a delocalization of the unpaired electron. Another role of’ this aromatic l&and might be to stabilize the adenine ring of ATP by stacking interactions. It is suggested that “Co(llI)-(phen)-ATP” attaches itself to the enzymes through an axial position. which is first occupied by 0; and exchanged for a ligand from the ATPases.
92
J. GRANOT, M. M. WERBER and A. DANCHIN
REFERENCES 1. 2. 3. 4. 5.
A. A. A. A. A. 6. A_ 7_ M.
Kowalsky.1 BioZ. c7rem. 244,6619 (1969). Danchin, ComptesRend. Acad. Sci Paris.. Ser. D 273,1636 (1971). Danchin. Biocidmie 54,333 (1972). Danchin. Biochimie 55.17 (1973). Danchin. Biochimie 57,875 (1975). Danchin and H. But: /. Biol. Chem. 248,324l (1973). M_ Werber, A. Oplatka, and A. Danchin. Biochemistry 13.2683 (1974). 8. M. M. Werber. A. Danchin. Y. Hochman. C. Carmeli, and A. Lanir, in Metal Ligund Inreractions in Organic Chemisrry and Biochemistry (B. Pullman and N. Goldbtum. edr). Reidel Publisbiug Company, Dordrecht/Boston (1977). Pt. 1, pp. 283-290. 9. S. J. D. Karlish and M. M. Werber. manuscript in preparation (1978). 10. M. M. Werber and A. Danchin, in Methods in Enzymology. Vol. 46 (B. Jakoby and M. Wilchek. eds.). Academic Press. New York (1977). pp. 312-321. 11. A. M. Michelson, Biochimie 55,925 (1973). 12. J. W. Hartz and H. F. Deutsch. 3. Biol. Chem. 244,456s (1969). 13. J_ P. Henry, M. F. Isambert. and A. M. Michelson, Biochim. Biophys. Acta 205,437
(1970). 14. R. D. Cornelius, P. A. Hart, and W. W. CIeiand, Inorg. Chem., 16.3299 (1978). 15. M. Sundaralingam, Biopolyme;s 7,821 (1969). 16_ H. Sternhcht. R. G. Shuimaa, and E. W. Anderson. J. C?rem. Phys. 43.3123 (1965). 17. H. Stemlicht, R. G. Shulman. and E. W. Anderson, f. C&em. Phys. 43,3133 (1965). 18. J. Grzzot and D. Fiat, J. AM. Chem. Sot. 99-70 (1977)_ 19s. Y. Yonetani, H. Yamamoto, and T. Iizuka, L Biol. aem_ 249.2168 (1974). 19b. J. 5. Griffith. The Theoor of Trartsition MetalIons. Cambridge Univ. Press, Cambridge. U.K. (1964). pp_ 360-363. 20. S. Koda. A. !%sono. and Y. Uchida. Bull. Chem. Sot. Jup. 43,3143 (1970). 21. A. -4bragam, The Principles of Nuclear Magnetism. Oxford Univ. Press, Oxford, U.K. (1961), Chap. 8. 22. Y. Lam, G. P. P. Kuntz. and G. Kotowyn, J. Am Ckem Sot. 96,1834 (1974). 23. hf. Cohn and T. R_ Hughes, Jr-.I BioL Ckm. 237,176 (1962). 24. M. M- Werber.J. 17reorer. BioL 60.51 (1976).
