Oxygen activation by the iron(II)-2-mercaptobenzoic acid complex. A model for microsomal mixed function oxygenases.

Full text of DOI:10.1515/znb-1969-0606

OXYGEN ACTIVATION BY THE IRON (II)-2-MERCAPTOBENZOIC ACID COMPLEX
6 9 9
Oxygen Activation by the Iron(II)-2-Mercaptobenzoic Acid Complex.
A Model for Microsomal Mixed Function Oxygenases
V o l k e r U l l r ic h
Physiologisch-chemisches Institut der Justus Liebig-Universität Gießen
(
Z. Naturforschg. 24 b , 699—704 [1969] ; eingegangen am 17. Januar 1969)
Es wird ein neues Hydroxylierungsmodell beschrieben, das molekularen Sauerstoff durch einen
Eisen (II) -2-mercaptobenzoesäure-Komplex in wäßrigem Aceton reduziert.
Dieses Modellsystem wurde aus den bisher bekannten Modellen zur mischfunktionellen Oxy-
genierung entwickelt, um eine Zweielektronen-Übertragung auf den Sauerstoff zu ermöglichen, wie
sie bei vielen Monooxygenasen anzunehmen ist.
Das neue Modellsystem hydroxyliert Aliphaten, desalkyliert aromatische Äther und substituiert
Aromaten nach einem elektrophilen Hydroxylierungs-Mechanismus. Aus Naphthalin entsteht das
1
.2-Dihydro-diol-Derivat.
Da diese Reaktionen in ihren chemischen Eij ;enschaften genau den mikrosomalen mischfunk-
tionellen Oxygenierungen entsprechen, wird ein ähnlicher oder sogar gleicher Mechanismus der
Sauerstoffaktivierung angenommen.
Mixed function oxygenases catalyse the introduc­ tion oxygenases. These prim arily are concerned
tion of one oxygen atom of molecular oxygen into
an organic substrate. The following stoichiometry
has been established 1 3:
with the stoichiometry and introduction of mole­
cular oxygen into the substrate. In addition, several
chemical aspects of the mixed function oxygenation
reactions have to be considered.
RH + DH2+ 180 2= R18OH + D + H20 .
DH2 = reduced electron donor.
The broad substrate specificity of the microsomal
mixed function oxygenation system turned out to be
of great advantage for investigation of the chemical
properties of enzymatically active oxygen. A large
number of organic compounds have been studied as
substrates, thus providing valuable inform ation
about the chemistry of mixed function oxygenases.
Four reactions can be regarded as typical:
The main problem in understanding the mechanism
of these enzymes is the activation of the oxygen
molecule. A direct investigation of this process is
hampered by the lack of pure enzyme preparations.
For several mammalian steroid hydroxylases and
for the mixed function oxygenation system in liver
microsomes it has been established that cytochrome
P­450 is involved in the reduction and activation of
molecular oxygen4. Some spectral investigations
have been made with respect to a possible oxy­
genated intermediate5. However, an interpretation
of any spectral data will only be meaningful if the
chemistry of oxygen activation is fully understood.
Therefore, this problem has been extensively
studied in chemical model systems for the activation
of molecular oxygen6,7. A large number of hydroxy­
lating systems have been described but, so far, none
1. Hydroxylation of a CH­bond at a saturated
carbon atom;
2
3
. Dealkylation reactions at heteroatom s;
. Hydroxylations of aromatic rings at positions
of high electron density;
4
. Epoxidation of double bonds.
Recently, special interest has been paid to the
last reaction because epoxides probably are the com­
mon intermediates for different pathways of mixed
of them could be regarded as a true model simu­ function oxygenation as depicted in the following
lating all characteristic features of the mixed func­
scheme:
1
2
H. S. M a s o n , Advances in Enzymol. 19, 79 [1957].
S. o r r e n iU s , J. Cell. Biol. 25, 713 [1965].
S. k a U f M a n n , J. biol. Chemistry 226, 511 [1957].
R. W . e s t a b r o o k , J. B. s c h e n k M a n , W. c a M M e r , H.
r e M M er , D. Y. c o o p e r , S. n a r a s iM h U l U , an d O . r o s e n ­
t h a l , in : B io lo g ic a l an d C h em ica l A sp ec ts of O x y g e n a ses,
5
R. W. E s t a b r o o k , A. H il d E b r a n d t , and V. U l l r ic H , Sym-
posium on “Mechanisms of Mixed Function Oxygenation”,
Konstanz, Germany. Hoppe-Seyler’s Z. physiol. Chem.
