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Elimination of Hydrogen Sulfide from Ferredoxin and Cysteine Methyl Ester.

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Crystals formed at cool end of transport tube.
Starting
materials
Ti
1 1
Time
(days)
Crystals found
Approx. vol.
(mn,,)
+ Si [b]
1j ::?. 1j
Structure
NbSi2 [a]
TaSiz [cl
NbSiz
1
i r l TaSi,
Cr i- Si [a]
hexag.
(D:)
c
hexag.
a = 4.78
c = 6.58
~
(D:)
i x l
I
’
6.36
a = 4.77
(D:)
c = 6.55
hexag.
a = 4.42
(D:)
c
CrzSi
10
cubic
a=
6.35
4.55
a
=
3.03
c =
3.23
=
(0;)
TiB2
tiny needles
[cl
=
10
7
1
VB2
(A)
a = 6.24
b = 4.77
c = 8.52
a 4.56
-..
1
Lattice
parameters
a = 3.17
c = 3.53
a = 3.00
hexaa.
tiny prisms
1
I I
Max. temperature ( “ C ) : [a] I050 ( A T
[cl l l O O ( A T % 120-150).
(D:hi
orthorh.
= 200);
1
c
=
3.07
a = 2.97
b = 7.86
c = 2.93
[bl 900 ( A T m 150);
At the hot end, powders of Ti&, VSi2, and CrSi2 were partly
converted into well-formed crystals, which were larger than
those grown at the cool end. When massive chromium and
silicon were used, well-formed Cr3Si crystals grew from both
elements; the same crystals grew at the cool end. No mineralization was observed with boride powders.
Crystals grown at the hot or the cool end have highly reflecting faces of low index. Only with TiSi2 was there a
tendency t o formation of stepped (111) faces (Hopper effect).
Transport of the borides was more than about five times
slower than that of silicides.
Received: July 61h, 1966
German version: Anjew.
Chem.
[ Z 293 IE]
78, 822 (1966)
Elimination of Hydrogen Sulfide from Ferredoxin
and Cysteine Methyl Ester‘ *
By Prof. Ernst Bayer and W. Parr
Chemisches Institut, Universitat Tubingen (Germany)
After we had shown that hydrogen sulfide is easily eliminated
from cysteine esters and cysteinyl-peptides in the presence of
iron salts [I], we attributed the “labile sulfur” in ferredoxin which is regarded by Rabinowitz et al.[21 as “inorganic
sulfide” - t o elimination of HIS from the cysteine residues in
the ferredoxin. Malkin and Rabino witz [31 have recently
opposed this view because, contrary t o our findings, they
failed to obtain H2S from cysteine methyl ester or from
glutathione and, moreover, could not detect dehydroalanine
in the ferredoxin. - This contradiction is hard t o understand,
since p-elimination of H2Sfrom cysteine methyl ester is a very
simple experiment and we identified dehydroalanine in
ferredoxin after the elimination of H2S by three independent
methods.
840
Under the following conditions [**I cysteine methyl ester, in
the presence of iron salts, eliminates 1 mole of H2S per mole
of iron salt:
For formation of the cysteine methyl ester-iron complex,
solutions of 17.1 mg of cysteine methyl ester hydrochloride
in 1 ml of oxygen-free distilled water and of 19.6 mg of
Mohr’s salt in 1 ml of oxygen-free distilled water are mixed
in a 25-ml round-bottomed flask. After addition of 5 ml of
0.5 M Tris hydrochloride buffer (pH 7.30), the mixture is
boiled for 15 to 20 min under a reflux condenser t o which is
attached a Peligot tube containing 7 ml of p-amino-N,Ndimethylaniline reagent (0.5 g of the amine hydrochloride in
500 ml of 5.5 N HCI). For determination of the hydrogen
sulfide [41, the Peligot tubs is removed and the condenser
rinsed down with 15 ml of distilled water. The cooled sample
(solution and precipitate) is placed in a 500-ml volumetric
flask containing 260 ml of 1.3 % zinc acetate solution and
10 ml of 1 2 % sodium hydroxide solution. Then 50 ml of
p-amino-N,N-dimethylanilinium
chloride solution containing
the reagent solution from the PCligot tube is added, and the
flask is stoppered and shaken until the precipitate has
dissolved. After about 10 min, 10 ml of a 0.023 M solution
of FeC13 in 1.2 N HCl is added. The mixture is set aside for
15 min and then made up to the mark with distilled water.
After a further 30 min the extinction at 670 m p is determined.
In more than 50 experiments, 1.60 j, 0.10 mg of H2S was
found, corresponding t o a molar ratio of added iron salt:
H2S = 1:0.94 j, 0.06. This is exactly the ratio of iron to
“labile sulfur” found in ferredoxin.
