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ESR Studies of Model Complexes for Non-Heme Iron Proteins.

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2 CH3- CO--CH2-CO-CH,
i4 H2S
+ 4 FeIIIC14- --+
t 3 FeW142- i 2 H3O+
+ 2 H20
( 1)
The violet crystals dissolve in water at pH < 3 to give a
colorless solution as described by equation (c).
they appear only on reduction of the complex. The electron
balance of these reductions is thus determined not only by
the central atom, but also by the acceptor ligand, a fact
which should always be borne in mind when the stoichiometry of electron uptake by iron proteins is discussed.
Received: January 26th, 1967
[Z 432 1EI
German version: Angew. Chem. 79. 273 (1967)
[*I Dip1.-Chem. K. Knauer and Doz. Dr. P. Hemmerich
Institut fur anorganische Chemie der Universitgt
SpitalstraBe 51
Basel (Switzerland)
Doz. Dr. J. D. W. van Voorst
Institute for Physical Chemistry of the University
The Fez+ formed was determined colorimetrically as the
Amsterdam (Netherlands)
bipyridine complex, whereas (CsH7S2)+ was found to have
[**I Financial support by the Swiss National Foundation for
the structure (2), R1 = Rz = CH3; the first stable dithiolium
the Advancement of Natural Science as well as the Netherlands
cation with only aliphatic substituents. It is stable towards
Organization for the Advancement of Pure Research (Z.W.O.).
hydrolysis at p H < 4. Structure (2) is evidenced by 1H-NMRThe elementary analyses are a generous contribution by the
spectroscopy [2 peaks only, 8.4 and 3.1 ppm, of intensity
microanalytical laboratory CIBA Ltd., Basel (Dr. W. Padowetz).
ratio 1:6], by means of UV-spectra (Imax
= 280, 265 nm;
[l] E. Buyer and W. Purr, Angew. Chem. 78,824 (1966); Angew.
E = 9300,7200 mol-lcm-1
compared to Amax = 356,287 nm;
Chem. internat. Edit. 5, 840 (1966); B. B. Buchanon: Structure
E = 19000, 3800 mol-lcm-1
of the known (2), R1 = C6H5,
and Bonding. Springer-Verlag, Berlin 1966. p. 109.
R2 = H)[61 and by isolation of the picrate C ~ I H ~ O ~ N ~ S ~ ,
121 Cf. E. C. Slater: Flavins and Flavoproteins. Elsevier, Amn1.p. 113OC.
sterdam 1966, BBA-Library Vol. 8 ; P . Hemmerich, C . Veeger,
and H . C. S. Wood, Angew. Chem. 77,699 (1965); Angew. Chem.
internat. Edit. 4 , 671 (1965).
[31 H . Brintzinger, G. Palmer, and R . H . Sands, Proc. nat. Acad.
Sci. USA 55, 397 (1966); J . D . W. van Voorst and P . Hemmerich
in “International Conference on Magnetic Resonance in Biological Systems”, Stockholm 1966, Pergamon, in press.
[4] P. Hemmerich, H . Beinert, and T. Vanngard, Angew. Chem.
78, 449 (1966); Angew. Chem. internat. Edit. 5, 442 (1966);
C. I Hemmerich
The complex ( / I might have the structure ( C S H ~ S ~ ) ~ [ F ~ ~ I P
in P . Aisen, W. E. Blumberg, and J . Peisack,
which is in agreement with its elementary composition. Its
“Biochemistry of Copper”. Academic Press, New York, 1966,
p. 1s.
very intense color, however, suggests Fe-SS-coordination,
[ S ] Dithioacetylacetone arises from acetylacetone and HzS, but
since CxH7S2@as well as the almost non-polarisable FeC142dimerizes instantaneously and irreversibly if not trapped as
a r e colorless. Therefore, we propose the structures i(Iu) or
monomer by chelating metals. Chelates with other metals ( N P ,
(16) as the most probable ones.
Coz+, Pdz+, Pt2+) have been announced by R . L. Martin and
J . M . Stewart, Nature (London) 210, 522 (1966).
