Advances in Molecular Relaxation and Interaction Processes, 16 (1980) 29-39 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

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PARAMAGNETIC ION BINDING TO AMINO ACIDS : THE STRUCTURE

OF THE MANGANESE (II)-L-PROLINE COMPLEX FROM CARBON-13

RELAXATION DATA,

BERNARD HENRY, MICHEL RAPPENEAU, JEAN-CLAUDE BOUBEL,

and JEAN-JACQUES DELPUECH

Laboratoire de Chimie Fhysique Organique, ERA CNRS 222,

Universite de Nancy I, C.O. n”l40, 54037 Nancy Cedex (France)

(Received 30th. May 1979)

ABSTRACT

Carbon-13 longitudinal relaxation times T, of aqueous solutions of proline at pH=ll containing 10 -4-10-5M manganese(I1) perchlorate are measured at 62.86 MHz and 6O’C. Under these conditions, the Mn 2+ cation is bound to three proline molecules in their dibasic form to form the complex p(L-PRO-G - . The relaxation of carbons c(, B, y, 6 in this complex is shown to be dipolar. The relevant correlation time is rotational ~~‘4.3~10 -lls

(at 6O'C). A method is given to compute the Mn2+-13C distances in the complex from the paramagnetic relaxation rates l/T,M of carbons CL to 6 and an assumed geometry of the proline molecule. The manganese (II) cation may be positioned with respect to each proline ligand, thus determining the structure of the hexacoor- dinated complex. The sites of coordination are the uncharged nitrogen and one carboxylic oxygen atom of the proline molecules, their distance to the Mn 2+ cation are respectively 2~2.2 and 1.57 i.

INTRODUCTION -

Nuclear magnetic resonance spectroscopy has been widely used in the determination of metal-ligand structures, and is also capable of yielding kinetic data about these interactions (1).

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Several methods have been devised for this purpose. One of them applies to paramagnetic cations and is currently used for obtai- ning structural and kinetic information in biochemical systems (2-4).

A qualitative procedure consists in observing the selective line-broadening in the NMR spectrum of compounds such as nucleic acids and bases, DNA, proteins and enzymes, nucleotides and nucleosides, in the presence of a paramagnetic cation used as either a natural or artificial probe. This allows to locate the cation binding site within the biological system.

This method however should be exercised with some caution if it is used to determine the accurate geometry of a metal ion complex. Even if the chemical exchange between free and bound ligand molecules is fast on the nmr time-scale, the observed linewidths are not necessarily a function of the distance between the cation and the binding site, more especially for probes such as Cu2+ and Mn2+ (S-6).

However.this difficulty may be overcome in some instances by studying nuclear relaxation effects induced by the paramagnetic cation. This paper is devoted to a study of a manganese(II)-L- Proline complex in aqueous solution by measuring carbon-13 longi- tudinal relaxation times T,. Investigations using amino-acids as ligands are relatively rare in the literature (6-8) in spite

of the importance of these compounds in the building of complex biochemical and biological molecules. Moreover this example has been chosen so as to test an original variant for computing the cation-nuclei distances in a paramagnetic complex. In this respect, kinetic information from studying line-broadening and paramagnetic shifts is deferred to a later publication.

THE METHOD

In water, L-Proline is a diacid pK,=2.03 and pK2=10.64

at 37OC (9). Above pH~l1, the dibasic form L-PRO- alone is

present in solution. Several complexes have been detected and their stability constants have been measured by poten-

tiometry at 37’C, as shown on the following equilibrium

reactions (10)

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Mn 2+ +L-PRO- g kn(L-PROg+ K,=,9-2*84

Mn 2++2L-PRO-: Mn(L-PRO-)2 K2',()_5.53

Mn2+ +3L-PRO-Z b(L-PRO-)]- K3=10-6'74 -

Mn2+ +L-HPRO 3 r Mn(L-HPRO)-2+ _I K4=,0-"50

-

where L-HPRO is the amino acid in its zwitterionic form. C_onsidering the concentration range used in this work

