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Electrochemistry as an Investigation Tool in Bioinorganic Chemistry: from Toxicology to the Simulation of Enzyme Catalyzed Reactions

Florinel Gabriel Banica, and Ana Ion




1 Department of Chemistry, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway.

2 Department of Analytical Chemistry and Instrumental Analysis,  "Politehnica" University of Bucharest,
  Bucharest, 1 Polizu Street, 78126,  Romania

Key words: Nickel, amino acids, cysteine, penicillamine, complexes, detoxication, hydrogenase, electrocatalysis, hydrogen
 

For longtime, the interest in the bioinorganic chemistry of nickel was connected only to its toxic effects and to the attempt at finding complexing agents that are able to perform an efficient detoxication. An important reorientation has been occurring in the last 20 years, when the new research field of nickel-enzymes emerged. In this regard, a special emphasis is put on nickel-hydrogenases, a class of enzyme that are able to catalyze the reversible reaction H2 = 2H+ + 2e-.

Nickel complexation by the SH group belonging to a cysteine residue in hydrogenases is well documented [1] and forms the basis for the electrochemical modelling of hydrogenase activity by low molecular complexes of nickel with cysteine and its derivatives [2,3].

3]. Making a direct correlation between the structure and the stability of nickel complexes with cysteine (Cys), on one side, and nickel behavior in living organism, on the other side, appears as a promising approach for understanding some aspects of bioinorganic chemistry of nickel. Unfortunately, the coordinating scheme in this system is very intricate and not surely defined, as it involves some polynuclear species in addition to the simple [NiL] (log b1 = 15.485) and [NiL2]2- (log b2 = 20.158) forms [4]. Conversely, penicillamine (Pen, Scheme 1), that includes similar binding sites, forms only two mononuclear complex species with the formation constants not too much different from that of cysteine analogous forms (log b1 =10.749; log b2 = 22.886 ) [4].

therefore, Pen appears as a suitable ligand for investigating nickel ion interaction with alpha-thioamino acids. In addition, Pen is a common pharmaceutical product whose effect depends in many instances on its complexing capacity. In this connection, is important to mention the use of Pen for curing nickel intoxication. It is only one of several chelating agents used to this end. Selection of such agents is mostly done in an empirical way, taking into account only the high formation constant of the relevant nickel complexes. Nevertheless, the formation constant alone cannot account for the detoxifying action and there are not yet a certain correlation between some chemical properties of the nickel complexes and the detoxifying capacity of the ligand [5].

Starting from the above considerations, a polarographic investigation of the Ni2+ - Pen system was undergone in the pH range around 7 (0.025 M CH3COONa, 0.025 M Na2HPO4 with appropriate additions of HClO4). Complex species concentrations were calculated as a function of pH, Ni2+ and Pen concentrations, using available equilibrium constants [4]. In view of the very low formation constants, the formation of nickel complexes with buffer components was neglected. Species concentrations were correlated with the parameters of the polarographic curves in order to assess which complex is responsible for each electrochemical process.

As shown in Fig. 1, nickel diffusion wave (A, on curve 1) is depressed after adding Pen (curve 2). Moreover, two additional waves appears on curve 2: wave C that is due to the catalytic reduction of nickel ion [6] and wave D that is ascribed to the catalytic hydrogen evolution, by analogy with the catalytic hydrogen pre-wave (CHP) produced by Cys [2].
 
 

FIG. 1.

Polarographic catalytic waves in the Ni (II) –Pen system at pH 6.52 and [Ni (II)] = 1 mM. [Pen]:1 0; 2 0.48 mM . Start potential –0.4 V; potential scale –0.2 V/division. Inset: a comparison of the Pen and Cys effects (0.2 mM each). 3 Cys; 4 Pen, pH 6.52, [Ni (II)]  = 2 mM ; the potential axis as before.




The decrease in the wave A current in the presence of Pen results from the conversion of [Ni(H2O)6]2+ into the complex form [NiL2]2-, that is not reducible. In addition, this complex undergoes an extremely slow decomposition when the complex equilibria are disturbed by the reduction of [Ni(H2O)6]2+ and may be assumed as chemically inert [7]. This behavior strongly contrasts that of the Cys analogous, which is labile and quickly decomposes during the reduction of [Ni(H2O)6]2+.

