Oct. 5 , 1956
CYSTEINECOMPLEXES WITH COBALT(III) ION [CONTRIBUTION FROM
THE
4891
DEPARTMENT OF CHEMISTRY, UNIVERSITY O F OREGON]
Cysteine Complexes with the Cobalt(II1) Ion. I. The Mononucleate Structure of Cobalt (111)Bis-cysteinate BY ROYG. NEVILLE’~~ AND GEORGE GORIN RECEIVEDAPRIL17, 1956 Cobalt(II1) bis-cysteinate has been prepared by the original method of Schubert and by a new method from hexarnminecobalt(II1) chloride. The results of analysis, titration and molecular weight determination indicate that the products obtained by both methods are identical and that the complex possesses a mononucleate structure of empirical formula, HCoCyz.2Hz.0. No evidence was found for the binucleate structure suggested by earlier investigators.
Complexes of cobalt(II1) formed in the reaction of cobalt(I1) ion with alkaline solutions of cysteine in the presence of atmospheric oxygen were first studied by Harris, Michaelis and his co-workers, 4-6 and Kendall and Holst.7 The reaction is complicated, and many contradictory results exist in the earlier literature. Attempts to isolate the complexes met with little success until 1931-1933 when Schubert8 described the preparation of several cysteine complexes of di- and trivalent cobalt. The isolation of these complexes in the pure state was found to be strongly dependent upon the concentration of reactants, the pH and the cobalt to cysteine molar ratio. Schubert described three interrelated cysteine complexes of cobalt(III), namely, a dark-brown complex of cobalt: cysteine ratio 1:2, and two isomeric complexes, green and red, in which the ratio was 1:3. In the course of a study of the metal-catalyzed oxidation of cysteine by gaseous oxygen the cysteine complexes of cobalt(II1) have been reinvestigated. Results significantly different from those reported by Schubert, and other workers, have been obtained. This paper is concerned with the formation and structure of cobalt(II1) biscysteinate. Historically, the cobalt(II1) complexes of cysteine and thioglycolic acid are closely related. On mixing cobalt(I1) ion with thioglycolic acid a t pH 7-8, Michaelis and Schubertgfound that, in entire absence of oxygen, a pale-green complex was formed in which the cobalt: thioglycolic acid ratio was 1:2. One-quarter of a mole of oxygen converted the green to an intensely dark-brown cobalt(II1) complex, which was found to be dimeric by cryoscopic measurements carried out on the potassium salt. A binucleate structure, containing two hydroxo bridging groups, was therefore assigned to cobalt(II1) bis-thioglycolate (Fig. 1). In the formation of cobalt(II1) bis-cysteinate, from cobalt(I1) ion and cysteine a t p H 7-8, one-quarter of a mole of oxygen was absorbed, and the composi( I ) Research Laboratory, Monsanto Chemical Company, 911 Western Avenue, Seattle, Washington. (2) Papers I, I1 and 111 of this series are based on a portion of the thesis submitted by Roy G . Neville in 1954 to the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy. (3) L. J. Harris, Biochcm. J . , 18, 739 (1922). (4) L. Michaelis, J . Biol. Chcm., 84, 777 (1929). ( 5 ) L. Michaelis and E. S. G. Barron, ibid., 89, 191 (1929). (6) L. Michaelis and S. Yamagtrchi, ibid., 83, 367 (1929). (7) E.C.Kendall and J. E. Holst, i b i d , , 91, 435 (1931). ( 8 ) M. P. Schubert, THIS JOURNAL, I S , 3851 (1931); 65, 3336 (1933). (9) L. Michaelis and M. P. Schubert, :bid.. 62, 4418 (1930).
tion of the dark-brown complex was found to be CoHCy2.5H20, where “cy” represents SCH2CH(NH2)COO. Since the absorption spectra of the bis-thioglycolate and bis-cysteinate of cobalt(II1) possessed a general similarity, Schubert tentatively formulated the bis-cysteinate as a binucleate structure, [CoH2Cy20Hl2.8H20, containing two hydroxo bridges, analogous to the bis-thioglycolate, Although quite insoluble in water, the complex dissolved in hydrochloric acid, and Schubert isolated a hydrochloride, [CoH2Cy20H]2.3HC1~H20.This observation, coupled with the spectral data, suggested that the amino group was not bound and that coordination to cobalt occurred by means of the sulfhydryl and carboxyl groups of cysteine.
r
Fig. 1,-Structure
7-4
A
of cobalt( 111) bis-thioglycolate.