2+ I1 Mn =10w4M and L-PRO-=1.28 M allows us to specify that the manganese(I1) cation is completely bound to the amino acid under the form of a tris-Proline complex (95 %) and a bis- Proline complex (5 90). We shall then consider that the observed relaxation rates l/T, of the carbon nuclei of the proline arise from a weighted mean between free proline molecules (l/T,D in the diamagnetic site D) and the proline ligands in the tris- proline complex (l/T,M in the paramagnetic site M). This assumes a fast microscopic equilibrium between sites D and M, which is achieved on the NMR time scale at temperatures slightly higher than room temperature. Experiments were there- fore performed at 6O'C.

Relaxation rates may thus be expressed as (13)

where pM is the mole fraction of the bound amino acid,

3CMn2+ PM = -----<<l 'PRO

CMn2+ and CpRO are the analytical concentrations of the cation and of L-Proline, respectively. The relaxation rate in the paramagnetic site is thus obtained from the equation

1/T jM= $(l/T,-J/TID) M

where T,D refers to the value of l/T, measured in the absence of the manganese(I1) cation.

Now the paramagnetic relaxation rate l/T,M is the sum of two contributions, scalar and dipolar (12). The scalar contri- bution is expressed as (13)

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U/TIM)RS=KRS(-$+ l+w T e c

where

KRS=8nZAZS(S+1)/3, with A : hyperfine coupling constant and S=5/2 the electronic spin number of the Mn2+ cation. ‘C is a correlation time, itself arising from two contributions :

l/rc=l/Tle+l/~M=l/T1e

where T leyb” -7-10-gs is the electronic relaxation time (14)

and ~~~10 s is the mean lifetime of a proline molecule in the complex (a value resulting from variable temperature experiments). oe=l.03x10 12 rad.s-’ is the electron resonance frequency in an induction field of 5.87 T. This contribution is in fact negligible for two reasons : the paramagnetic shifts of the carbon nuclei are weak (a few Hz), this results into small A and KRS values (KRS<ti8 .lO 1 2s-2) ; the factor

We-r,=10 3 -10’ is clearly larger than unity and the correlation function T~/(~+LI:~:) thus amounts to (u~T,) -1 $%10-‘5-10-‘7.

The upper limit found for the scalar contribution

(l/TIM)RS”~10 -zs-1 cannot account for the experimental values 3 4 -1 l/T,M%lO -10 s .

The longitudinal relaxation rate is therefore purely dipolar in character (15) and may be expressed as (16-17)

1 /TIM=KDD. k. (5,; KDD %’ r r

C c c’ C

with

KDD= 15 6x10-l4 U;Yf

pM=5.92 Bohr magnetons is the electronic spin moment of the Mn 2+

cation and yc =6.72x107rad.s-’ . 1s the magnetogyric ratio of the 13 C nuclei. rc is the distance between the manganese(I1) cation and the examined carbon nucleus. TC’ is a correlation time which is the sum of three contributions

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where rr<<T ,e,~~ is the rotational correlation time of the complex. ~c=3.9Sx108rad.s-1 . 1s the resonance angular frequency of the 13c

nuclei. These equations cannot yield the two unknowns rc and ?r if

only one carbon nucleus is considered. However the problem of determining the structure of the complex may be solved if several carbon nuclei are examined in the same experiment. Then we may write as many expressions (4) as there exist magnetically non- equivalent carbon nuclei C,,C2...C,, whose ratios allow to compute quantities ~~,h2...h, such as

, i=3 to n-l

assuming that rr is the same for all nuclei. It is therefore

possible to locate the manganese(I1) cation with respect to one

proline molecule if the carbon atoms C,,C2... occupy known positions in this molecule. Four carbons may be used for this purpose in the pyrrolidine ring of the proline : Ca,CB,CY, CA.

rC2 A ratio such as XI=7 (where Cl and C2 stand for C C ) a' 8

indicates 5

that the manganese nucleus should lie on a sphere S, whose diame- ter M1M2 is determined by the two points M,M2 which share the internuclear vector C,C2 in the aboc, ratio x,. The position of the manganese nucleus results from the intersection of spheres s,,s2...

