Oct. 5 , 1956
NATUREOF
THE
COORDINATION OF CYSTEINE-CO(III) COMPLEXES
pared from cobalt(I1) ion and cysteine in the presence of oxygen, as originally described by Schubert or by the metathetical reaction of cysteinate ion with the hexamminecobalt(II1) ion. These results exclude the possibility, in the case of cysteine, of a desmotropic relationship between the cobalt (111)-sulfhydryl and cobalt (11)-disulfide structures suggested by Michaelisg for the darkbrown thioglycolic acid complex with cobalt. The difference between a hydroxo complex, such as that suggested by Schubert, and a hydrate lies in the number of replaceable hydrogen atoms. The empirical formulas for the bis-cysteinate may be written as HCoCyz.H20, or H ~ C O C ~ ~ O acH, cording to whether the complex contains one or two hydrogen atoms which can be titrated in water, and hence are more strongly acidic than water. Titration of the acid form of the complex with base shows only one point of inflection, corresponding to only one replaceable hydrogen, and a neutralization equivalent of 326. For HCoCy2.2H20, the figure is 334. This evidence, coupled with the formation of only two ions on dissociation of the
[CONTRIBUTION FROM THE
4893
1
I-
H+
L
Fig. 2.--Structure
of cobalt( 111) bis-cysteinate.
sodium salt, corresponds with a mononucleate structure (Fig. 2 ) and eliminates the possibility of a binucleate structure possessing two hydroxo bridges. The complex may be represented in the conventional manner in which the central cobalt atom is surrounded octahedrally by two bidentate cysteine ligands, “cy,”’and two water molecules. Acknowledgment.-The authors wish to express their thanks to the Research Corporation for financial assistance throughout this work. EUGEXE, OREGON
DEPARTMENT O F CHEMISTRY,
UNIVERSITY OF OREGON]
Cysteine Complexes with the Cobalt (111) Ion. 11. Spectrophotometric Study of the Nature of Coordination in the Complexes of Cysteine with the Cobalt(II1) Ion BY ROYG. K E V I L L E AND ’ ~ ~ GEORGEGORIN RECEIVED APRIL 17, 1956 The three cysteine complexes of cobalt( 111) first isolated by Schubert have been reinvestigated, their interrelationship demonstrated and their structures proved. Green cobalt(II1) tris-cysteinate was prepared from hexamminecobalt(II1) chloride, as in the case of the bis-cysteinate. This complex is stable only above about PH 11; lowering the PH to about 8 causes decomposition to the bis-cysteinate and cysteine. The bis- and green tris-cysteinates were shown, spectrophotometrically, to be quantitatively interconvertible by raising or lowering the pH value. The nature of coordination in these complexes was deduced from a comparative study of the absorption spectra of the cobalt(II1) chelates with thioglycolic acid, 2-mercaptoethylamine, aminoacetic acid and N-formylcysteine. Coordination in the bis- and green tris-cysteinates occurs through the sulfhydryl and amino groups of cysteine. I n the isomeric red tris-cysteinate coordination to cobalt occurs via the sulfhydryl and carboxyl groups. Sodium cysteinate a t pH 12 converts the red tris-cysteinate irreversibly to the green isomer.
Although three complexes of cysteine with the cobalt(II1) ion were isolated in the solid state by Schubert3 as long ago as 1931, no study of the nature of coordination in these compounds has been carried out. On the basis of a general similarity in the absorption spectra of the brown bisthioglycolate and bis-cysteinate of cobalt(III), Schubert concluded that the sulfhydryl and carboxyl groups of cysteine were coordinated to the cobalt atom. The dissimilarity in absorption spectra of the red tris-cysteinate and tris-alaninate complexes of cobalt(II1) appeared to support the same conclusion in the case of the red complex. No study was made with the green tris-cysteinate of cobaIt(II1). Martell and Calvin4 later suggested that coordination in these complexes probably oc(1) 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) M . P . SChubmt, T H I S J O U R N A L , 58,3851 (1931);6 6 , 3 3 3 6 (1933). (4) A. E. Martell and M . Calvin, “Chemistry of the Metal Chelate Compounds,” Prentice-Hall, New York, N. Y., 1952, p. 382.
