Nonenzymatic NADH-Dependent Reduction of Keggin-Type 12-Tungstocobaltate ( III ) in Aqueous Medium

The kinetics of the electron transfer reaction of NADH with 12-tungstocobaltate(III) has been studied over the range 5.07 ≤ 10 [NADH] ≤ 15.22 mol dm, 7.0 ≤ pH ≤ 8.0 and 20 ≤ t ≤ 35 C in aqueous medium. The electron transfer reaction showed first-order dependence each in [NADH]T and [12-tungstocobaltate(III)]T. The products of the reaction were found to be NAD and 12-tungstocobaltate(II). The activation parameters ∆H (kJ mol) and ∆S (JKmol) of the electron transfer reactions were found to be 64.4±1.8 and -48.86±6.0. Negative value of ∆S is an indicative of an ordered transition state for the electron transfer reaction.


Introduction
Nicotinamide adenine dinucleotide (NADH) is a coenzyme.It is oxidized to NAD + in presence of enzymes.In metabolism NAD + is involved in redox reactions, carrying electrons from one reaction to other.The coenzyme is therefore found in two forms in cells: NAD + as an oxidizing agent and NADH as a reducing agent.The redox potential of the NAD + /NADH couple is -0.32 volts 1 , which makes NADH a strong reducing agent.The reaction is reversible 8 , this means the coenzyme can continuously cycle between NAD + and NADH forms.Studies on the electron transfer reactions of NADH and its synthetic analogues have been reported [2][3][4][5][6][7][8] .Reduction of methemoglobin 9 , orthovanadate and vanadyl sulphate 10 , ferric siderophores 11 and heteropolytungstovanadophosphates 12 by NADH has been reported.
The oxidant Keggin type 12-tungstocobaltate(III) (Co III W 12 O 40 5-or Co III W 5-) cluster has a reduction potential of 1.0V 13 , hence it can accept electrons from NADH.The above cluster is known for photochemical hydrogen generation and is involved in multielectron reduction P. MOHANTY et al. processes [14][15][16] .The redox behavior of the above cobalt cluster with few biomolecules like ascorbic acid 17 , glutathione 18 , citric acid 19 , DL-methionine 20 and cystine 21 have been investigated.In order to examine the redox behavior of 12-tungstocobaltate(III), its reaction with NADH has been investigated.

Experimental
[Co III W 12 O 40 ] 5-was prepared by the reported method 17 and was characterized spectrophotometrically 23 (at 388 nm ε 388 = 1150±2 dm -3 mol -1 cm -1 ).The chemicals used were of A.R grade and doubly distilled water was used throughout the experiment.The electron transfer reaction was studied at pH = 7.5±0.01using a pre-standardised Elico (India) digital pH meter equipped with glass electrode.The pH of the solution was maintained using phosphate buffer.
The absorbance measurements for the kinetic studies were done using CECIL CE 7200 UV-VIS scanning spectrophotometer equipped with CE 2024 thermoelectric controller.Reaction progress was monitored at 380 nm, pseudo first order conditions were maintained throughout the runs by using large (≥ five fold) excess of [NADH].The rate constants (k obs ) were obtained from the slope of -ln(A t -A ∞ ) versus 't' (min) plots (Figure 1).(1) Where A t and A ∞ are the absorbance of the reaction mixture at time 't' and at equilibrium respectively.The reported rate data represented as an average of duplicate runs were reproducible to within ±3%.The correlation coefficient of the plots used to determine k obs were found to be 0.99 in most cases.

Results and Discussion
Co III W 12 O 40 5-has absorbance maxima at 388 nm and on mixing with NADH the peak is found to be decrease with time (Figure 2) with simultaneous increase of new peaks at 625 nm (Figure 3) due to the formation of Co II W 12 O 40 6-and a peak at 260 nm (Figure 5) due to the formation of NAD + is observed.The decrease of absorbance at 380 nm is followed for the reduction of Co III W 12

Effect of [NADH] on reaction rate
The effect of varying [NADH] on the reaction rate was studied at fixed pH (7.5) and between 20-35 o C. The concentration of Co III W 5-was kept constant and 10 4 [NADH] was changed from 5.07 to 15.22 mol dm -3 .
The plot of k obs /s -1 vs.
[NADH] T is linear at four different temperatures (20-35 o C) (Figure 4).This fact indicates that the reaction is first order with respect to [NADH] T .Since the second order rate constant does not change appreciably at a given temperature (Table.1).The overall order of the electron transfer reaction is two.Hence the order with respect to the [Co III W 5-] T must be one.

Rate dependence on pH
At 298K with [Co III W 5-] T = 9.93×10 -5 and [NADH] T = 5.07×10 -4 mol dm -3 , the pH was varied from 7.0 to 8.0 using phosphate buffer(KH 2 PO 4 + Na 2 HPO 4 ) and 10 4 k obs (s -1 ) was found to be fairly constant (1.11±0.38).The electron transfer reaction does not show any pH dependence in the pH range 7.0 to 8.0.The electron transfer reaction could not be studied at higher pH because oxidant cobalt(III) clusters will undergo base hydrolysis.

Rate dependence on temperature
The electron transfer reaction was followed in the temperature range 20-35 o C. The oxidant concentration was kept constant at 9.93×10 -5 mol dm -3 and the reductant NADH concentration was varied from 5.07×10 -4 to 15.22×10 -4 mol dm -3 .The k obs was found to increase with increase in temperature (Figure 4).The second order rate constant 10 2 k 2 changed from 5.7±0.4 to 21.2±5.0 when temperature was changed from 20-35 o C. The values of ∆H # and ∆S # were calculated using eyring equation and were found to be 64.4±1.8 kJ mol -1 and -48.86±6.0JK -1 mol -1 .The negative value of activation entropy indicates an ordered transition state for the electron transfer reaction.

Polymerization study
The participation of free radicals was examined as follows.The reaction mixture, to which a known quantity of acrylonitrile scavenger had been added initially, was kept in an inert atmosphere for 5 h.When the reaction mixture was diluted with methanol, a white precipitate resulted.The above experiment supports the formation of free radical.Blank experiments with either [Co III W] 5-or NADH with acrylonitrile did not give polymerization under the same conditions as those used for the reaction mixture.

Stoichiometry and characterization of product
The stoichiometry of the reaction was studied at 30 o C where [Co III W 5-] T was kept constant at 9.93×10 -5 mol dm -3 and [NADH] T at 5.07×10 -4 mol dm -3 .Formation of a peak at 260 nm after 3 half lives (Figure 5) indicates the formation of NAD + .The formation of a peak at 625 nm (Figure 3

[Figure 1 .
Figure 1.Determination of k obs value -ln(A t -A ∞ ) = k obs t + C(1) Where A t and A ∞ are the absorbance of the reaction mixture at time 't' and at equilibrium respectively.The reported rate data represented as an average of duplicate runs were reproducible to within ±3%.The correlation coefficient of the plots used to determine k obs were found to be 0.99 in most cases.

Figure 4 .
Figure 4. Rate dependence on temperature