Received 21 August I977
CHEMLSTR Y 9,81-90
81
(I 978)
Structural and Exchange Properties of “Co(III)-PhenanthrolineATP”:
A Labeling Reagent for the Active Site of ATPases
J. GRANOT and M. M. WERBER*
Structural Chemis?q~ and Po&mer Deparhnenrs. The I~‘eizmann Institure of Science. Rehovot, Israel A. DANCHIN Dkpartement de Biolo,oiee-Pllol~cuiaire, Insritut Pasreurand Insrirur de Bisiogie Physico-chimique, Paris, France
ABSTRACT
This paper reports
on 1H and 3lP NMR as well as EPR measurements of This complex is reagent of ATPase sites. “Co(III)_(phen)_ATP~” found to be paramagnetic. as deduced both from its EPR spectrum and from the significant broadening, though almost unshifted. proton and phosphorus resonances. This paramagnetism is a result of the incorporation of the superoxide free-radical anion in the coordination sphere of the trivalent cobalt ion. Evidence for the presence of superoxide in the complex is based on competition experiments with cyanide, which is able to displace the superoxide anion. The latter was identified by its inducing effect on the photoreactivity of luminol. The displacement of superoxide by cyanide was accompanied by the abolition of the paramagnetism of the complex. The relative distances between the protons and phosphorus atoms of ATP and the superoxide anion in the complex were caiculated using the NhiR line-broadening data. Structural models compatrbie with the experimental results are proposed. Under conditions of excess of adenine nucieotides or phenanthroline, the coordinated ATP molecule becomes exchangeable_ This phenomenon is attributed to the labihzation of the cobaltic ion ligands induced by the superoxide anion.
the labeling
INTRODUCTION The potentiality of the use of transition-metal ions in the d3 or d6 low-spin state for the affinity labeling of Mg” or Mg*+ -substrate binding sites in proteins and nucleic acids, has previously been described previously [l-5] _ Recently, several Co(IJI) complexes of nucleotides have been studied in detail as labeling reagents in several different systems:‘glycogen phosphorylase [6] , rabbit-muscle l Present address: Washington 99164.
@ Elsevier North-Holland,
Biochemistry/Biophysics.
Inc.. 1978
Washington
State
University,
Pullman,
J. GRANOT, M. M. WERBER and A. DANCHlN
82
myosin 17, 81, coupling factor 1 ATPase [8] and (Na+ + K+) ATPase [9] . Thus, this method has been shown to be useful for the labeling of a variety of specific sites, especially nucleotide binding sites [lo] , such as allosteric regulatory sites or Gatalytic sites. Cobalt(II1) complexes are usually inert to ligand substitution for either S,! or SN2 reactions. However, when a Co(Ill) complex having the same stereoconfiguration as that of the Mg2+ complex is allowed to bind at a specific site on a protein, both the entropy and energy of activation for a substitution reaction may be considerably lowered. This in turn will favor a ligand exchange, causing a former ligand of the complex to exchange for a new ligand from the protein, and thus a stable substrate-metal-protein complex will be formed. Since the Co(III) analog of a substrate-magnesium complex might be a “blueprint” of the site to which it can rigidly bind, we were prompted to investigate the structural features of the complex *‘Co(lH)-(phen)-ATP” that has been used for the labeling of ATPases. Magnetic cesonace techniques have proved to be excellent tools for the elucidation of geometrical (e.g., interatomic distance) and exchange properties of paramagnetic complexes. In the present study it was found that the complex “Co(III)(phen)-ATP” is paramagnetic, due to the presence of the free-radical 0; anion as a ligand. The characterization of the complex has thus been carried out by means of EPR as well as lH and 31P NMR measurements. EXPERIMENTAL Materials The BDH analytical reagents used were CoC12*6H20 and +phenanthroline (phen). Sodium salts of ATP and ADP were obtained from Sigma. Hydrogen peroxide (30%) was obtained from Merck. Deuterium oxide (99.710) was purchased from Carl Roth KG, Germany. All other reagents were of analytical grade. Double distilled water was used throughout. Preparation of Complex The “Co(III)-(phen)-ATP” was prepared by H,Oa or 02 oxidation ofCo(l the presence of phen and ATP at pH 10. as previously described [ 101 . Its molecular weight, as determined by chromatography on Sephadex G-10, was found to be 850 2 40, which is compatible with a 1 : 1 : I ratio of the initial components. It was lyophilized and redissolved in DzO, pH 10.2 ? 0.1 (rcfcrring to the pH-meter reading without correction for isotope effect). Complex c‘onc‘entrations were determined by atomic absorption of the cobalt, and nucleotidc concentrdtions were obtained from their 259~nm absorption.