curs via the amino and sulfhydryl groups of cysteine, since both nitrogen and sulfur are strong,electron donors showing marked tendency to combine with cobalt. The carboxylate group of cysteine was thought not to be coordinated with cobalt for steric reasons. In the study of the metal-catalyzed oxidation of cysteine with oxygen it was of great interest to establish the interrelationship and nature of coordination in the three complexes of cobalt(II1). This paper presents the results of a detailed spectrophotometric study of cobalt(II1) with cysteine, thioglycolic acid, aminoacetic acid, 2-mercaptoethylamine and N-formylcysteine. By comparing the ultraviolet absorption spectra of complexes in which the nature of coordination is unambiguous, the structures of the cysteine complexes of cobalt(111) were established. In addition, it has been shown that the brown bis- and the green tris-cysteinates are quantitatively, and reversibly, interconvertible. Evidence is also presented to show that the green and red tris-cysteinates of cobalt(111) are structural isomers and not stereoisomers.
4804
ROYG. NEVILLEAND GEORGEGORIN Experimental Materials and Methods
Materials.-The chemicals listed were supplied by Eastnian Organic Chemicals, Rochester, New York. LUl were of C.P. grade, a n d no further attempts at purification were made: cysteine hydrochloride, thioglycolic acid, aininoacetic acid, alanine, cystine, glutathione, tyrosine, 1hexanethiol. S-Methylcysteine was prepared by methylation of cysteine with dimethyl sulfate in alkaline solution, according t o the method of du V i g n e ~ d . T ~ h e recrystallized compound turned brown at 235-240", and melted with decomposition a t 248'. N-Formylcysteine was prepared by the method of Fruton and Clarke,B by treating cystine in 90% formic acid with acetic anhydride and reducing the diS-formylcystine with zinc in dilute formic acid. Decomposition of the copper(1) salt with hydrogen sulfide yielded N-formylcysteine a s colorless crystals, m.p. 87-89' dec. -1small sample of 2-mercnptoethylamine, prepared from ethyleneimine,' was kindly donated by Oregon State College, Corvallis, Oregon. All compounds containing sulfhydryl groups were assayed for free -SH b y the iodometric method of Lavine,* and solutions used in quantitative work corrected accordingly. Apparatus and Procedure.-Absorption spectra were determined using a Beckman spectrophotometer model D U , equipped with hydrogen and tungsten lamps. Stoppered quartz cells, of thickness 10 m m . , were used throughout. All solutions for spectrophotometric determination were made u p directly by weight, with subsequent dilution if necessary. General Qualitative Survey.-In cysteine three possibilities exist for the octahedral coordination of groups about the central cobalt atom: namely, coordination of -KH? and -COOH, -NH2 a n d -SH,or -SH and -COOH. T o establish which of these possibilities holds for the cysteine coniplexes of cobalt(III), a series of experiments was carried nut using compounds containing either one or two of the above functional groups. At a given p H value, the ease with which e?ch of these compounds formed a cobalt(IT1) complex with either cobalt(I1) or cobalt(II1) ion was determined. One-ml. aliquots of 0.01 A I cobalt(I1) sulfate were treated at 20' with excess of a n amino acid or thiol compound. Each solution was made progressively more alkaline by addition of 7.5 M potassium hydroxide, no attempt being made t o exclude atmospheric oxygen. similar series of evperirnents was carried out using 0.01 Af hexamminecobalt (111) chloride. I n alkaline solution, compounds possessing L: free -SH group immediately formed a highly colored Co (111) complex. Compounds possessing -NH2 or -COOH, or blocked sulfhydryl (e.g., -SCH3), gave no reaction. By boiling solutions of methionine or aminoacetic acid for several hours low yields of Co(II1) complex were produced. Since complexes form immediately on mixing either Co(I1) or Co(II1) ion with cysteine, it appears t h a t coordination occurs through the -SH group. Interconversion of Cobalt(II1) Bis- and Green Tris-cysteinates.-A solution of the green tris-cysteinate, on exposure t o air, rapidly turns brown by formation of biscysteinate. T o establish whether this reaction is reversible, 1 g. of doubly recrystallized his-cysteinate was mixed with a tenfold excess of cysteine hydrochloride dissolved in a small amount of water. T h e mixture was titrated t o pH 10 with 8 AI potassium hydroxide, using a Beckman pH meter. T h e bis-cysteinate was completely soluble at this pH value. A brisk stream of oxygen-free nitrogen was passed through the solution t o expel dissolved oxygen. The temperature was gradually raised t o 60°, then the whole allowed t o cool, with the nitrogen still passing. T h e spectrum of this deep-green solution was identical with t h a t of the green triscysteinate prepared b y t h e method of Schubert.s Addition of a few drops of 30% hydrogen peroxide caused the solution t o change t o the intense yellow characteristic of the sulfone. T o establish t h a t these complexes were quantitatively interconvertible, their spectra were examined in t h e region 320-620 mp. T o 10 ml. of 0.05 M cysteine hydrochloride 0.2 M sodium hydroxide was added t o pH 8. T h e mixture was added t o 5 ml. of 0.01 M hexamminecobalt(II1) chloride, and the whole diluted t o 50 ml. On warming t o 50" the (5) V. du Vigneaud, 13. S. Loring and H. A. Craft, 1.B i d . Chem., 101, 481 (1934). (6) J. S. Fruton and H. T. Clarke, ibid., 106, 667 (1934). (7) E.J. Mills and M. T. Bogert, TEISJOURNAL, 62, 1173 (1940). (8) T.F. Lavine, J . Biol. Chem., 109, 141 (1935).