The inert character of the above Pen complex was also demonstrated in the case of the catalytic nickel reduction (wave C on curve 2, Fig. 1). It was proved that the reducible species in this case is the [NiL] form and, by analogy, it was asserted that a similar species is involved in the reduction of nickel ion catalyzed by Cys [6]. The above conclusions emerged from the correlation between wave C current and nickel species concentrations. The catalytic character of this process results from the regeneration of [NiL] by the reaction of the excess [Ni(H2O)6]2+ with Pen molecules released by the reduction of the complex.

This huge difference in the kinetic behavior of the analogous Pen and Cys complexes was explained by some marked differences in the chemical structure that occurs despite the identical combining ratios [8]. As shown in Scheme 1, whereas the Pen complex (b) has a cis configuration, its Cys analogue (a) shows a trans arrangement. In addition, the disposition of the anionic carboxyl groups in the Cys complex (a) offer more favorable conditions for the axial binding of a water molecule as a first step in the decomposition process. Furthermore, after the breaking of a –NH2 Ni2+ bond, the splitting of the second coordination bond occurs much more faster in the case of the Cys complex owing to the well-known trans effect [9]. Both above outcomes are much less effective in the case of the Pen complex (b). This accounts for its outstanding kinetic stability, as demonstrated by the polarographic data [6, 7] and, also, provides an explanation for the higher detoxifying capacity of Pen as compared to Cys, already noticed in ref. [10].
 
 

Scheme 1

As far as the catalytic hydrogen evolution is concerned (Fig. 1, wave D), it is clear that it involves a transient Ni(0) complex that is able to coordinate the hydride anion as an intermediate in H+ reduction to H2 [2]. One crucial problem in this connection is the structure of the Ni2+ species that generate the Ni(0) complex by electrochemical reduction. Fig 2 shows that, at constant Pen concentration, the CHP current variation parallels the change in the concentration of the [NiL] species.

Conversely, the CHP is absent in the region of the low [Ni2+]/[Pen] ratio, although the [NiL2] complex is present in a significant concentration. It is evident that the species responsible for the occurrence of the catalytic hydrogen evolution in the potential range of the CHP is the mono-ligand complex. The effect of other parameters (pH, Pen concentration, edta as a competing ligand) points to the same conclusion.

Clearly, the same kind of complex functions as the precursor of the catalytic species in the case of the Cys as well. This assumption is supported by the full analogy between the CHPs produced by either Pen or Cys. It results, therefore, that the electrochemical processes that may simulate the enzymatic hydrogen ion reduction [2] starts up with the reduction of the mono-cysteineate nickel(II) complex.

In conclusion, it may be pointed out that, although the thermodynamic data are extremely useful for understanding the behavior of the metal ions in living organisms, the kinetic behavior of the relevant complex compounds is sometimes decisive in this respect. Polarography, as well as other electroanalytical methods are particularly advantageous in this respect.


 FIG. 2.

Effect of Ni(II) at constant Pen concentration. pH 6.5.a) CHP (wave D) current vs. the [Ni(II)]/[Pen] ratio. Ni(II), mM:
(1) 0.5; (2) 0.2. b) Species concentrations (in mM), for 0.5 mM Pen.

References (with links to some reprints available in  NTNU Library, Trondheim)

1.  M. Frey, Struct. Bonding, 90 (1998, 90,) 97.
2.  A. Calusaru and V. Voicu, J. Electroanal. Chem., 43 (1973) 257; F. G. Banica, Bull. Soc. Chim. Fr.,
    128,  (1991) 697.
3.  M. A. Halcrow, G.Christou, Chem. Rev., 94 (1994), 421.
4.  D. D. Perrin and J. G. Sayce, J. Chem. Soc. (A), (1968) 53.
5.  S. K. Tandon, S. Singh, V. K. Jain and S. Prasad, Fund. Appl. Toxicol., 31 (1996) 141.
6.  F. G. Banica, A. Ion, Collect. Czech. Chem. Commun., 63 (1998) 995.
7.  A. Ion, F. G. Banica, C. Luca, Collect. Czech. Chem. Commun., 63 (1998) 187.
8.  N. Baidya, D. Ndreu, M. M. Olmstead and P. K. Mascharak, Inorg. Chem., 30 (1991) 2448;. N. Baidya, M. M. Olmstead
    and  P. K. Mascharak, Inorg. Chem., 30 (1991) 3967.
9.  F. Basolo and R. G. Pearson, Mechanisms of Inorganic Reactions, John Wiley, New York, 1967, p. 417.
10. M. M. Jones, M. A. Basinger and A. D. Weaver, J. Inorg. Nucl. Chem., 43 (1981) 1705.