STRUCTURAL
AND
I’XCltANGE:
Superoxide-Induced
PROPERTIES
Photoreactivity
OF “Co(II1)-(phen)-ATP”
83
of Luminol
The procedure was csscntially as given in Michelson [ 1 11 To 2 ml ofO.1 M potassium phosphate hul‘t’cr at various pH values (7.5-10) 30 t_tl of 10 mM luminol and 0.5 ml of 1 rt1.11 o< comples. followed or prececicd by 0.5 ml of 10 mM of KCN, were added. The enzyme superoxide dismutase from bovine blood [ 121 (a gift from Dr. A. M. Michelson) was added (10 I.cg/ml final) in either native or heat-denatured form before the addition of cyanide. The light emission was monitored as described by Michelson et al. [ 13) . Nuclear
Paramagnetic
Resonance
Spectra
The ‘H NMR spectra were recorded on a Bruker HFX-IO spectrometer operating at 00 Mf11. The experimental solutions contained a trace of dioxane, which served as an internal reference for shift measurments. Due to the low scjluhility of phenltnthrolinc. titration experiments with it had to be performed in the Fourier transform mode employing a Niculet 1080 computer. The ,jlP NMR spectra were recorded un a Bruker WH-270 spectrometer. equipped with a Nicoiet I IX0 computer. operating at 109.3 MHz in the Fourier transform mode. ShiI‘ts were measured relative to orthophosphvric acid as external reference. All the measurements \vere performed at tin ambient probe temperature of ‘7 ?I 1”. Electron
RESULTS
Paramagnetic
AND
Characterization
Resonanw
Spectra
DISCUSSION of
the “Co( I II )-( phen)-ATP”
complex
C!nlike many other (‘o( III) complcses. such as those with amines. pyrophosphates. ~rtpolypl~c)sph~tes. and adenine nuclcotides [h. 141 . the “Co(lll)-(phenjATP” complex was found to be paramagnetic. Both the ‘11 (Fig. 1 ) and 31P (FIf_. 2) NhiK spectra 01’ the i~~nples in quc~~us soiutions rrvruled strongiy I‘he phcn~ntlirc,linc ~t’\crn;tnc’es \vrre actualI: hr~~~denCd hro:rdcnrd rcsonanccs beyond detcclion. lniply~ng the‘ proUmi1y 01’ this molrculr tcl the paramagneiic center. The more sipnificant broadcnmg 01‘ the H8 lint of ATP as compared to the t 1, ,dnd HI’ lines indicates that Ha is closer to the param~gnetic center.
J. GRANOT,
84
-6
-5
.d PPM
M. M. WERBER
-3
and A. DANCHIN
-2
FIG. I. Proton spectra at pH 10.2 of: (a) “Co(III)_(phen)-ATP” complex (45 mM); (b) complex (45mM) + ATP (80 mhi); (c) complex (45 mM) + ATP (207 mM); (d) complex (4.5 mM) + CN- (188 m,M); (e) free ATP (44 mhi). Chemical shifts are relative to dioxane. This is consistent with the preferred conformation of ATP (i.e., anti) about the glycosidic bond [ 15]_ Comparison of the spectra of the complex with those
of free ATP at the same pH and concentration revealed only small proton shifts (e.g.. 36 Hz and 11 Hz. upheld, for the Ha and HI’ resonances, respectively) and no phosphorus shifts (within experimental error). This suggests that the paramagnetic properties of the complex are not due to the presence of Co(R) impurities, since the direct binding of Co(II) to the phosphate moiety of ATP allows the transfer of unpaired spin density from the metal ion to the
phosphorus nuclei. consequently inducing large contact shifts [16] _ Similarly, the ATP protons in the Co(I1) complex are also shifted considerably, the shifts being partiahy due to contact, and to a larger extent to pseudocontact hyperfine interactions [I7, 18]_ The absence of these shifts in the “Co(lII)rphen)-ATP” complex implies that its paramagnetism arises from a hgand coordinated to the
metal ion. This conclusion is substantiated by the EPR spectrum of the complex (Fig. 3) A broad line (line width 275 G) centered around g = 2 is observed_ The va!ue is very different from those obtained in Co(H) complexes (e.g.. gi = 2.3, gr = 2.0 [Isa] or g = 4.3 [19b] _ However, the splitting of the
STRUCTURAL
AND EXCHANGE
PROPERTIES
.?