A computer program has been written for this purpose. The proline atomic coordinates are taken from crystallographic data relative to the Cu '+-proline or Pd '+-proline complexes (38-20) and are expressed in an orthonormal basis. The coordinates of couples of points such as MIM2 are computed through some straight- forward algebra, thus yielding the centers 011,02,0S and radii RI,

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R2,R3 of the spheres S,, S2,S3 (obtained by considering the couples C&: c c B Y, CYC, of 13C nuclei, respectively). The coordinate basis is thenchanged so as to bring the new origin at the center

0, of S,' the new x axis along the vector 0,02, and the new xy plane into plane 0,0203. The intersection of spheres Ss,S2 is contained in a plane P, parallel to the yz plane, and the inter- section of S2,S3 in a plane P2 p arallel to the z axis, The inter- section of P,P2 is a straight line A parallel to the z axis. The cation nucleus is located at the common intersections of A with spheres S,,Sz,S3. Two positions are obtained in this way, which are symmetrical about the xy plane. One of these positions could be discarded on account of a too close proximity of the manganese(I1) cation to the pyrrolidine ring. All carbon-manga- nese(I1) distances are computed from the rectangular coordinates, this allows in turn to compute the correlation time tc from equation (4).

EXPERIMENTAL SECTION

L-Proline and hydrated manganese(I1) perchlorate Mn(C104)2,6H20 were obtained from Bachem Feinchemikalien and Alfa Inorganics, respectively, and were used without further purification. Stock solutions of 0.01 M Mn(C104)2 in deuterated water were diluted to the desired concentration. Aliquots were added with L-proline in computed quantity and with deuterated soda NaOD to obtain pH 11. Potentiometric measurements were carried out with a Methrom EA436 potentiometer and a combined glass-calomel Ingold microelectrode. All samples were prepared in a nitrogen atmos- phere using water which had been purged with nitrogen and 8 mm 0.D NMR tubes equipped with tight-fitting caps. Relaxation times of 13 C nuclei at 62.86 MHz are obtained from partially relaxed Fourie Transform spectra using a CAMECA RMN 250 spectrometer, D20 as an internal heteronuclear lock and 180°--r-90° pulse sequences with the fast inversion-recovery variant (21). 328 FID's over 36 K poin

were accumulated in each run. All calculations were performed on a Texas Instruments 980A minicomputer equipped with a digital plotter, Hewlett-Packard 7210A.

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RESULTS AND DISCUSSION

Four concentrations CMn2+ = 0 ; 5.0 ; 10.0 ; 20.0 JO-‘M of the manganese(I1) cation were used in this study. Values of the para- magnetic relaxation rate 1 /T,M are obtained from the slope of the least squares line representing the relaxation rates l/T, measure1 as a function of CMn2+ (Table l), with an error of cu. 5 %. Ratio such as x, are then computed with an error ~?,,/~,=2x5/6=1.5 %. Values xl?Ahl,h2’Ax2,13?Ax3 were then introduced into the compute program to derive the whole range of distances rc. Each of these distances is given in Table 2 as the mean of values resulting fro] all the combinations of the A,,A~,x~ ratios. The corresponding error is estimated as the difference between the maximum and

TABLE 1. The longitudinal relaxation times of carbons a,@,~,6 of a 1.28 M L-Proline solution in D20 at pD=11.4 ; 62.86 MHz and 6O“C as a function of the concentration of the manganese(I1) perchlorate, and the corresponding relaxation rates l/T,M in

the paramagnetic site

0" 7.04 6.13 7.63 5.81

5.0 1.56 4.00 4.95 1.60

10.0 0.90 2.76 * 3.53 1.04

20.0 0.47 2.15 2.70 0.53

“TIM(s-‘) 4230 656 509 3625 +210 k33 +22 ?181

x In agreement with values from the literature (22).