349, 1605 [1968].
G. A. H a m il t o n , J. Amer. chem. Soc. 8 6 , 3391 [1964].
V. U l l r ic H and H j. s t a U d in g E r , in: Biochemie des Sauer-
stoffs (H Ess, s t a U d in g E r , Eds.), Springer-Verlag, Berlin
1968, p. 229.
3
4
6
7
(
b l o c h , h a y a is h i, E d s.) M aruzen Comp. LTD., T o k y o ,
J ap an , 1966, p. 153.
Unauthenticated
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7
0 0
V. ULLRICH
mechanism of peracid oxidations which proceed in
R ’> = < 2
OX
H
a concerted reaction with the OH® ion (the pro­
tonated oxygen atom) as the attacking species. This
mechanism, however, is hardly in accordance with
the activation of molecular oxygen by cytochrome
P 450 and does not seem feasible under physiologi­
cal conditions.
R 1
r
2
,R 2
>
=
<
^
X
H
DT +H20
R 1
r
2
R 1
r
2
Recently we have reported the existence of a new
hydroxylation mechanism which is observed during
the reduction of molecular oxygen by stannous phos­
phate complexes. The stoichiometry of the hydroxy­
lation is described by the equation 14:
X^­­­­<H
OH
>
OH
= = <
OH
X
X = H, Halogen
Which pathway will be followed depends prim arily
on the structure of the substrate. Reaction III in­
volves the m igration of a substituent to an adjacent
position of the arom atic ring and is known as the
Sn2®+ RH + 180 2+ H® ­> Sn4®+ R180H + 18OH°.
By this reaction it was first shown that a two­elec­
trans reduction of the oxygen molecule can lead to a
high­energy precursor of hydrogen peroxide able
to hydroxylate aliphatic and aromatic compounds.
With regard to oxygen activation this system re­
presents the best model. However, the hydroxyla­
tion reactions of aliphatic and aromatic compounds
are highly unselective and, therefore, lack the elec­
trophilic properties of enzymatically active oxygen.
In order to approach more closely to the enzy­
matic systems it was desired to reduce the oxygen
molecule by iron (II)­complexes instead of the stan­
nous phosphate complex. It seemed probable that
iron (III)­complexes could stabilize the active oxy­
gen as in the peroxidase reactions16-11 and thereby
lessen its reactivity.
Hydroxylating model systems with iron (II)­com­
plexes are well known but they usually generate OH­
radicals by a sequence of three one­electron reduc­
tion steps 9’ 18, 19. Since we know that oxygen reduc­
tion to an oxenoid species must proceed by a two­
electron transfer, we have tried iron (II)­complexes
with a thiol group as ligand in order to get a two­
electron donating entity. In this paper, mixed func­
tion oxygenations by the iron(II) ­2­mercaptobenzoic
“
NIH­shift” 8. Compatible with the chemistry of
these reactions is the assumption of an active oxygen
species with six electrons formed by a two­electron
reduction of the oxygen molecule. Only a system
generating such an “oxenoid” species being able
to perform the above mentioned reactions can be
regarded as
a true model for microsomal mixed
function oxygenases and monooxygenases in general.
Three different chemical hydroxylation mecha­
nisms have been investigated and compared with the
microsomal hydroxylating system: hydroxylations
by O H ­radicals9’ 10, by peracids11 and by an
“
oxenoid" hydroxylation mechanism found in
systems with reduced metal ions and molecular oxy­
gen 12~ 14. Model systems involving OH­radicals are
unable to hydroxylate aliphatic CH­bonds and do not
form epoxides or dihydrodiols at a double bond 12.
Also, no m igration of substituents is observed during
the hydroxylation of aromatic compounds 8. In ad­
dition, OH­radicals are formed by a one­electron
reduction of hydrogen peroxide which does not par­
ticipate in the enzymatic reactions 15.