161 E. Klingsberg, J. Amer. chem. SOC.83, 2934 (1961).
ESR Studies of Model Complexes €or Non-Heme
Iron Proteins
If the colorless aqueous solution of ( I ) (cf. eq. (c)) is reduced
by Na2S204 or NaBH4, red -brown crystalline FeI1I(C5H7S2)3
is obtained, which is soluble in CHC13 and may be further
reduced by shaking with aqueous Na&04 to give the
autoxidable Ferr(C5H7S2)2. The compound FeIII(CgH7S2)3
shows the ESR properties expected for an asymmetrical low
spin d5-octahedron (Fig. I), while Fe(C5H7S2)z and ( I ) d o
not show a n ESR absorption at T 3 170°K.
g=ZlL A
L qauss
/ V
The formal oxidation state of the iron is increased on
reduction of complex ( I ) . This system may be a model for
redox-active iron in proteins in which the ESR signals at
g < 2 appear to belong to a n “oxidized” d5-state, even though
Airgew. C h e m . intermit. Edit. 1 Val. 6 (1967)
No. 3
By A . Roder and Ernst B a y e r r * ]
It is difficult to decide the valence of the iron in redoxactive non-heme iron proteins since the ligand, as well as
the iron, is redox-active. In the reduced state many of these
iron proteins have ESR spectra with g values below that
of a free electron 111. The ESR spectrum of reduced xanthine oxidase contains a broad signal with g = 1.94
(-175 C) “1; the g values for reduced ferredoxin from
CIostridium pasfeuriunum are 1.89, 1.96, and 2.05 (15 OK) (2,
31. Bonding of the iron to cysteinyl groups appears to
be certain for ferredoxinr41. Thus iron complexes of low
molecular weight involving sulfur-containing ligands should
also give g values below 2.0 at corresponding redox states,
and it should be possible to determine the oxidation state of
the active centre in the reduced non-heme iron.
Under the following conditions iron complexes of cysteine,
cystine, and cysteamine gave g values at 1.94:
(a) Oxidation of the previously described 141 iron(I1) complex
of cysteine methyl ester:
1. 392 mg of Mohr’s salt is added, under nitrogen, to a solution
of 342 mg of cysteine methyl ester hydrochloride in 1CO ml of
0.5 M tris-HCl buffer (pH = 7.0). The cysteine methyl esteriron(i1) complex that precipitates immediately is dissolved
by the addition of 1 N NaOH (final p H = 9.5). The solution is
shaken for 1 min with an equal volume of a 1 solution of
iodine i n benzene in an ESR sample tube, then frozen and
measured [**I.
2. 227 mg of cysteamine and 392 mg of Mohr's salt are
dissolved in 50 ml of 1 M tris-HC1 buffer (pH = 7.0). The p H
is then adjusted to 9.5 by 1 N NaOH, and a solution of
504 mg of the sodium salt of riboflavin monophosphate
(FMN) in 25 ml of H 2 0 is added. After 5 minutes the mixture
is transferred to a n ESR sample tube, frozen, and measured.
In this case absorption by the flavosemiquinone at g = 2.00
also occurs (see Fig. lb).
(b) By oxidation of tricarbonyl(cystine dimethyl ester)iron(0) [51:
0.2 ml of a 10-2 M ethereal solution of the cystine dimethyl
ester-iron(0) complex is treated in an ESR sample tube with
5 mg of solid iodine, frozen after 20-seconds' shaking, and
Received: December Zlst, 1966; revised: January 18th, 1967 [Z 434 IEI
German version: Angew. Chem. 79,274 (1967)
[*I Dip].-Chem. A. Roder and Prof. Dr. Ernst Bayer
Fig. 1. a) ESR spectrum of the cysteine methyl ester iron(rl) complex
after oxidation by method (all, and of cysteine methyl ester after
reaction with iron(nr) chloride by method (c) (Mod. 1000, SL 1000,
SR 200 gausslmin).
b) ESR spectrum of the radical complex (gll = 1.94; g l = 2.3) obtained
by method (a)2, and of the flavosemiquinone radical ( g
2.00) (Mod.
1000, Ampl. 1000, SR 250 gaussjmin).
The ESR spectrum reproduced in Fig. l a has the shape that,
according to Kneuenbiihl's calculations [61, is characteristic
of axial symmetry in complexes. A similar symmetry was
observed with the reduced non-heme iron protein from
Azofobacter vinelandii [I].
The oxidation states of the initial complexes used for (a) and
(b) are given in the formulae ( I ) and (2). The iron(1r)
complex ( I ) of cysteine methyl ester is equivalent to the
tricarbonyliron(0) complex (2) of cystine dimethyl ester with
respect to its total oxidation state when the oxidation stAes
of the ligand and the central atom are considered together.
Both complexes are converted into ESR-active substances
on oxidation. In so far as oxidation involves a oneelectron step the same ESR spectrum should result if the
iron(r1r) salt is allowed to react with cysteine methyl ester;
one can therefore formulate complex (3).