minimum values thus obtained. A good agreement is observed betwee these distances and those obtained in the solid state for the Cu2+-Proline complex (19) . These results allow to position the Mn 2+ cation with respect to carbons C,,CB,CY,6& of the proline ligands. The distances to the other atoms of the proline molecule i.e. nitrogen and atoms C(O;), 0,,02 of the carboxylate group are

86

o

TABLE 2. The distances (A) between the manganese(II) cation and the atoms of L-Proline in the complex Mn(L-PRO-) 3 , and the corresponding values for the Cu 2+ proline complex in the solid state (]9)

Mn2+-L-Proline Cu2+-L-Proline Atom complex complex C 2.87±0.28 2.85

C B 3.92±0.41 3.98 C 4.08±0.36 4.]2 Y

C6 2.95±0.3] 2.95

N 2,22 ].95 C(o) 2.86 2.82 01 1.97 2.04 02 3.99 4.04

also computed by using the geometry of the proline molecule in the solid state and the position of the Mn 2+ cation described abo~e. The internuclear distances N-Mn 2+ and Oi-Mn 2+ (2.22 and 1.97 A)

are slightly smaller than the sums of the co~responding Van der . ° O 2 - Waals or ionic radii (N : 1 5 A ; : 1.32 A ; Mn 2+ : 0.8 ),

thus showing that the coordination sites of the proline ligand are the uncharged nitrogen atom and an oxygen atom of the carbo- xylate anion, as in the solid state proline-Cu 2+ complex, or as the histidine-Mn 2+ complex in aqueous solution (8).

We may then suggest a hexacoordinated structure of the whole complex ~Mn-(L-PRO)~3 - by assembling three proline units where the six ~-'coordinatio-ff sites are three nitrogen and three carboxylic oxygen atoms in an octahedral arrangement, as represented in Figure J, obtained from a computer using program ORTEP (23).

The rotational correlation time obtained at 60°C : z r = (4.33±2,00)~]0-]Is has an expected order of magnitude if compared to values of J.6×]0-]Js at 65~C for the smaller complex Mn(H20)62+ (24) and of J.52~]0-]0s at 70~C for the Mn2+-histidine

complex (8). In conclusion, let us recall the conditions or assumptions

which are necessary for obtaining structural information by this

method.

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(a) The relaxation of the investigated carbon nuclei must

be dipolar

(b) There should exist a known basis of four carbon-13 atoms at least in the ligand molecule

(c) This basis should be rigid or semi-rigid within the

complex, so that internal rotation is negligible with respect to

the tumbling motion of the complex (T r must be the same for all

the carbon nuclei of the basis, at least for a system where T1e>Tr ).

Figure I. The structure of the manganese(II)-Proline complex in aqueous solution at pH=11 and 60°C.

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The latter condition was not fully respected for the proline molecule, since the gauche structure of the pyrrolidine ring may have internal inversion. However it should be noticed that the computation is feasible (i.e. spheres S,,S2,S3 are actually in- tersecting) only when using the solid state geometry of the pro- line-Cu2+ complex (in which the corner C C C

BY& of pyrrolidine is

pointing to the manganese(I1) cation). This result suggests that the conformation of the proline ligand is unchanged in aqueous solution, probably on account of the presence of strongly solva- ting water molecules around and/or within the complex framework.

Acknowledgments. Financial support from the Centre National de la Recherche Scientifique is gratefully acknowledged. All NMR spectra were recorded on spectrometers of the Groupement R6gional de Mesures Physiques de l'Acad&mie de Nancy-Metz. We thank M. Dumortier for his technical assistance, Pr. Roques and Castro for stimulating discussions, and Pr, Protas and Dr, Lecomte for the ORTEP program.

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