Oxidations by peracids with very strongly pola­
rized O —O bonds as in trifluoroperacetic acid ex­
hibit chemical properties very sim ilar to enzymati­ acid complex are investigated and compared with
cally active oxygen. This can be understood by the
the corresponding reactions in liver microsomes.
8
G. G U r o f f , J. W . D a l y , D . J er x n a . J. r e n s o n , b . W it k o p ,
and S. U D e n f r ie n D , Science [Washington] 157, 1524
13 V. U l l r i c h , E . A m a d o r i, and Hj. S t a u d i n g e r , Z. Natur-
forschg. 22 b. 226 [1967],
14 V. U l l r ic H , Z. Naturforschg., in print.
15 J. R. g il l E t t E , J. J. k a m m , and b. b . b r o d iE , Federat.
Proc. 18.394 [1959],
16 P. G e o r g e , J. biol. Chemistry 201, 427 [1953],
17 B. c H a n g E , H. t H E o r E l l , and A. E H r E n b E r g , Arch. Bio-
chem. Biophysics 37, 237 [1952].
18 Hj. S t a u d i n g e r and V. U l l r i c h , Z. Naturforschg. 19 b, 409
[1964],
[
1967].
r. b r e sl o W and L. N. l U k e n s . J. biol. Chemistry 235. 292
I960],
9
[
10
R . O. C. n o r M a n and G. k . r a D D a , Proc. chem. Soc. [Lon-
don] 1962. 138.
J. R. l in D s a y s M it h and R. 0 . C. n o r M a n , in: Oxidases
and Related Redox Systems (k in G , M a s o n , M o r r is o n ,
E ds.), Wiley, N ew York 1965, p. 131.
V. U l l r ic h and H j. s t a U D in G e r , in: Biological and Che-
mical Aspects of Oxygenases (b l o c h , h a y a is h i, Eds.),
Maruzen Com. Ltd., Tokyo 1966.
1
1
1
2
19 Hj. S t a u d i n g e r and V. U l l r i c h , Z. Naturforschg. 19 b, 887
[1964],
Unauthenticated
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OXYGEN ACTIVATION BY THE IRON (II)-2-MERCAPTOBENZOIC ACID COMPLEX
701
Materials and Methods
Cyclohexanol Cyclohexanone
Substrate
£h /^D
[
/<Moles]
[//M oles]
2
­Mercaptobenzoic acid (thiosalicylic acid) was ob­
Cyclohexane
D odecadeutero-
cyclohexane
6.9
2 , 8
tained from Fluka AG, Buchs S.G., Switzerland. Do­
decadeuterocyclohexane (“Uvasol'’ grade) was a pro­
duct of Merck AG, Darmstadt, Germany.
8 . 0
1 , 0
1,08
The standard incubation mixture contained the fol­
lowing concentrations in an aqueous acetone medium:
Table 1. The hydroxylation of cyclohexane by the model
system. Concentrations and incubation conditions are de-
scribed under “Methods”. Cyclohexane and dodecadeutero-
cyclohexane 2 M. Acetone was removed by evaporation and the
residue was dissolved in 1 ml of 1 N NaOH. Cyclohexanol was
extracted into 1,5 ml of ethyl acetate. 1,0 ml of the organic
phase was concentrated to 0,1 ml and 3 u \ aliquots were ana-
lysed by GLC (gaschromatograph F 6 . Perkin-Elm er). 1st
column: 15% trimethylolpropane tripelargonate /Celite 545,
60 —100 mesh, 200 cm. 2nd column: 15% polyethylene gly-
2
­mercaptobenzoic acid 10” 1M, ferrous sulphate hepta­
hydrate 10­2 M, sodium hydroxide 5 x 10­3 M. The con­
centrations of substrates varied between 0,8 and
,0 and are given in the legend to the tables. Fer­
M
2
M
rous sulphate and sodium hydroxide were added in
water so that the total water content of the incubation
mixture was 10 per cent. For the incubation in non­
aqueous medium, anhydrous ferrous chloride * and
sodium methylate were used.