OF “Co(iiI)-(phen)-ATP”
85
_.= 27
-
e _.~L.__-‘w_L-___.
_-
L.._ : -
;-
-__-..-_r.C.
._,..
_-
-_.L_.___-._
I_
:i___
_-..
.._
_.:
--- . _.._
_
c.‘--i.-,_
..\
J-
FIG. 2. Phosphorus-3 1 spectra at pH IO.2 of: (a) “Co(III)-(phen)-ATP” complex (13 mM); (b) complex (6.5 mhi) + phenanthroline (10 mM): (c) complex (13 ?nM) + ATP (65 mM); (d) complex (13 mM) + CN(42 mhi); (e) complex (13 nhl) + CN- (I 50 mM): (f) free ATP (15 mM).
resonance to eight/lines, which corresponds to hyperfine interaction via spin transfer, is consistent with the hypothesis that 3 paramagnetic ligand whose spin is f is directly linked to the central Co(II1) ion (nuclear spin $)_ The paramagnetic ligand of the “Co(III)(phen)-ATP” complex thus possesses 3 single unpaired electron partiahy deIocaIized over the metal ion, but apparently not over the nuclei of the other ligands coordinated to Co(II1). It has been previously noted that the formation of the “Co(Il1)-(phen)-ATP” complex requires the presence of O2 [7] or Hz02 18) and pH values higher than 9.5. These ObSerVdtiOnS, in conjunction with the magnetic resonate data (see
also Koda et al. [ZO) ) led to the conclusion
that
the paramagnetic
ligand
in
J. GRANOT.
86
M. M. WERBEK and A. DANCHIN
of the “Co(lll)FIG. 3. Electron paramagnetic resonance powder spectrum 2 was determined (phen)-ATP” complex (SO mg) in X band. The position of g = _ -___ using DPPH as an external marker. Arrows indicate sites that might revcal hyperfine coupling between delocalized unpaired electron and nucleus of 59C’o central atom.
the complex can be identified as the superoxide anion. Oz, whose formation and decomposition are strongly pH-dependent [ Eq. (I)] , as is often the case for OS-con:alning complexes [I I]. alkaline
phen
Co(il)
pH
ATP-
l
phcn
Co(lll)--ATI’
ptf
acidic
0 2
(1)
d 2
Under the experimental conditions (pH - IO), the right-hand side of Eq. (I ) would predominate. The above results are in somplete agreement with this assertion. Further evidence for the presence of the superoxide anion in the campIes is given below. The magnetic resonance results imply that the line broadening (Jv~~~) of both the proton and the phosphorus resonances of ATP can be attributed solely to the dipolar interaction between the magnetic mome;Its of the nucleic and the paramagnetic center. This broadening, which is directly related to the spin-spin relaxation of the nuclei, can be used to calculate the distance (R) between the paramagnetic center and the nucleus under observation by means of the relation [ 2 1] Av
where
K is a constant
containing
2M
nuclear
=
KU--= parameters.
