Thiol/disulfide formation associated with the redox activity of the [Fe3S4] cluster of Desulfovibrio gigas ferredoxin II. 1H NMR and Mossbauer spectroscopic study

NIGMS NIH HHS (GM-41482)Desulfovibrio gigas ferredoxin II (FdII) is a small protein (α4 subunit structure as isolated; M(r) ≃ 6400 per subunit; 6 cysteine residues) containing one Fe3S4 cluster per α-subunit. The x-ray structure of FdII has revealed a disulfide bridge formed by Cys-18 and Cys-42 approximately 13 Å 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, FdII(ox), the 1H NMR spectra, exhibit four low-field contact-shifted resonances at 29, 24, 18, and 15.5 ppm whereas the reduced state, FdII(R) (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, FdII(int), that yields 1H resonances at 24, 21.5, 21, and 14 ppm. This intermediate appears in the potential range where the cluster (E'0 ≃ -130 mV) is reduced from the [Fe3S4]1+ to the [Fe3S4]0 state. FdII(int) is observed during reductive titrations with dithionite or hydrogen/hydrogenase or after partial oxidation of FdII(R) by 2,6- dichlorophenolindophenol or air. Our studies show that a total of three electrons per α-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 FdII(int) is at the same oxidation level as FdII(ox), 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 FdII(ox) and FdII(int) 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.publishersversionpublishe

Magnetic Circular Dichroism Spectroscopy of Metalloproteins

Metals and metal clusters in proteins typically serve as important structural/functional motifs. Because of this reason, there is a wide range of techniques that specifically probe the structure and energy levels of metals in metalloproteins. One technique, magnetic circular dichroism (MCD) spectroscopy, is the focus of this chapter. MCD spectroscopy monitors the circular dichroism spectrum induced by a magnetic field and is an effective way of obtaining electronic and structural information of paramagnetic metal ions or clusters. The basic methodology of this technique is discussed along with examples of how MCD spectroscopy can be used to elucidate typical metal clusters in proteins. Special emphasis is placed on iron-sulfur (FeS) clusters