When the reaction was carried out without Mohr’s salt, only
0.38 + 0.05 mg of H2S was found. This shows that the reaction is catalysed by formation of the iron complex. Since
ferredoxin is an iron complex of a cysteine-rich polypeptide,
ail analogous reaction must be assumed t o occur. H2S is also
eliminated if the iron complex of cysteine methyl ester is
prepared without boiling, namely by mixing the solutions
and setting them aside in a beaker at room temperature for
16 t o 24 hr; however, as a result of incomplete formation
of the chelate, and also as a result of side reactions, yields
are then only about 20 % of those obtained by the procedure
given above. Elimination of H2S from cysteine methyl ester
does not require base catalysis and occurs even in a neutral
medium.
On elimination of H2S from ferredoxin a dehydroalanylpeptide is formed, from which pyruvic acid should be
obtained on total hydrolysis. Malkin and Rabinowitz [31 were
unable t o detect pyruvic acid after ferredoxin had been
heated with 3 N HC1 on the water bath for 1 hr. However, if
ferredoxin is treated - as is usual in protein hydrolyses with 6 N HCI under nitrogen for 24 hr at 110 “C, then 1 mg
affords 27.5 yg of pyruvic acid, which can be determined
enzymatically with lactate dehydrogenase and NADH. In
view of the lability of dehydroalanine under the conditions
of hydrolysis the yield of about 30 % of pyruvic acid (calculated for 5 moles H2S eliminated per mole of ferredoxin
of molecular weight 6000) is satisfactory, although it permits
no quantitative conclusions t o be drawn.
A substantially better method for detection of dehydroalanine
consists in reduction of the dehydroalanyl-peptide to the
alanyl-peptide with sodium borohydride and subsequent
amino-acid analysis [5961. As the cysteine content decreases, SO
the alanine content must increase.
If 1 mg of ferredoxin from Clostridium yasteurianum is treated
with sodium borohydride under the conditions described by
Pigmnn et al. (61, then treated with performic acid by Hirs’s
techniqueL71, and hydrolysed with 2 ml of 6 N HCI for 24 hr
at 110 “C, the following values are obtained for cysteine and
alanine:
1 mg of ferredoxin, 5 days at 22OC in 1 ml of 0.2 N NaOH
containing 11.3 mg (0.3~10-3 mole) of NaBH4, then oxidation with performic acid and subsequent hydrolysis:
cysteic acid, 0.176 ymole; alanine, 1.410 ymole.
1 mg of ferredoxin, 8 days at 5 “ C in 1 ml of 0.1 N NaOH
containing 11.3 mg of NaBH4 (0.3~10-3 mole), then oxidaAngew. Chem. internat. Edit. ] Vol. 5 (1966)
1 No. 9
tion with performic acid and subsequent hydrolysis:
cysteic acid, 0.246 pmole; alanine 1.308 pmole.
1 mg of ferredoxin, only treated with performic acid and
then hydrolysed :
cysteic acid, 0.772 pmole, alanine 0.974 pmole.
It can be seen that about 0.4 pmole of cysteine is converted
into alanine during the elimination of H2S. The other amino
acids are unaffected by these processes.
Received: July 7th, 1966
[Z292 I E ]
German version: Angew. Chem. 78, 824 (1966)
Experiments with compounds of type R(R)CHX, which can
be considered as carbene precursors, lead analogously to
ketene 0,N-acetals [31.
Experimental: 0.1 mole of carboxylic acid N-halogenoamide
is mixed with 0.2 to 0.3 mole of dimethylformamide 0,Odiethyl acetal or with 0.1 mole of the latter and 0.1 mole of
triethylamine, with exclusion of moisture and with stirring
and cooling (10'C). An insoluble oil first formed soon
dissolves while the solution becomes warm. The mixture is
then heated for 2 hr at 8OoC and fractionated in vacuum.
Received: July 6th. 1966
[Z 283 IEI
German version: Angew. Chem. 78, 826 (1966)
Pa-t 1 :[I].
[*I Part 2 of Ferredoxin.
[**I We thank students in the organic chemistry undergraduate
~
courses for series experiments.
[l] E. Buyer, W . Parr, and B. Kazmaier, Arch. Pharmaz. Ber.
dtsch. pharmaz. Ges. 298, 196 (1964).
[2] W . Lovenberg, B. B. Bucltanan, and J . C . Rabinowitz, J. biol.
Chemistry 238, 3899 (1963).
[3] R. Malkin and J. C. Rabinowitz, Biochemistry 5 , 1262 (1966).
141 J. K . Fog0 and H . Popowsky, Analytic. Chem. 21, 732 (1949);
cf. also[zl.
151 R. Carubelli, V . P. Bhavanandan, and A . Gottschalk, Biochim. biophyisca Acta IOI, 67 (1965).
[6] K . Tanaka, M . Bertolini, and W . Pigman, Biochem. biophys.
Res. Commun. 16, 404 (1964).