(‘1 In the absence
of a precise
STRUCTURAL
AND EXCHANGE
PROPERTIES TABLE
R,,
Y’
c.fil’
R,,,‘R,,,tHs
F
relative
I’ Distances
mrasurcd
C Model distances
HI3
H2
4’
I
1.4
5.2 1
to H8. calculated
a Distances;
and Various
cr-P
c?-P
y-P
1.6
0.71
0.71
0.72
7.6
8.0
4.’
4.0
4.0
1.5
1.5
0.81
0.77
0.77
according
13~ means of a space-filling
rclativc
87
I
and Model Distances Between Superoxide Anion Nuclei of ATP in “Co(III)-(phen)-ATP” Complex
Experimental
R,:RJH,
OF “Co(lIl).(pi~en)_ATP“
to Eq. (2 1. atomic
model.
IO H,.
knowledge of the constant K, it is possible, under the reasonable assumption that the relasation of ~111the nuclei in the
J. GRANOT,
88
M. M. WERBER and A. DANCHIN
comparing the solvent (HDO) signal in pure water with that in the complex solutions. It is thus conchided that no water molecules are bound in the inner coordination sphere of the complex. This is aIso consistent with the stability of the complex on lyophilization to complete dryness. Effect of Cyanide Ion
To confirm the presence of 0; in the Co(III) complex, competition studies with CN- were undertaken_ Under conditions of several-fold excess. the stable CN- donor is able to displace 0, from the complex: phen-Go3+-ATP
+ CK- -
b,
3+-ATP phen-::
+ 0,
(3
CN-
If the pH is not sufficiently alkaline, the main pathway is internal electron transfer to form 02 and Co*+ [Eq. (I)]. In any case, the spontaneous dismutation of O,, which considerably reduces its lifetime, has to be taken into account_ The superoxide ions displaced by CN- were monitored by making use of their inducing effect on the photoreactivity of luminol. The specificity of this reaction is limited by spontaneous photoemission, which occurs at pH vaIces higher than !?.O [ 1 I] _ To avoid this effect and since lower pH values would have favored internal electron transfer and hence destruction of 0, [Eq. (I)] _ the experiments had to be performed at pH 8.5. It was found that the time course of 0, liberation in the medium was of biphasic nature; 3 flash of emission was first observed, followed by a continuous slow emission (FigA). This can tentatively be interpreted to represent two kinds of displacement mechanisms by cyanide. The fast and slow emission phases may be the result of different positions of cyanide attack. To ascertain that the effect on luminol is really caused by O,, experiments were also performed in the presence of either native or heat-denatured superoxide dismutase. In addition, to ensure that CN- acts as a displacing agent. experiments were performed with injection of CN- followed by complex and with pre-injection of complex followed by CN-. The results of such experiments (Fig. 4) support the assumption that 0; is a ligand of the cobaltic ion and that it can be displaced by a stable donor, such a CN--. Further evidence is provided by the changes observed in the proton and phosphorous spectra of the complex on addition of CN-. The resulting sharp lines (cf. Figs. 1 and 2) demonstrate the displacement of the paramagnetic ligand by CN-. Effect of Nucleo tides
Addition of ATP to an aqueous solution of both the proton and the phosphorus narrowing was found to be proportional
of the complex caused the narrowing resonances of ATP. The e.xtent of to the ATP concentration. A single
STRUCTURAL
AND EXCHANGE
PROPERTIES
OF “Co(II1)-(phen)-ATP“
89
iiME
FIG. 4. Liberation of 0; on addition of excess cyanide to “Co(III)-(phen)0, was monitored by photoemission of ATP” comolex. The presence of luminol at pH 8.5 as described by Michelson [ 11 I _ Either CN- or the complex were added first: only the presence of both components induces photoemission. Denatured superoxide dismutase (10 pg/ml) does not affect the emission, whereas the native enzyme (10 pg/ml ) reduced the photoemission by a factor larger than 100. signal was observed for each resonance. indicating an exchange. which is fast on the NMR time scale, between the free and the bound ATP molecules. The lability of the ATP molecule in this complex is in contrast with the observations that Co(II1) complexes are usually substitution inert. It is suggested that the presence of a free radical as a ligand in the complex causes the labilization of other ligands. The smaller effect of added ATP on the line widths of the phosphorus as compared to those of the proton resonances may be attributed to different exchange rates of the phosphate and adenine moieties of the coordinated ATP. Whereas the binding to the cobaltic ion of all the ATP molecules involves coordination through phosphate groups, it may be that only part of the molecules are linked through the adenine ring. A similar finding was reported for the
J GRANOT.
