POSTER SESSIONS: I. METALLOPROTEINS
and is reducible with dithionite. This result is contrary to previous studies which have been interpreted in terms of center S2 being either a low potential [2Fe-2S] cluster [3] or non-existent [4]. EPR
power saturation studies confirm that centers SI
and S2 are magnetically interacting in the dithionite-reduced enzyme. However, center S2 is not
EPR silent but can be detected as weak shoulders
to high and low field of the reduced S1 EPR spectrum at temperatures below 20 K in dithionite-reduced Complex II, as well as reconstitutively
active and inactive SDH. This new signal is most
clearly observed in dithionite-reduced Complex II
and difference spectra under conditions where S1
is power saturated show the principal g-values to
be 2.06, 1.95, and 1.85. These g-values are characteristic of [4Fe-4S] 1 + centers in bacterial ferredoxins.
S1 and S2 are present in both reconstitutively
active and inactive SDH. The third cluster, S3, is
only present in significant amounts in reconstitutively active enzyme. In both succinate and dithionite-reduced reconstitutively-active SDH both the
form of the MCD spectrum and the magnetization
data identify this cluster as a paramagnetic, EPR-silent reduced [3Fe-xS] center. This result confirms the recent linear electric field effect EPR
measurements on the isotropic g = 2.01 signal
observed in oxidized Complex II [5], which indicated that this signal originates from an oxidized
[3Fe-xS] center rather than a tetranuclear high potential Fe-S cluster, [4Fe-4S] 3 +. Moreover the
MCD data suggest that center S3 is a necessary requirement for reconstitutive activity and indicate
that bulk conversion of this center to a [4Fe-4S]
cluster does not occur on addition of substrate.
Although the results do not completely rule
out the possibility of partial [3Fe-xS] to
[4Fe-4S] conversion on addition of substrate, it
seems likely that the [3Fe-xS] center in SDH is
able to sustain Q-reductase activity.
These results resolve many of the long standing
controversies concerning the Fe-S cluster content
of SDH and for the first time enable rationalization of all the published spectroscopic data in
addition to the analytical and core extrusion studies [6].
184
REFERENCES
[1] B.A.C.
ACKRELL, E.B. KEARNEY, C.J. COLES, J. Biol.
Chem., 252, 6963-6965 (1977).
[2] K.A. DAVIS, Y. HATEet, Biochemistry, 10, 2509-2516
(1971).
[3] T. OHNISHI, J.C.
SALERNO,
in T.G.
SPIRO (ed.),
«Iron-
-Sulfur Proteins», John Wiley and Sons, New York, 1982,
pp. 285-327.
ALBRACHT, Biochim. Biophys. Acta, 612, 11-28
(1980).
[5] B.A.C. ACKRELL, E.B. KEARNEY, W.B. MIMS, J. PEISACH,
H. BEINERT, J. Biol. Chem., 259, 4015-4018 (1984).
[6] C.J. COLES, R.H. HoLM, D.M. KURTZ, W.H. ORME-JOHNSON, J. RAWLINGS, T.P. SINGER, G.B. WONG, Proc. Natl.
Acad. Sci. U.S.A., 76, 3805-3808 (1979).
[4] S.P.J.
6?)
PS I.28 — TU
K. NAGAYAMA
Biometrology Lab, JEOL Ltd.
Nakagami, Akishima, Tokyo 196
Japan
REDOX STATE STUDIES
OF TWO IRON-SULFUR CENTERS
IN 7Fe FERREDOXINS
BY PROTON MAGNETIC RESONANCE
1) Two types of iron-sulfur clusters, [3Fe-XS]
and [4Fe-4S], were identified by 1 H-NMR in the
intact ferredoxins (Fd) extracted from Thermus
thermophilus, Mycobacterium smegmatis and
Pseudomonas ovalis. The [4Fe-4S] clusters always
showed the redox couples which had potentials lower than that of the [3Fe-XS] clusters.