Vol. 78
bis-cysteinate formed rapidly. T h e spectrum of this soluill. tion was determined at a Co concn. of 5 X The same experiment was repeated a t pH 11. T h e spectrum was t h a t of the green tris-cysteinate. On lowering the p H t o 8 ( b y addition of a trace of concd. sulfuric acid t o maintain constant volume), the brown bis-cysteinate was produced, the spectrum being identical with t h a t of the biscysteinate originally prepared at p H 8. T h e bis- and green tris-cysteinates are thus quantitatively interconvertible. Conditions of Formation of Green Cobalt( 111) Tris-cystehate.-To investigate the conditions of formation of these complexes, in particular the green tris-cJ-stein:tte, a n apparatus was constructed in which the coniplexes were prepared in absence of oxygen, T h e :IppariLtus consisted essentially of a Thunberg tube connected with a system of six gas-washing bottles. Solutions for investigation were prepared a s follo\v~. Aliquot5 of 0.5 ml. of 0.005 ' A hexamminecobalt(I1 I ) chloride were placed in the side-arms of a number of Thunberg tubes. Appropriate amounts of cysteine, previously titrated with sodium hydroxide to PH 10.5, v e r e placed in each of the tubes and the t o t d volume made up t o 5.0 ml. to give solutions 5.33 X 1 0 - 4 -1Iin Co and of PH about 1 0 . I n this way, Co:cysteine ratios varying from 1: 1 t o 1: 17 were prepared. Dissolved oxygen in tlie solutioiis was removed b>-means of a water-pump connected t o the tube z'io a t h r e e - m y stopcock. Sitrogen from a cylinder was purified from traces of oxygen by passage through a wash-bottle containing alkaline pyrogallol, then thrtrugh five tubes containing Fieser's solution , 9 This nitrogen was admitted to the solutions, which were then mixed a n d allowed to st;lnd a t 20' for two hours. T h e spectrum (between 380 :inti 620 m p ) of each solution \vas then determined by p1:icing the Thunberg tubes directly into the Beckman spectr(rp1iotonieter, n o correction being made for the roundness of the tubes. I n this region, the spectra were identical with those obtained using optical!y-flat quartz cells. Solutions of ratio 1 : 1 to 1 : 3 were brown. hut ratios from about 1 : 3 t o 1 : 17 were progressively greener in color, possessing ahsorpt ion maxima at 440 and ,580 nip, characteristic of the green triscysteinate. . i t mtios greater than 1: 5 the spectrum was t h a t of the pure tris-cysteinate. No change in tliese spectra mas observed even on standing for 168 hours. I n entire absence of oxygen, therefore, the green tris-cgstein:ite is stahlc. Cobalt(II1) Tris-cysteinate (Green Isomer).-Specinlens of this complex, prepared from cobalt(I1) ion and cysteinc by the method of Schubert,3 were found d w a y s to be cnntarnitiated with his-cysteinate and were thus unsuit:ttrle for spectral work. Solutions for spectrophtrtometric use n w e prepared directly from hexarnminecobalt(111) chloride. Cobalt(II1) Tris-cysteinate (Red Isomer) .-This complex was prepared using a nidification of the Schubert inethod. Potassium hydroxide ( 7 . 5 .If,10 nil.) in I d nil. of water W:LS added t o 10 g. of cysteine hydrochloride dissolved in -10 nil. of wzter. This mixture was added to 33 nil. of 1 91 cobalt (IT) sulfate containing 9 ml. of 7.5 ill potassium hydroxide, the whole was quickly stirred then filtered. Air was pnssetl through the filtrate for one hour. After 24 hours, the precipitate was filtered, washed with witer followed h y ethanol, then dried in the air; yield 90%. Owing to its insolubility in dilute acids and water. this complex W:LS n('t recrystallized. The complex was soluble in dilute sodium hydroxide, b u t heating this solution resulted in parti:il decomposition t o the his-cysteinate. Cobalt determination by electrolytic deposition cnrresponded t o the arihy(lrous tris-cysteinate. A n d . Calcd. for Hs[Co(SCH2(XH2)CHCOO)a]: Co, 14.10. Found: Co, 14.15, 14.08 ( 2 detns.). Substitution of hexamminecobalt( 111) chloride for cobalt( 11) sulfate in the above preparation gave bis-cysteinate only. On treating the tris-cysteinate with excess hot sodium cysteinate a t pH 12 the green isomer formed rapidly. Lowering the pH t o 6 gave t h e bis-cysteinate. Cobalt(II1) Tris-N-formylcysteinate .