[7] C . H . W . Hirs, J. biol. Chemistry 219, 611 (1956).
[l] H. Bredereck, G. Simchen, and E. Goknel, Angew. Chem. 76,
861 (1964); Angew. Chem. internat. Edit. 3, 704 (1964).
[2] H. Bredereck, G. Simchen, and S . Rebsdat, Angew. Chem. 77,
507 (1965); Angew. Chem. internat. Edit. 4, 523 (1965).
131 P. Horn, Dissertition, Technische Hochschule Stuttgart, i n
preparation.
Triazidomethylium Hexachloroantimonate,
[C(N3)3] -+ [SbC161By U. Muller and Dr. K. Dehnicke
Laboratorium fur Anorganische Chemie,
Technische Hochschule, Stuttgart (Germany)
Preparation of 0,N-Acetals of Acyl Isocyanates
By Prof. H. Bredereck, Dr. G. Simchen, and H. Porkert
Institut fur Organische Chemie,
Technische Hochschule, Stuttgart (Germany)
Numerous classes of compound that form addition compounds with tertiary amines can substitute dialkylformamide
acetals on the formyl-carbon atom 21.
We have treated N-halogenocarboxamides (as nitrene precursors) with dialkylformamide acetals and have obtained
good yields of 0,N-acetals ( I ) of acyl isocyanates in an
exothermic reaction that is, formally, an electrophilic
substitution of the formyl-hydrogen atom. For complete
reaction one mole of N-halogenocarboxamide requires
2 moles of dialkylformamide acetal, of which, however,
1 mole may be replaced by another tertiary amine. Side
reactions occasionally afford, alongside ( I ) , 0,O-acetals (2)
of the acyl isocyanates.
Because of the energy-rich azide groups, heavy-metal azide
halides can undergo various reactions "1. The compound
[SbC14N3]2, which is accessible from SbCl5 and trimethylsilyl
azide [21, chlorazide [31, or hydrogen azide [41, reacts when
left in boiling CC14 (1 hr) with formation of the hitherto
unknown triazidomethyl cation as hexachloroantimonate in
90 % yield. The salt, pale yellow, slightly hygroscopic needles,
insoluble in nonpolar solvents and sensitive to shock or
rapid heating, m.p. 145 "C (decomp.), is isolated by filtration.
3 SbC14N3
0
+
R-C:
NHX
R'
E
Ai
AI
= C2H,
E
OR'
+
HCON(CH3)z
RCON=C:
(2) OR'
+
R'OH + R'X
HN(CH3)z
+
f
IIJ
R
Yield
9.p.
Yield
( %)
( %)
-
75
Br
Br
Br
39
44
41
67
51
53
I/
9.p.
( "C/inm)
52/0.1
-
72-75/0.1
45-48/0. I
58-60/0.I
56jO.i
52/0.I
-
Angew. Chem. internat. Edit.
1 Vol. 5 (1966) f No. 9
[C(N3)3]-[SbChj]
+ 2 SbClS
Characteristic frequencies (in cm-1) of the CN3 grouping in
[ C W 3 h I + and I C ( N H h 1 ' .
Class
OR'
2 HC:-OR~
N(CH3)2
+
The IR spectrum indicates that the cation [C(N,),]+ has C3"
symmetry. The three a-N atoms and the carbon atom are
coplanar and the azido groups are bent in the same sense at
the a-N atoms. The planar arrangement results from sp2
hybridization at the carbon atom, the resonance stabilization
corresponding to that of the guanidinium cation. Accordingly, the characteristic IR absorption of the CN3 grouping
agrees substantially with that of the guanidinium ion [51.
OR'
-t R'X
RCON=C:
+ 2 R'OH
( I ) N(CH3)z + HCON(CH3),
/
+ CC14
I Type
I [C(N3)31+
1565
1030
788
529
I [C(NH2)3I'
1670
1010
720
530
The four azide stretching vibrations of the ion [C(N3)31f
expected because of its C3v symmetry are 2285 (v,,, Al),
2190 (v,,, E), 1221 (v,, Al), and 1050 cm-1 (vs, E). The IRactive, very characteristic absorption bands [61 of class F1,
for the [SnCI& anion occur at 339 (v,,) and 172 cm-1 (8,s).
A compound N =C-N=C(N&, which is isoelectronic with
[C(N3)31+ and probably has a n analogous structure, has been
obtained from BrCN and NaN3 [7,81.
Received: July IIth, 1966
[Z 284 IE]
German version: Angew. Chem. 78, 825 (1965)
[l] K . Dehnicke, Angew. Chem. internat. Edit., in press.
[2] N . Wiberg and K . H . Schmid, Angew. Chem. 76, 380 (1964);
Angew. Chem. internat. Edit. 3, 444 (1964).
84 1
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elimination, hydrogen, methyl, sulfide, esters, cysteine, ferredoxin
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