The incubation was carried out at 25° under
vigorous shaking with air as the gas phase. After about
0 minutes the color of the mixture turns from blue
to yellow indicating the end of the reaction. The iso­
lation and identification of the products are given in
the legend of each table. Concentrations were deter­
mined by means of calibration curves obtained by in­
cubating the pure products with an oxidized sub­
strate­free assay mixture. All values represent aver­
ages of 10 experiments.
col 1500/Celite 545, 60 —100 mesh, 200 cm.
T
=
115 °C.
carrier gas: N2 , detector: FID. Retention times: cyclohexa-
none 1 2 , 8 min., cyclohexanol 16,0 minutes.
2
.
The O-dealkylation of N-acetylphenetidin
phenacetin )
6
(
Lipid soluble organic compounds with alkyl
groups at heteroatoms like oxygen, nitrogen or
sulphur, are readily dealkylated by the mixed func­
tion oxygenation system in liver m icrosom es21­23.
The alkyl group is found as the corresponding al­
dehyde in the reaction medium. It is generally ac­
cepted now that the main mechanism leading to de­
alkylation involves hydroxylation at the a­carbon
atom of the alkyl group. In one case of a TV­dealky­
lation this unstable intermediate could be identi­
fied24. For A^­acetylphenetidin, dealkylation is the
m ajor route of metabolism in the body 25. Hydroxy­
lation at the aromatic ring occurs to a m inor extent
only.
Results
7
. The hydroxylation of cyclohexane
Recently we have shown that cyclohexane is
hydroxylated to cyclohexanol with high specific ac­
tivity by the mixed function oxygenation system in
liver microsomes 20. In principle the same hydroxy­
lations at alicyclic carbon atoms occur with the
various steroid hydroxylases. We, therefore, have
studied the hydroxylation of cyclohexane as a model
reaction for enzymatic hydroxylations at a saturated
carbon ato m 7,12. The results with the system Fe20/
OH­radicals, as well as the “ oxenoid” mechanism
in the system Sn2®/HPO420/O 2 are also able to de­
alkylate phenacetin2(i. The hydroxylation at the
aromatic ring, however, is always favored. Table 2
contains the yields and ratios of the phenolic pro­
ducts obtained when phenacetin is used as a sub­
strate in the Fe2®/2­mercaptobenzoic ac id /0 2
system.
2
­mercaptobenzoic a c id /0 2 are shown in Table 1. At
the same time the existence of kinetic isotope
a
effect with dodecadeuterocyclohexane was in­
vestigated :
The system hydroxylates cyclohexane with good
yields. As in the microsomal mixed function oxy­
genation of cyclohexane and dodecadeuterocyclo­
hexane no isotope effect could be detected 20.
The mixed function oxygenation of phenacetin in
this system yields a higher ratio of dealkylation:
hydroxylation than any other model system in­
vestigated.
*
This compound was kindly supplied by Dr. H . J. s e if e r t ,
Institute for Inorganic and Analytical Chemistry, Gießen.
23 P. M a z e l , J. F. h e n D e r s o n , and J. a x e l r o D , J. Pharma-
col. exp. Therapeut. 143, 1 [1963].
24 h . k e b e r l e , W. r ie ss, k . s c h M iD , and k . h o f f M a n n ,
Arch. int. Pharmacodyn. Therap. 142, 125 [1963].
25 b. b . b r o D ie and J. a x e l r o D , J. Pharmacol, exp. Thera-
peut. 97,58 [1949].
20 V. U l l r ic h , Hoppe-Seyler’s Z. physiol. Chem. 350. 357
[1969],
21
J. a x e l r o D , R. s c h M iD , and L. h a M M a k e r , Nature [Lon-
don] 180. 1426 [1957].
22
J. a x e l r o D . Biochem. J. 63, 634 [1956],
26 V. U l l r ic h , D. h e y , h J. s t a U D in G e r , h . b ü c h , an d W .
r U M M e l , B ioch em . P h arm acol. 16, 2237 [1967].