-This complex was prepared b y a procedure similar t o that described above, using 0.22 g. of ?r-formylcysteine, in 1 ml. of water. After adjusting t o pH 6 with 7.5 A[ potassium hydroxide, cobalt(I1) sulfate (0.77 ml., 1.0 M ) was added. (9) I,. F. Fieser, "Experiments in Organic Chemistry," Heath and Co., Boston, hlass., 19-11,p . 395.
Oct. 5 , 1956
NATUREOF
THE
COORDINATION OF CYSTEINE-CO(III) COMPLEXES
A n a l . Calcd. for H ~ [ C O ( S C HNH,CHO)CHC00)3] Z( : Co, 11.71. Found: Co, 11.73, 11.74 (2 detns.). Cobalt(II1) Tris-aminoacetate.-Two isomeric forms of this coniplex exist, a violet dihydrate and a rose-red monohydrate.'O T h e violet isomer was prepared by the method of Ley and Winkler," from cobalt(II1) hydroxide and aminoacetic acid, and was obtained as deep-violet crystals of composition [ C O ( O O C C H ~ N H .2H20. ~)~] From the filtrate the rose-red monohydrate was obtained in small yield. For spectrophotometric determinations, the violet complex was recrystallized twice from boiling water then dried at 80'. T h e red isomer was prepared by a new method from hexamminecobalt(II1) chloride. Aminoacetic acid (2.0 g., 0.0263 mole) and hexamminecobalt(II1) chloride (2.34 g., 0.0087 mole) were dissolved in 150 ml. of water. Potassium hydroxide (6.6 M ) was added to p H 10, the solution boiled under reflux for three hours, then evaporated to the point of crystallization. The deep rose-red crystals of tris-aminoacetate monohydrate, after recrystallizing twice from water, were dried a t 80" and used without further purification. The spectrum of this complex was identical with that of the monohydrate prepared by the method of Ley and Winkler. Cobalt(II1) Bis-thioglycolate .-The potxssium salt of this complex was prepared using a modification of the method of Michaelis and Schubert.l2 For spectrophotometric use, solutions were prepared by direct weighing. Complexes of Cobalt(II1) with 2-Mercaptoethy1amine.Owing to the difficulty of isolation of pure specimens of cobalt( 111) complexes with 2-mercaptoethylamine, solutions for spectrophotometric use were prepared from cobalt(I1) ion as described below. Spectrophotometric Determinations.-Bis-complexes of cobalt(II1) with cysteine, 2-mercaptoethylamine and thioglycolic acid were prepared from 1:2 molar ratios of 0.01 M cobalt(I1) sulfate and 0.01 M solutions of chelating agent, dilute potassium hydroxide being added to raise the PH to 8. After air oxidation for five minutes, spectra were determined a t a Co concn. of 5 X 10-4 M . The green tris-complexes of cysteine and Z-mercaptoethylamine were prepared by mixing 1 ml. of 0.01 M hexamminecobalt(II1) chloride with from 3 to 10 ml. of chelating agent, dilute potassium hydroxide being added to pH 11.5. The solution was then diluted almost to 100 ml. with water, heated t o 60" to effect formation of the complex, cooled, and the solution made up t o 100 ml. to give a Co concn. of 1 X l o w 4hl. For convenience, spectral measurements were
made at a Co concn. of 1 X 10-2 M in the case of the violet and red tris-aminoacetates. Solutions of the red triscysteinate and tris-N-formylcysteinate 5 X 10-6 M in Co, at p H 10, were prepared by direct weighing. All spectra were measured in the range 210-700 mp. Absorption maxima and extinction coefficients are listed in Table I.