As the structure of cobalt(II1) bis-cysteinate could not be regarded as having been firmly established, this paper describes work carried out to learn more about this complex. On the basis of analysis, titration, molecular weight determination and formation from hexamminecobalt(II1) chloride, the structure of the bis-cysteinate is shown to be mononucleate. Determination of the nature of coordination in this complex will be described elsewhere. lo
Experimental Materials and Methods Materials.-Cysteine hydrochloride of C.P. grade was supplied by Eastman Organic Chemicals, Rochester, New York, and was used without further purification. Assay of fresh samples iodometrically, by ferricyanide, and by iron-catalyzed oxidation with gaseous oxygen, showed it to contain about 94% free sulfhydryl group. Throughout the investigation, standard solutions of cysteine were always stored under nitrogen and discarded after two days. Assays of four-day-old solutions showed that, even on storing under oxygen-free nitrogen, the percentage of free -SH group had fallen to 89. Cobalt(I1) sulfate hexahydrate of C.P. grade was supplied by the J. T. Baker Chemical Company, Phillipsburg, New Jersey. Hexamminecobalt(II1) chloride was prepared by the method of Inorganic Syntheses.l1 All other reagents employed were of C.P. grade. (IO) Paper I1 of this series, R. G. Neville and G Gorin, ibid., 78, 4893 (1956). (11) “Inorganic Syntheses,” Vol. 11, John Wiley and Sons, Inc., New York. N.Y.,1948, p. 217.
3592
ROYG . NEVILLEAND GEORGEGORIN
1-01, 7s
Analyses for Cobalt.-Cobalt(I1) sulfate was oxidized of 9570 ethanol was added and the whole set on ice. The with hydrogen peroxide in dilute acid solution and precipicrystals were filtered, mashed with 2 little cold water fol!owed tated as cobnlt(II1) hydroxide with sodium hydroxide.'Z by ethanol, then dried in the air. The complex was reThe cobalt( 111) hydroxide was dissolved in glacial acetic crystallized b y dissolving in potassium hydroxide mid reticid and sufficient 1-nitroso-2-naphthol (in 5070 cetic aacid) precipitating with hydrochloric acid, followed by ethanol, as acldcd to precipitate the cobalt as Co( C ~ I , H ~ O ? N ) ~ . ~ Habove. ~O. Calcd. for C o S O 4 6 H 2 0 : Co, 22.40. Found: Co, 22.44. Analysis by the electrolytic deposition method gave Ci1, For the determination of cobalt in the cysteine complexes 17.617' ( 3 detns.). Calcd. for CoHCy2.2H20: Co, 17.63. of cobalt( 111). a weighed amount of complex was decomposed 2. Formation from Hexamminecobalt(II1) Chloride.t o cobalt(I1) ion by heating with a few ml. of concentrated Hexamminecobalt(II1) chloride (10.0 g., 0.0373 mole) mas nitric acid. Excess nitric acid was removed by heating with dissolved in the minimum amount of hot water (100 ml.). ;I small amount of concentrated sulfuric acid. On dilution with water, cobalt(I1j ion was determined gravimetrically as T o this solution were added 12.55 g. of cysteine hydrochloride (0.0746 mole, 94% -SHj in 35 ml. of water and with :rbove, or ele~trolytically.~3 stirring 8.37 g. of potassium hydroxide (0.1492 mole) in 15 Assay of Cysteine .-Three diff erent methods were used of water. The mixture was heated at 60-70" until all throughout to determine the amount of free sulfhydryl ml. evolution of ammonia had ceased (about 2 hr.), then concd. group in the cysteine hydrochloride. hydrochloric acid was added dropwise until the solution 1. Iodometric Method.-The iodometric determination reached pH 1.O. The micro-crystalline precipitate of bisa s tiormallp carried out tends to give high results, but Lacysteinate was filtered, washed several times with 50-ml. vine14obtained accurate values with a solution of iodine in 1 M hydriodic acid (actually an equimolar mixture of 1 M aliquots of cold water, then with 95% ethanol and dried in HCI and 1 M K I ) . This method was employed as follows. the air; yield 11.0 g. (88Yc). T h e complex was recrystalTwo 10-1111. aliquots of KI-HCl-I? mixture were taken, the lized b y dissolving in potassium hydroxide and precipitating with concd. hydrochloric acid, as described above. solution being 0.025 A7 in iodine. T o one aliquot 10 ml. of Analysis by the electrolytic deposition method gave Co, approximately 0.02 .ll cysteine solution was added. T h e 17.64. Calcd. for CoHCy2.2H20: Co, 17.63. other solution served as a control. Each solution was titrated with standard 0.026 N sodium thiosulfate, the differDetermination of Ammonia Liberated in Preparation from ence between the two titration values being due to the iodine Hexamminecobalt(II1) Chloride .-To establish t h a t the consumed by the cysteine. Starch was used as indicator. complex prepared by both methods was identical, it was Found: -SH group, 95a/c. necessary to demonstrate t h a t in the preparation of the biscysteinate from hexamminecobalt(II1) ion and cysteine hy2 . Ferricyanide Oxidation.