J. Biol. Chem., Vol. 269, Issue 11, 8052-8058, 03, 1994
Thiol/disulfide formation associated with the redox activity of the [Fe3S4] cluster of Desulfovibrio gigas ferredoxin II. 1H NMR and Mossbauer spectroscopic study
AL Macedo, I Moura, KK Surerus, V Papaefthymiou, MY Liu, J LeGall, E Munck and JJ Moura
Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Monte da Caparica, Portugal.
Desulfovibrio gigas ferredoxin II (FdII) is a small protein (alpha 4
subunit structure as isolated; M(r) approximately 6400 per subunit; 6
cysteine residues) containing one Fe3S4 cluster per alpha-subunit. The
x-ray structure of FdII has revealed a disulfide bridge formed by Cys- 18
and Cys-42 approximately 13 A away from the center of the cluster;
moreover, the x-ray structure indicates that Cys-11 forms a disulfide
bridge with a methanethiol. In the oxidized state, FdIIoxm the 1H NMR
spectra, exhibit four low-field contact-shifted resonances at 29, 24, 18,
and 15.5 ppm whereas the reduced state, FdIIR (S = 2), yields two features
at +18.5 and -11 ppm. In the course of studying the redox behavior of FdII,
we have discovered a stable intermediate, FdIIint, that yields 1H
resonances at 24, 21.5, 21, and 14 ppm. This intermediate appears in the
potential range where the cluster (E'0 approximately -130 mV) is reduced
from the [Fe3S4]1+ to the [Fe3S4]0 state. FdIIint is observed during
reductive titrations with dithionite or hydrogen/hydrogenase or after
partial oxidation of FdIIR by 2,6- dichlorophenolindophenol or air. Our
studies show that a total of three electrons per alpha-subunit are
transferred to FdII. Our experiments demonstrate the absence of a
methanethiol-Cys-11 linkage in our preparations, and we propose that two of
the three electrons are used for the reduction of the disulfide bridge.
Mossbauer (and EPR) studies show that the Fe3S4 cluster of FdIIint is at
the same oxidation level as FdIIox, but indicate some changes in the
exchange couplings among the three ferric sites. Our data suggest that the
differences in the NMR and Mossbauer spectra of FdIIox and FdIIint result
from conformational changes attending the breaking or formation of the
disulfide bridge. The present study suggests that experiments be undertaken
to explore an in vivo redox function for the disulfide bridge.
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