THEOXIDATION OF CYSTEINE AND GLUTATHIONE BY MOLYBDENUM(VI)
2863
The Oxidation of Cysteine and Glutathione by Molybdenum(V1) by J. F. Martin' and J. T. Spence Chemistry Department, Utah State University, Logan, Utah 84331
(Received March 10, 1970)
The oxidation of L-cysteine and glutathione by illo(V1) in phosphate buffer at pH 7.50 has been investigated as a possible model for similar reactions in molybdenum enzymes. KO evidence was found for complex formation between Mo(V1) and either cysteine or glutathione; Mo(V), however, forms a strong l :2 complex with cysteine, and a very weak monomeric, esr-active complex is formed with glutathione. The oxidation of cysteine was found to be third order, first order in Mo(V1) and second order in cysteine. The rate of the reaction with glutathione was found to be independent of Mo(V1) concentration at high Mo(V1) concentrations (lo+ M ) and dependent to the first power at low concentrations M ) , and dependent on glutathione to the first power of all concentrations. Mechanisms for both reactions have been postulated. Redox reactions between the R!Io(V)/hlo(VI) couple and the thiol-disulfide system, 2RSH/RSSR, are of interest as possible models for such reactions in molybdenum enzymes, where the metal ion appears to be bound to a cysteine side chain.2 A previous paper reported the study of the oxidation of a simple thiol ligand, thioglycolic acid, by R'Io(VI).~ This paper deals with the oxidation of the amino acid L-cysteine and the tripeptide glutathione (a-glutamylcysteinylglycine), ligands which should more closely approximate the metal binding site of the enzymes. Complexes of Mo(V1) and Mo(V) with cysteine have been studied by a number of ~ o r k e r s ~and - ~ the X-ray crystal structure of a solid 1: 1 dimeric ?tlo(V)cysteine complex has been determined.' Aqueous solutions of both cysteine and glutathione with Mo(V) have been reported to give an esr signal indicative of the presence of a small amount of monomeric species, with parameters similar to the signal observed with the enzymes.* It has also been reported that cysteine is oxidized by R/Io(VI), but the reaction was not studied in detail.6
Results It has been shown that the oxidation of the thiol
Furthermore, although X max is the same, e for the 1 :2 complex in solution is considerably greater than that reported for the 1 : l complex (16.4 X lo3 os. 12.8 X 103).4 I n an attempt to resolve this difficulty, the solid 1 : 1 complex was prepared, as described by R/Ielby6 and l ' l i t ~ h e l l ,and ~ their values of E were verified. When the solid 1 :1 complex was added to a solution of excess cysteine at pH 7.50, no increase in the absorbance at 305 mp was observed, indicating the 1 : 1 complex, once formed, is inert to the addition of more ligands. A possible explanation for the different results may be in the somewhat different conditions of formation. The 1 :2 complex is formed in solution in the presence of excess ligand. Upon precipitation, perhaps because of solubility considerations, the solid 1 : 1 complex is obtained. Once formed, this 1 : 1 complex appears to be very stable to substitution in solution even in the presence of excess cysteine, possibly because of its dioxobridge structure.' The 1 : 2 complex, as with similar species in the literature6~g~'0 most likely is a monobridged structure, with the cysteine bound through the thiol and either the carboxy or amino groups.6 All attempts to obtain a solid 1 : 2 complex gave the 1 : 1 complex.
group by R!Io(VI) produces a di~ulfide,~ in this case cystine. Because of its zwitterion nature, cystine is quite insoluble at pH (1970). ~ The Journal of Physical Chemistry. Vol. 7 4 , No. 16, 1970
2864
J. F. MARTINAND J. T. SPENCE
Os2
Os' 0
A
Y t
1
I
1
2
3 [CY$]
I
I
4
5
I 6
7
8
/[Mo(V)],
Figure 1. Molar ratio plot for Mo(V)z-cysteine complex. Absorbance at 303 mp is plotted us. ratio M. [ c y s ] / [ M o ( V ) ] ~ .[Mo(V)] = 7.50 X
It should be pointed out that the question regarding the structure of the complex is unresolved. If the C above interpretation is correct, the 1: 1 dioxobridged complex is not in equilibrium with the 1:2 species in 100 gauss solution at this pH. Figure 2. Esr spectra: A, M O ( V ) ~ O ~ ( C ~3.00 S ) ~x~ -10-2 , M; Solutions of Mo(V)Z and cysteine exhibit an esr gll = 2.015, 91 = 1.959, 0 = 1.978; S L = 100; B, [Mo(V)] = signal a t this pH. The signal is small, however, 3.00 X M, [GSH] = 5.00 x 10-lM. 811 = 2.010, accounting for less than 1% of the total No(V). The g l = 1.979, 0 = 1.987, g = 1.910; S L = 100; C, [Mo(V)] = g values of the signal were determined to be glI = 3.00 X lo-* M; g = 1.921; SL = 250; all spectra 2.015, gl = 1.958, 0 = 1.978 (Figure 2). The g value recorded in 1.00 M phosphate, pH 7.50, 77". M A = 4000, attentuation = 5.90. agrees reasonably well with that reported by Huang and Haight for the system.8 As with most n!Io(V)thiol systems, the g values are quite high, indicating Since the molar ratio study indicated essentially all considerable electron delocalization. the Mo(V) is present as the 1:2 complex and as no Solutions of glutathione and either 1\IoOCIj2other species absorb in this region, the increase in (which is dimerized at this pH) or lI004~-gave no absorbance at 305 mp was used t o follow the kinetics. spectral evidence of complex formation a t pH 7.50 in Trial and error plots of the rate data, for runs with phosphate buffer. A small amount of a monomeric excess cysteine, indicated the reaction was first order in 3Io(V) complex (