Discussion of Results The interconversion of the bis- and green triscysteinates may be explained in the following manner. When hexamminecobalt(II1) ion and cysteine M ) in the are mixed in dilute solution (about region of pH 10, there exists an equilibrium comprising green tris-cysteinate, bis-cysteinate and sodium cysteinate, which constitutes a buffer sysr
Complex of Co(II1)
Bis-cysteinate
Bis-(a-mercap toethylamine)
(moles/l
x
10-6)
2 2 2
5 5
5 Bis-thioglycolate Tris-aminoacetate (violet) Tris-aminoacetate (red) Tris-(2-mercaptoethylamine) Tris-cysteinate (green)
5 50 50 50
5 5 1 1 1
Tris-cysteinate (red)
0.5
.5 Tris-( N-formylcysteinate)
.5 0.5
.5 .5
Absorption max., mfi
Extinction coefficient ( X IO')
280 350 440 280 350 440 350 380 540 540 340 580 280 330 570 270 410 505 270 420 490
7.50 5.51 2.25 6.8 4.8 1.8 4.3 0.134 0.108 0.050 4.18 0.34 18.5 3.68 0.30 4.9 3.4 8.8 3.2 2.9 5.2
(10) H.Krebs and R. Rasche, 2. anorg. Chem., 216, 236 (1954). (11) H.Ley and H. Winkler, Ber., 42, 3894 (1909). (12) L. Michaelis a n d M. P. Schubert. THISJOURNAL, 62, 4418 (1930)
1
coo
-
I
"2
H+
coo L
J
Fig. 1.-Bis-cysteinatodiaquocobaltate(II1).
c 00
TABLE I SPECTRAL DATAFOR COBALT(III)COMPLEXES Concn.
4895
-3
Hi+
I
coo
L
Fig. 2.-Tri~-cysteinatocobaltate( 111), green isomer.
-3
H
+;
Fig. 3.-Tris-cysteinatocobaltate(III),
red isomer.
R. W. GREENAND H. K. TONG
4896
tem. At 1:1-1:3 ratios, the equilibrium lies in favor of the bis-cysteinate but as the cobalt: cysteine ratio is increased above 1:3 progressively more of the green complex is formed. At ratios greater than about 1:5 the equilibrium is shifted totally in favor of the tris-cysteinate. Above p H 11, a 1 :3 ratio gives pure tris-cysteinate showing that chelation of the third cysteine residue is strongly OH-dependent. From these observations it is clear that the third cysteine molecule is not so tightly bound to cobalt as are those in the biscysteinate, which is not decomposed in strong acid. Lowering the p H from 11 to 8, even in concentrated solutions, causes immediate dissociation of the tris-cysteinate to the bis-complex and cysteine. The corresponding cobalt(II1) chelates with -3rnercaptoethylamine behave in the same manner. Comparison of the spectra of the bis- and green tris-complexes a t p H 8 and 11 show that these compounds are quantitatively interconvertible. The marked instability toward oxygen of the green tris-complexes of cysteine and 2-mercaptoethylamine may be explained by the ease of dissociation of the third ligand, which is then oxidized to the corresponding disulfide and does not recombine with the cobalt. The very close similarity in the spectra of the
[CONTRIBUTION FROM THE
Irol. Th
bis- and green tris-complexes of cysteine and 2 mercaptoethylamine, together with the quantitative interconversion data, lead to the formulation of the bis- and green tris-cysteinates as sulfhydryland amino-coordinated complexes (Figs. 1 and 2 ) .l'i In N-formylcysteine, the formyl group renders the nitrogen atom less basic than it is in cysteine resulting in sulfhydryl- and carboxyl-coordination. The similarity in spectra of the tris-complexes of cysteine and N-formylcysteine indicates that this mode of coordination occurs in the red triscysteiiiate (Fig. 3 ) . Treatment of the red tris-cysteinate with hot sodium cysteinate a t pH 11-12 results i s rapid formation of the green isomer. Lowering the p H to C, gives the bis-cysteinate. The red tris-cysteinate is actually prepared a t pH 6, from cobalt(II1 ion and cysteine, but the above observation shows that, once formed, sulfhydryl- and amino-coordination is favored. The red and green tris-cysteinates are thus not interconvertible. Acknowledgment.-IVe wish to express our thanks to the Research Corporation for financial assistance in this work. (13) Paper I of this series discusses the structure of the his-cy