20
V. U l l r ic h , Hoppe-Seyler’s Z. physiol. Chem., in print.
Unauthenticated
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7
0 2
V. ULLRICH
System
2-H vdroxy-
phenaeetin
3-H vdroxv-
phenacetin
4-H ydroxy-
acetanilide
Yield
[
//M oles]
[
%]
[%]
[% ]
F e2®/2 -M ereaptobenzoic Aeid/C^
R at Liver M ierosom es/N A D P H /02 27
1 2
1
31
0.3
57
98.7
1 . 1 0
Table 2. Hydroxylation and dealkylation of phenacetin in the model system. Concentrations and incubation conditions are
described under “Methods”. Phenacetin: 0,8 M. 2,0 ml of 0,5 M NaOH was added to the incubated mixture which subsequently
was neutralized by adding ammonium acetate and extracted with 10 ml of ether. The products were isolated and determined
as described previously 27.
3
.
The hydroxylation of acetanilide and toluene
the microsomal mixed function oxygenation system
was found to be around 0,01 29. When the incuba­
tion was carried out in a nonaqueous medium at
conditions described under “Methods” about the
same yields and an identical hydroxylation pattern
are obtained.
Aromatic compounds with one first order sub­
stituent (ortho-para directing) are hydroxylated by
the unspecific liver microsomal system at positions
of high electron density 28, 29. This leads to a favored
attack at the ortho- and para-positions, indicating
that we were dealing with an electrophilic attacking
species. In general the orfAo­position always is sub­
ject to considerable steric effects so that the meta:
para-ratio is a better parameter for expressing the
selectivity of electrophilic properties of a substitu­
tion mechanism30. In contrast, the active oxygen
species in the system Sn20/HPO420/O2 attacks the
aromatic nucleus completely unselectively yielding
meta: para ratios of about two which would be ex­
pected for a random substitution.
In order to further establish the electrophilic pro­
perties of the active oxygen in microsomes and our
model system we have determined the pattern of
phenolic isomers obtained by the hydroxylation of
toluene in both systems (Table 4).
Cresol
ortho- meta- para-
Y ield
m eta:
para
System
[
//M oles]
[
% ]
[% ]
[% ]
F e2®/2-Mercapto-
benzoie A c id /0 2
Rat Liver M icro-
som es/N A D P H / 0 2
The distribution pattern of phenolic products ob­
tained with acetanilide in the Fe20/2­mercapto­
benzoic acid/02 system can be seen from Table 3.
61
59
13
13
26
28
0.50
0,47
1 .1
1.9
Table 4. Ring hydroxylation of toluene in rat liver micro-
somes and the model system. Microsomes of phenobarbital-
treated male rats (Wistar AF/Han) were u sed 20. The incu-
bation mixture contained in a total volume of 2 0 ,0 mg of tris
H ydroxy-
acetanilides
ortho- meta- para- para
System
m eta:
Y ield
[
//M oles]
[
%]
[% ]
[%]
buffer (0,05 m pH 7.5) the following concentrations: micro-
somal protein 2 mg/ml. NADPH 5 x 10- 4 M, glucose-6 -phos-
phate 2 x 1 0 ~ 3 m, glucose-6 -phosphate dehydrogenase 10 U,
toluene 10~ 2 m , added as 1 M solution in ethanol. The incuba-
tion was carried out for 30 min. at 25 °C. Protein was re-
moved by addition of uranium acetate, subsequent heating to
F e2® 2-M ercapto-
benzoic A cid /0 2
R at Liver Micro-
so m es/N A D P H /0 229
57
5
3
1
40
94
0,08
3.2
0 . 0 1
8
0 °C, and centrifugation. The supernatant was extracted
Tab. 3. Hydroxylation of acetanilide in the model system.
Acetanilide 1,25 M. All conditions and procedures are the
same as used for phenacetin (see legend to table 2 ).
with 5 ml of ethylacetate. 3 ml were concentrated to 0.1 ml
and aliquots were separated by gaschromatography using two
2 0 0 cm columns (10% tri-2,3-xylyl phosphate). 7 = 140 °C,
carrier gas: N , detector FID. Retention times: benzaldehyde
8,4 min., benzyl alcohol 30,4 min., ortho-cresol 71,5 min.,
para-cresol 91.5 min., m eta-cresol 100,1 minutes. The same
separation method was applied for the model hydroxylations.
Toluene 2 M. The isolation of the products was the same as
described for cyclohexanol (see legend to table 1 ).