-Cysteine (0.04 -4lj was tidrochloride under basic conditions all the ammonia co6rditrated with 0.04 M potassium ferricyanide. The end-point nated to cobalt was quantitatively replaced by water. T h e mas located by making the solution alkaline with dilute ammonia,? then adding a few drops of 10% sodium nitroprusfollowing equation w a s assumed t o hold in the formation side. Ferricyanide was then added dropwise until the deep- of this complex purple nitroprusside-sulfhydryl complex disappeared. Found: -SH group, 947,. [Co(SH3)6]CI, BKOH 2[HSCH?CHI?Hq) According t o Shinohara and co-workers,'"16 and to ArCOOH]Cl - = K[Co(SCH~CH(~H2)COO)2!H20)21 46KCl 6hH3 4H?O nold," the use of nitroprusside for sulfhydryl determination gives low values. I n this work the results obtained agreed Bccording to this equation, six moles of ammonia is liberwithin 15; with those found by other methods. per mole of hexammineci,bnlt(III) chloride. Two 3 . Iron-catalyzed Oxidation by Oxygen.-Cysteine is ated series of experiments were carried out. In one series, six completely oxidized to cystine by gaseous oxygen in the moles of potassium hydroxide w a ~ used: and in the other, presence of catalytic amounts of iron salts in alkaline solu- seven moles of potassium hydroxide per mole of hesammmetion.'* This reaction was used as a n assay method. cobalt(II1) chloride. One-g. s:imples of cohalt(II1) comT v e n t y rnl. of 0.04 AT cysteine in phosphate buffer ( p H plex were used in every case. The mixture was boiled and 7.0) was mixed with 2 nil. of 0.01 1 16 ferrous ammonium su!the evolved ammonia swept out with a stre:im of nitrogen fate in the oxygen-absorption apparatus described else- and absorbed in 200 ml. of stant1:irti (0.2500 S)hydrochloric where.'g T h e rate of oxygen uptake was very slow, and acid. T h e residual acid was bnck-tltrated with standard after 24 hr. only about one-half of the cysteine had been N j sodium carbonate. Found: 5.97 moles of amoxidized. Another 2 ml. of ferrous ammonium sulfate was (0.1000evolved per gram atom of Co ( 3 detns.). added and the whole allowed to stand, with stirring, for a monia Titration of Cobalt(II1) Bis-cysteinak--The number of further 24 hourr. Oxygen uptake was then complete since replaceable hydrogen atoms per atom of Cn was determined no more absorption occurred during a further 24-hr. period, by converting air-dried samples of his-cystcinn te (prepared even on adding 2 ml. of the ferrous solution. by both methods) to the sodium derivative, by addition of The results of the three methods of sulfhydryl determinatwo equivalents of standard bnse, then back-titrating with tion in cysteine may be summarized: Iodometric method, standard hydrochloric acid. T h e titration was conducted 95Ch; Ferricyanide method, 94r; ; Iron-catalyzed oxidation under nitrogen. I n a typical experiment. 0.0334 g . of conimethod, 947,. plex was equivalent to 1.02 atoms of Iiytlrogen. For F: Cobalt(II1) Bis-cysteinate.-This complex was prepared mononucleate structure (see later) of mol. rvt. 334.3. this from cohalt(1I) ion by a method similar t o that employed corresponds to a H : Co ratio of 1 : 1. by Schuberrs and by a new method from hexamminecobaltCryoscopic Determination of Molecular Weight.-The (111) chloride. form of the bis-cysteinate was too insoluble for direct 1. Formation from Cobalt(I1) Ion.-Cysteine hydro- acid determination of molecular weight. l d d i t i o n of onc equiv:ichloride ( 5 g . ) w a s dissolved in 20 ml. of water, and 7.5 ml. lent of sodium hydroxide gave the monosodium %It, K a of 7.5 Ji potassium hydroxide was added. This solution [Co(Cy),(H20)2], which possessed markedly greater soluwas then poured into 16 ml. of 1 M cobalt(I1) sulfate conbility. I n solution, two ions are present per molecule of taining 4.5 ml. of 7.5 M potassium hydroxide. T h e mixture was stirred, filtered and air passed through the filtrate for 1 complex, S a + and [Co(Cy)2(H.10)?1-. Cobalt(II1) bis-cysteinate (0.3160 g.) was titratpd under hr. Concentrated hydrochloric acid was then added dropnitrogen t o p H 7.0 with 0.104 N sodium hydroxide. The wise until the dark-brown complex crystallized. Fifty ml. solution was diluted to 25.00 ml. with distilled mater and the freezing point determined using a standard Beckmann (12) A . I . Vogel, " hText-book o f Quantitative Inorganic Analysis," apparatus. -1ssuming the complex t o be mononucleate with I,ongmans, Green and Co.. London, 1947, p . 547. a molecular weight of 334.3, the expected depression of (13) W. F. Hillebrand, "Applied Inorganic Analysis," John Wiley freezing point for 0.3343 g. per 25 ml. would be 0.1484' and Sons, I n ? , , New York, N. Y.,1953, p . 419. (;.e., 0.0742' per ion). I n two determinations, using (14) T. F. Lavine, J . Bioi. Ckem., 109, 141 (1935). 0.3160 g. of complex, the depressions were 0.139 and 140°, 115) K . Shinohara and M. Kilpatrick, ibid., 105, 241 (1934). corresponding to 1.98 and 2.00 ions, respectively.
+
+
+
( 1 0 ) I