90
TABLE
hl. M. WERBER 2
Line fiaoadening of ATP Resonances as Function Total ATP Concentration Ratio
(.4TPlb/‘[ATPI,“ 1 .oo
and A. DANCIIIN
of Bound:
H8
H2
HI’
Cr-P
S60*
90
43
540
580
540
47
‘7
0.50
0-P
--
Y-P
0.36
160
33
17
520
560
520
0.25
110
18
8
480
5’0
490
410
430
410
0.20
X0
12
4
0.17
f)O
8
3
D Ratio of bound
to total
ATP concentrations.
* Average value calculated by means resonance of ATP hound in the complex
of proton line-broadening data. The Ha was hroadencd hcyond detection.
manganese(ll)-ATP complex (221, in which the metal ion was ftlund to associate directly with the phosphate groups and N;7 of the adenine ring. Addition of ADP tv solutions of the complex res:llted in similar. though weaker. effects as compared to those induced by ATP. l’vcn LLlarge csccss CI! ADP could not displace a11 the ATP from the comples. indILating that AlIP cannot compete as effectively as ATP tar the metal ion. The Lompetition studies with ADP supports our suggestion that Co(lll) may be chelated either by two or three phosphate groups. However. the effect of ATP indicates that the species that involve ring c*oordination are less abundant than those involving chelation by all three phosphate groups. Effect of Phenanthroline
Addition of phenanthroline to an aque~~us solution of the CompIcs resulted in considerable narrowing of the proton and the phosphorus (Fig 2) rcsonances. In addition. the signals cjf the added phcnanthrolinc ~c~l~~eti wilh those of the bound molecules. it appears that excess of rt~cnantllrc,lill~ ATP
from
the complex.
It has. however. IO be noted that narrowng
resonances may also be due phenanthroline.
Noting
to the displacement
that addition
of either
the signals of the coordinated phenanthrolinc, be rcgardcd as substitution inert.
of the supcroside
d~splaccs of the ATP anion by
or ADP has no cl‘t’ect on it is wncluckd that this ligand can ATP
STRUCTURAL CONCLUDING
AND EXCHANGE PROPERTIES
OF “Co(lII)-(phen)-ATP”
91
REMARKS
The complex “Co(,fll)-(phen)-ATP” is an analog of the M.;-ATP subtrate of ATPase and can act as a competitive inhibitor without being cleaved by the enzymes [S] . The present study has demonstrated that the metal is directly linked to three ligands in the complex: (1) o-phenanthroline, (2) the superoxide anion, and (3) ATP. With regard to the coordination of ATP, two species may be formed; one involving binding through the three phosphate groups only and the other through two phosphate groups and N7 of the adenine ring. It has been suggested that in Mg-ATP the metal ion is coordinated to the p- and y-phosphates [ 231 and possibly to the adenine ring [ 181 . It was found that the “Co(llI)-(phen)-ATP” complex is labile with regard to ATP. and ADP, in ATP substitution by chelators, such as phenanthroiine, decreasing order of efficiency. However, it should be noted that exchange of ATP takes place only in the presence of excess of chelating agents. The observation that water does not enter the complex provides evidence in favor of the inertness of “Co(lll)-(phen)-ATP” in aqueous solutions in the absence of chelators. The labilization effect is attributed to the presence o1’superoxide anion in the complex. Cobaltic complexes with amine and nucleotide ligands similar to those of the present complex bu: lacking the superoxide anion are known to be substitution inert [4. 141. A&hough the detailed mechanism of this labilization process is not known. it must be noted that the superoxide anion can impart to the metal ran a partial Co(ll) character. which might change its substitution properties. As for the role of the phenanthroline moiety, we would like to suggest that it stabihzes the superoxide anion in the complex by acting as an “zleztron sink’ ]24], which enables a delocalization of the unpaired electron. Another role of’ this aromatic l&and might be to stabilize the adenine ring of ATP by stacking interactions. It is suggested that “Co(llI)-(phen)-ATP” attaches itself to the enzymes through an axial position. which is first occupied by 0; and exchanged for a ligand from the ATPases.