The low meta: para ratio of the isomers clearly
points to an electrophilic and rather selective hy­
droxylation mechanism. The corresponding meta:
para ratio for the hydroxylation of acetanilide by
H. B ü c h , K. P f l e g e r , W. R u m m e l, V. U l l r i c h , D. hey,
and Hj. S t a u d i n g e r , Biochem. Pharmacol. 16, 2247
29 V. U l l r ic H , J. W o l f , E. a m a d o r i. and H j. s t a U d in g E r ,
Hoppe-Seyler’s Z. physiol. Chem. 349. 85 [1968].
30 J . H in e , Reaktivität und Medianismus, S. 353, Thieme-
Verlag, Stuttgart 1960.
[
1967].
D. V. P a r k E and R. T. W il l ia m s , Ann. Rept. diem. Soc.
5, 376 [1959].
5
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OXYGEN ACTIVATION BY THE IRON (II)-2-MERCAPTOBENZOIC ACID COMPLEX
7 0 3
It can be seen that an almost identical hydroxyla­
tion pattern is obtained in microsomes and the
model system. In both systems the hydroxylation of
the side chain is considerable. The relative yields of
benzyl alcohol are not given because benzoic acid
as a main secondary oxidation product could not be
determined in the same test.
hence the electrophilic properties of an oxenoid spe­
cies, we have studied naphthalene as a substrate in
the model system (Table 5).
The main product obtained under the conditions
of the assay and isolation procedure was identified
as the dihydrodiol compound by its /^/­values, ir­
spectrum and nmr­spectrum. According to the nmr­
spectrum the hydroxyl groups have the trans-con­
figuration.
4
. The mixed function oxygenation of naphthalene
Substrates containing double bonds are epoxidized
by the microsomal system and by mixed function
Discussion
oxygenases in general1131, 32.
The iron (II)­complex of 2­mercaptobenzoic acid
reduces molecular oxygen to an active oxygen spe­
cies which is capable of reacting with organic com­
pounds in a manner analogous to the microsomal
mixed function oxygenation system. The model
system can hvdroxylate even in the complete ab­
sence of water indicating that the oxygen atom of
the hydroxyl group must derive from the oxygen
molecule of the gas phase.
According to the scheme in Fig. 1 the epoxide
may undergo hydration to the dihydrodiol com­
pound.
Naphthalene can be regarded as an aromatic 10.r
electron system with a high degree of double bond
character in one ring. In liver microsomes in the
presence of NADPH and oxygen it is converted to
8
0% naphthalene­1,2­dihydrodiol and 20% a­ and
/
?­naphthol12. The Jrans­configuration of the two
Characteristics of this hydroxylation mechanism
are its electrophilic reaction towards substrates with
JT­electron systems, and that it also can hydroxylate
aliphatic CH­bonds. The active oxygen, therefore, is
clearly distinct from the OH­radical which does not
form alcohols or the dihydrodiol compound of
naphthalene. On the other hand it is also different
from the oxene­mechanism in the stannous­phos­
phate/oxygen system in being selective and less re­
active.
hydroxyl groups 31 and the fact that only one oxygen
atom derives from molecular oxygen33 are both
compatible with the assumption that the 1,2­epoxide
is the primary product of the attack by the enzymati­
cally active oxygen species. This was confirmed very
recently by the isolation and identification of
naphthalene epoxide from a microsomal system34.
As the formation of an epoxide or a dihydrodiol
compound gives direct proof for the addition and
1
-Naphthol
2-Naphtho]
Naphthalene
Y ield
System
1 .2 -dihvdrodiol
[
//Moles]
[
%]
[%]
9
[%]
F e2®/2-M ercaptobenzoic Acid/O -2
R at Liver M icrosom es/N A D PH /0 2 1 2
1 1
80
80
1.7
2 0
Table 5. Mixed function oxygenation of naphthalene in the m odel system. The standard incubation mixture was used. Naphtha-
lene 1 m . After completation of the reaction acetone was evaporated and 2,0 ml of 0,1 M NaOH were added. After neutralisa-
tion with solid ammonium acetate an excess of naphthalene was removed by extracting twice with 5,0 ml of petrol ether (b.p.