(c) 80 mg of anhydrous iron(I11) chloride is added, under nitrogen, to 100 ml of a 10-2 M solution of cysteine methyl ester
in 0.5 M tris-HC1 buffer (pH = 10.5). The p H value is then adjusted to 9.5 by addition of 1 N NaOH. After 10 min the
mixture is transferred to an ESR sample tube, frozen at
once, and measured. The same spectrum is obtained as under
(a)l (Fig. la).
The total oxidation state of the ESR-active complex is thus
proved. The complex can he formulated as iron(rr1) mercaptide (3), as iron(r1) radical complex (4), or as iron(r)
complex of the disulfide (5). The unpaired electron is
delocalized over the central ion and the ligand. Neither
the central ion nor the ligand can be assigned a specific
oxidation state.
The similarity between the ESR spectra of reduced nonheme iron proteins and synthetic iron complexes makes it
probable that formulations ( 3 ) to (5) can also be applied to
reduced non-heme iron. This is supported by ESR-spectroscopic studies of bis(hexamethylbenzene)iron(I) tetrafluoroborate [71, of Na3[Fe(CN)sNO] [a], and of iron-oxygen-radical
complexes [91. The oxidized form of non-heme iron
proteins is therefxe described by structure (6) of an iron@)
complex of the disulfide; this is in agreement with results
obtained by chemical methods.
Chemisches Institut der Universitat
Wilhelmstr. 33
74 Tiibingen (Germany)
[l] H. Beinert in A . San Pietro: Non-Heme Iron Proteins. The
Antioch Press, Yellow Springs, Ohio, 1965, p.23 and further
literature cited.
[2] G. Palmer, R. H. Sands, and L. E. Mortenson, Biochem.
biophys. Res. Commun. 23, 357 (1966).
[3] E. Bayer, W. Parr, G. Schworer, and A . Roder, unpublished.
141 E. Bayer, W. Parr, and B. Kazmaier, Arch. Pharmaz., Ber.
dtsch. pharmaz. Ges. 298, 196 (1965); cf. also Angew. Chem. 78,
824 (1966); Angew. Chem. internat. Edit. 5, 840 (1966).
[**I All measurements at -17OoC (V-4540 variable temperature
controller). Varian V-4502 X-Band spectrometer.
[ 5 ] E. Bayer and A . Roder, unpublished.
[6] F. K. Kneuenbiihl, J. Chem. Physics 33, 1074 (1960).
[7] H. Brintzinger, G. Palmer, and R . H. Sands, J. Amer. chem.
SOC.88, 623 (1966).
[S] H. Beinert, P . Hemmerich, and J . D . W. Van Vaarsf,Biochim.
biophysica Acta 96, 530 (1965).
[9] W. E. Bfumberg and J . Peisach in A . San Pietro: Non-Heme
Iron Proteins. The Antioch Press, Yellow Springs, Ohio, 1965,
p. 101.
Dimeric Complexes
By J . K . Nicholson[*][**I
In certain dimeric carboxylate complexes of ruthenium the
ruthenium atoms are formally in the oxidation states two
and three. Their unexpectedly high magnetic moments have
been ascribed to a spin free systemr11. We have now isolated
a tri-n-butylphosphine complex of ruthenium, [Ruz (11,Iir)Cls
(P(n-Bu)3)4] [21 which has the expected magnetic moment for
a spin paired dimer containing one unpaired electron per
molecule. When a concentrated ethanolic solution of ruthenium trichloride and tri-n-butylphosphine (molar ratio
1 :2.2) is allowed to stand at 2OoC under nitrogen for 72 h,
(l'),dark red crystals, m.p.
a complex [RuC13{P(n-B~)3)2]~
132-134 "C, separates. After filtration in air, the mother
liquor deposits [Ru~Cls{P(n-Bu)3)4](2'), dark red crystals,
m.p. 95.5-96 "C, over a period of 24 h.
Complex (I') has a magnetic moment of 1.93 B.M./Rur31, as
expected for a spin paired ruthenium(Ir1) complex [41. Molecular-weight determination (calculated 1224; found 1004;
thermoelectric method of Simon and Tomtinsun) suggests
that, in dichloromethane solution, the complex (1') is largely
dimeric. Complex (2') has a molecular weight of 1165 f 24[5]
(calc. 1189). The magnetic moment 0.75 B.M./Ru (31 indicates
that there is one unpaired electron per molecule. The infrared spectrum exhibits no absorption bands in the region
4.0-6.0 p (metal-hydride stretching frequency range).
Complex (2') is formulated therefore with the ruthenium
atoms formally in the oxidation states I1 and 111. It is not
Angew. Chem. internat. Edit.
Vol. 6 (1967) 1 No. 3
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esr, mode, iron, protein, non, complexes, heme, studies
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