2) The oxidizability of a redox couple, [4Fe-4S],
in a 7Fe ferredoxin extracted from P. ovalis was
monitored by 1 H-NMR. The iron-sulfur cluster in
the ferredoxin was not only reducible, but also
oxidizable in its native form. This result provided
the first verification of Carter's 3 redox theory for
a redox center in ferredoxin, 4Fe, in the native
form of the protein.
Rev. Port. Quint., 27 (1985)
2nd INTERNATIONAL CONFERENCE ON BIOINORGANIC CHEMISTRY
3) The redox couples in 7Fe ferredoxins treated
with ferricyanide were monitored by 1 H-NMR. An
excess amount of ferricyanide was found to effect
conversion of one of the two redox centers, the
4Fe core, to a 3Fe core in the ferredoxins extracted from T. thermophilus, M. smegmatis and P.
ovalis. On long term incubation in air, the converted 3Fe core showed even further change in NMR.
spectra.
4) With above three results the complicated
change observed during the reduction-reoxidation
process in NMR spectra of Thermus thermophilus
Fd was interpreted in the term of the redox reaction and 3Fe-4Fe interconversion.
PS 1.29 — TH
E. BILL
A.X. TRAUTWEIN
H. WINKLER
Physik
Medizinische Hochschule
2400 Lübeck I
W. Germany
F.-H. BERNHARDT
Physiologische Chemie
Rheinisch-WeSlfnliSChe Technische Hochschule
5100 Aachen
W. Germany
redox reaction
4Fe -.3Fe
conversion
3Fe
- 1V
-0.5
r
T.thermophilus
o
0.5
1V
.
4Fe
^
(4Fe
t
NITROSYL-BINDING
TO THE MONONUCLEAR NON-HEME IRON
OF PUTIDAMONOOXIN:
A MODEL FOR THE CORRESPONDING
PEROXO COMPLEX
3Fe
M.smegmatis
. '
V
4Fe
4Fe
3Fe
.r
P.ovalis
- 1V
' -0.5
4Fe
0
4
^
5
1V
Figure
Redox potentials of 3Fe and 4Fe redox centers and potential
range of 4Fe to 3Fe interconversion
REFERENCES
[1] K.
NAGAYAMA,
D.
OHMORI,
T. IMAI, T.
OSHIMA,
FEBS
Lett., 158, 208-212 (1983).
[2] K. NAGAYAMA, D. OHMORI, FEBS Lett. , 173, 15-18 (1984).
[3] K. NAGAYAMA, T. IMAI, D. OHMORI, T. OSHIMA, FEBS
Lett., 169, 79-84 (1984).
Rev. Port. Quím., 27 (1985)
Putidamonooxin (PMO), the terminal oxygenase
of a 4-methoxybenzoate monooxygenase enzyme
system (EC 1.14.99.15), consists of three identical
subunits, the active centers of which contain one
[2Fe-2S] cluster and one mononuclear non-heme
(cofactor) iron [1-6]. The activation of molecular
oxygen by the reduced PMO is achieved by binding 0 2 to the cofactor iron, and by successive
transfer of two electrons to the 0 2 -molecule, one
from the reduced [2Fe-2S] cluster and one from
the reduced non-heme iron, respectively. Because
of the relatively fast kinetics of this process (< 10
ms) we were so far not able to follow the stepwise
transfer of these electrons or to detect the "mononuclear non-heme iron peroxo complex" [Fe0 2 ] .
via ESR or Mássbauer spectroscopy. The corresponding nitrosyl-complex, however, is more stable
and therefore accessible for ESR and Mássbauer
measurements.
We have recorded MOssbauer spectra of the nitrosyl complex (using different substrates) in the
temperature range 1.5 K to 200 K with varying
applied magnetic fields Heil. One of our results is
that we did not observe any substrate dependence.
Additionally, we find from the spin-Hamiltonian
185