5
0 —60 °C ). The products were then extracted into 10 ml of ether. A 9,0 ml aliquot was evaporated and chromatographed by
two-dimensional TLC. 1st solvent: benzene 79, methanol 14, acetic acid 7 (v/v). 2 nd solvent: petrol ether 40, chloroform 55,
-propanol 5, half-saturated with ammonia. The spots containing a-naphthol and /?-naphthol, respectively, were collected in
2
centrifuge tubes. After addition of 1 ml 0.1 M NaOH, 1 ml N a2CO ;! and 1 ml of Folin-Reagent (Merck AG., 1: 5 dil.) the blue
color was developed at 37 °C for 30 minutes. The extinction was read at 578 mm in a photometer. Naphthalene-1,2-dihydro-
diol was first converted into a-naphthol by treatment with 0,9 ml 0,1 M HC1 for 60 min. at 100 °C. Then 0,1 ml 1 m NaOH was
added and the blue color with the Folin-Reagent was determined as described before.
3
1
P. s iM s, Biochem. J. 73, 389 [1959].
b . M . b lo o M and G. M . s h U l l , J. A m er. ch em . S oc. 77,
34 D . M . J e r i n a , J. W . D a l y , b. W it k o p , P. z a l t s M a n ­
n ir e n b e r G , and S. U D e n f r ie n D , J. Amer. chem. Soc. 90,
6525 [1968],
3
2
5
767 [1955].
33 J. L. h o l t z M a n n , J. R. G il l e t t e , and G. W . a . M il n e , J.
biol. Chemistry 242, 4386 [1967].
Unauthenticated
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7
0 4
OXYGEN ACTIVATION BY THE IRON (II)-2-MERCAPTOBENZOIC ACID COMPLEX
Most important is the finding of a system which
forms the 1,2­dihydrodiol compound from naphtha­ the system Fe2®/2­mercaptobenzoic acid/02 com­
lene. This led us to suggest an oxenoid­species as the
pared to that of the system Sn20/HPO42G/O2 may
active oxygen. Further studies on the chemical pro­ be caused by complex formation thereby stabilizing
The moderate reactivity of the oxenoid species in
perties of the mechanism are in progress in order
to strengthen this hypothesis.
the active oxygen at the ferric ion in a manner
similar to the various oxygen complexes of iron­
porphyrin enzymes 16‘17.
In summary our results indicate that the reaction
of the iron (II) ­2­mercaptobenzoic acid complex with
oxygen provides a suitable model for the activation
of molecular oxygen by cytochrome P­450.
Further investigations are in progress with a more
The sulfhydryl group was chosen as a ligand
for ferrous ion in order to provide a donor for
the second electron needed for the generation of an
oxenoid species. It is not known at present, how the
two­electron transfer to the oxygen molecule occurs
at cytochrome P­450. However, one may speculate
on the participation of a thiol group since cyto­ detailed study of the chemical and physical proper­
chrome P­50 instantaneously looses its characteristic
unusual spectral and chemical properties on treat­
ment with iodine, /V­bromosuccinimide 20,25 or
ties of this model system.
p­chloromercuribenzoate36. At the same time
a
conversion from a low spin cytochrome to a high
spin state is observed3'. The role of sulphur as a
ligand of iron has been established in the case of a
bacterial oxygenase 38.
The author wishes to thank Dr. H. a H l b r E c H t for
the recording and interpretation of the nmr­spectra and
Prof. Dr. Hj. s t a U d i n g E r for helpful discussions and
continued interest in this work.
3
5
V . U l l r ic h . B. co h e n , D. Y. co o p e r , and R. W . e s t a ­
b r o o k , S y m p o siu m on C ytoch rom es, O saka, J ap an , 1967,
U n iv ersity of T o k y o P ress, T ok yo 1968, p. 649.
h . s. M a s o n , J. c. n o r t h , and M . V a n n e s t e , Federat.
Proc. 24, 1172 [1965].
37 V. U l l r ic h and R. W . e s t a b r o o k , u n p u b lish ed resu lts.
38 s. s e n o h , h . k it a . and M. k a M iM o t o , Biological and
Chemical Aspects of Oxygenases (b l o c h , h a y a is h i, Eds.),
Maruzen Comp. Ltd.. Tokyo. Japan, 1966, p. 378.
38
Unauthenticated
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