Vol. 2, No. 2,April, 1963
COMPLEXES OF CYSTEINE WITH MOLYBDENUM(V) AND (VI)
319
CONTRIBUTION FROM THE CHEMISTRY DEPARTMENT OF UTAHSTATE UNIVBRSITY, LOGAN, UTAH
Complexes of Cysteine with Molybdenum(V) and Molybdenum(V1) BY J. T. S P E N C E
AND
H E L E N H. Y . CHANCY
Received July 30, 1962 Spectrophotometric studies have shown that cysteine forms complexes in acidic solution with molybdenum(V) and molyb denum(V1). The molybdenum(V1) complex has a cysteine:metal ratio that varies from 3 : 1 to 1:1, depending upon concentration. Studies with compounds similar t o cysteine have indicated that the ionized mercapto and carboxyl groups of cysteine are most likely involved in the complex. Molybdenum(V) forms a 1: 1 complex with cysteine that is unstable to atmospheric oxidation. Formation constants for the complexes have been estimated. The implications for sulfhydryl enzymes are discussed. of these compounds and Mo(V1) were cooled to 0” immediately The molybdo-flavoenzymes, xanthine oxidase and upon mixing and kept at this temperature until just before measnitrate reductase, have been reported to be sulfhydryl urements were made. They then were placed in a constant e n ~ y m e s . In ~ ~the ~ course of an investigation in this temperature bath, warmed to 2 5 O , and read immediately in the Laboratory of the biological function of molybdenum sgectrophotometer. This procedure was unnecessary in the it seemed of interest to study complexes of this metal case of cysteine. L-Cysteine, S-methylcysteine, L-cysteine ethyl ester hydrowith cysteine in aqueous solution, as a model for poschloride, L-cystine, P-mercaptoethylamine hydrochloride, and sible molybdenum-enzyme interaction. L-alanine were purchased from Nutritional Biochemicals Corp. I t has been reported that thioglycollic acid (merP-Mercaptopropionic acid was purchased from Eastman Kodak captoacetic acid) forms complexes with both molybCo. Methyl 0-mercaptopropionate was prepared according to denum(V) and (VI) and that this compound can be the method of Schmidt and Grob.’ All mercapto compounds were analyzed by the iodometric titration method of Neville and used as a reagent for the photometric determination of Gorin.8 Deaerated solutions of these compounds were prepared m ~ l y b d e n u m . ~It is also known that molybdenum just before use and kept under nitrogen. All other compounds forms complexes with various sulfhydryl c o m p o ~ n d s . ~ , ~ were used without further purification. However, no reports have appeared concerning comMolybdenum(V) and (VI) solutions were prepared and standplexes with cysteine or cysteine-containing peptides. ardized as described previously.9 All buffers used were prepared
from reagent grade chemicals.
Experimental Spectrophotometric data were obtained with a Perkin-Elmer Model 40004 recording spectrophotometer and Beckman Model DU and Model B spectrophotometers, using cells of the appropriate path length. A silica absorptioa cell, which could be evacuated, was joined to a specially constructed vessel for the molybdenum(V) measurements. This was necessary since molybdenum(V) is easily oxidized by the oxygen of the air unless kept in solutions 1A4 or stronger in hydrogen ion, The proper amount of molybdenum(V) (in 3 M HCl), buffer, and cysteine were added to the vessel under nitrogen or helium. A sample then was drawn off into the evacuated cell by means of a stopcock and the closed cell removed from the vessel and placed in the spectrophotometer for absorption measurements. The absorbance of the sample in the cell was constant for 3 hr., indicating no oxidation during this period. All solutions were kept in a constant temperature bath at 25 i:0.1’ for at least 30 min. before measurement and the cell compartment of the Beckman DU spectrophotometer was equipped with thermospacers which kept the temperature in the cell compartment a t 25 ;t 1’. Cysteine ethyl ester, 8-mercaptopropionic acid, and p-mercaptoethylamine were slowly oxidized by Mo(V1) a t p H 5 , forming molybdenum blue. In order to minimize this reaction, solutions (1) Abstracted from the thesis submitted by H. C. in partial fulfillment of the requirements of the M.S. degree a t Utah State University, 1962. Journal paper No. 228, Utah State University Agricultural Experiment Station. (2) H. R. Mahler, B. Mackler, and I). E. Green, J . Rid. Chem., 210, 465 (1954). (3) J. Hariis and L. Hellerman, “A Symposium on Inorganic Nitrogen Metabolism,” W. D. McElroy and B. Glass, Ed., The Johns Hogkins Press, Baltimore, Md., 19.56, p. 565. (4) A. I. Busev and F. Chzhan, Zh. A m l . Khim., 16,39 (1961). ( 5 ) T.W. Gilbert, Jr., and E. B. Sandell, J . A m . Chem. Soc., 82, 1087 (1860). (6) A. I. Busev and F. Chzhan, Zh. Anal. Khim., 18, 171 (1961).
Results Molybdenum(VI).-In the pH range from 4-6, a deep yellow color is formed immediately when molybdenum(VI) is added to a solution of cysteine, indicating complex formation. Molybdenum (VI) solutions are colorless, but exhibit “end absorption,” beginning a t about 350 rnp in the concentration range used, with the absorbance increasing sharply with decreasing wave length, The spectrum of the complex shows the same kind of absorption, without any maximum, but shifted bathochromically. The yellow color is due to the long shoulder that extends into the visible region. At 400 mp the absorbance of molybdenum(V1) is negligible, while that of the complex is sufficient for making measurements. This wave length was used for most measurements The effect of pH on the absorbance of the complex a t 400 mp is seen in Fig, 1. The rise of the absorbance below pH 4.6 is most likely due to a molybdenum(V)molybdenum(V1) compound formed when reduction of some molybdenum(V1) by cysteine a t this lower pH occurs. Ostrowetsky and Souchay have reported rnolybdenum(V)-molybdenum (VI) complexes that absorb a t 450 mp10 and 325 mp.“ As the pH is lowered (7) H. Schmidt and E Grob, Helv Chzm Acta, B l , 360 (1048) ( 8 ) K G Nevtlle and G Gorin, J A m Chem Soc , 78, 4891 (1956) (9) J T. Spence ana J Tocatlian, z b z d , 83, 810 (1961) (10) S Ostrowetsky and P Souchay, Comfit ! * e n d ,261, 373 (1960) (11) S Ostrowetsky, zbzd , a p t , 1068 (1960).
320 J. T. SPENCE .4ND HELENH . IT. CHANG
0.700
Inorganic Chemistry
,D'
,
0.400
0.600
0.500
0.300
6
2
5
0.400
i e, 0