92
J. GRANOT, M. M. WERBER and A. DANCHIN
REFERENCES 1. 2. 3. 4. 5.
A. A. A. A. A. 6. A_ 7_ M.
Kowalsky.1 BioZ. c7rem. 244,6619 (1969). Danchin, ComptesRend. Acad. Sci Paris.. Ser. D 273,1636 (1971). Danchin. Biocidmie 54,333 (1972). Danchin. Biochimie 55.17 (1973). Danchin. Biochimie 57,875 (1975). Danchin and H. But: /. Biol. Chem. 248,324l (1973). M_ Werber, A. Oplatka, and A. Danchin. Biochemistry 13.2683 (1974). 8. M. M. Werber. A. Danchin. Y. Hochman. C. Carmeli, and A. Lanir, in Metal Ligund Inreractions in Organic Chemisrry and Biochemistry (B. Pullman and N. Goldbtum. edr). Reidel Publisbiug Company, Dordrecht/Boston (1977). Pt. 1, pp. 283-290. 9. S. J. D. Karlish and M. M. Werber. manuscript in preparation (1978). 10. M. M. Werber and A. Danchin, in Methods in Enzymology. Vol. 46 (B. Jakoby and M. Wilchek. eds.). Academic Press. New York (1977). pp. 312-321. 11. A. M. Michelson, Biochimie 55,925 (1973). 12. J. W. Hartz and H. F. Deutsch. 3. Biol. Chem. 244,456s (1969). 13. J_ P. Henry, M. F. Isambert. and A. M. Michelson, Biochim. Biophys. Acta 205,437
(1970). 14. R. D. Cornelius, P. A. Hart, and W. W. CIeiand, Inorg. Chem., 16.3299 (1978). 15. M. Sundaralingam, Biopolyme;s 7,821 (1969). 16_ H. Sternhcht. R. G. Shuimaa, and E. W. Anderson. J. C?rem. Phys. 43.3123 (1965). 17. H. Stemlicht, R. G. Shulman. and E. W. Anderson, f. C&em. Phys. 43,3133 (1965). 18. J. Grzzot and D. Fiat, J. AM. Chem. Sot. 99-70 (1977)_ 19s. Y. Yonetani, H. Yamamoto, and T. Iizuka, L Biol. aem_ 249.2168 (1974). 19b. J. 5. Griffith. The Theoor of Trartsition MetalIons. Cambridge Univ. Press, Cambridge. U.K. (1964). pp_ 360-363. 20. S. Koda. A. !%sono. and Y. Uchida. Bull. Chem. Sot. Jup. 43,3143 (1970). 21. A. -4bragam, The Principles of Nuclear Magnetism. Oxford Univ. Press, Oxford, U.K. (1961), Chap. 8. 22. Y. Lam, G. P. P. Kuntz. and G. Kotowyn, J. Am Ckem Sot. 96,1834 (1974). 23. hf. Cohn and T. R_ Hughes, Jr-.I BioL Ckm. 237,176 (1962). 24. M. M- Werber.J. 17reorer. BioL 60.51 (1976).
Received 21 August I977