Samples of the dithionite-reduced FeFe protein (the dinitrogenase component of the Fe-only nitrogenase) from Rhodobacter capsulatus have been investigated by 57Fe Mössbauer spectroscopy and by Fe and Zn EXAFS as well as XANES spectroscopy. The analyses were performed on the basis of data known for the FeMo cofactor and the P cluster of Mo nitrogenases. The prominent Fourier transform peaks of the Fe K-edge spectrum are assigned to Fe-S and Fe-Fe interactions at distances of 2.29 A and 2.63 A, respectively. A significant contribution to the Fe EXAFS must be assigned to an Fe backscatterer shell at 3.68 A, which is an unprecedented feature of the trigonal prismatic arrangement of iron atoms found in the FeMo cofactor of nitrogenase MoFe protein crystal structures. Additional Fe...Fe interactions at 2.92 A and 4.05 A clearly indicate that the principal geometry of the P cluster is also conserved. Mössbauer spectra of 57Fe-enriched FeFe protein preparations were recorded at 77 K (20 mT) and 4.2 K (20 mT, 6.2 T), whereby the 4.2 K high-field spectrum clearly demonstrates that the cofactor of the Fe-only nitrogenase (FeFe cofactor) is diamagnetic in the dithionite-reduced ("as isolated") state. The evaluation of the 77 K spectrum is in agreement with the assumption that this cofactor contains eight Fe atoms. In the literature, several genetic and biochemical lines of evidence are presented pointing to a significant structural similarity of the FeFe, the FeMo and and the FeV cofactors. The data reported here provide the first spectroscopic evidence for a structural homology of the FeFe cofactor to the heterometal-containing cofactors, thus substantiating that the FeFe cofactor is the largest iron-sulfur cluster so far found in nature.
The prismane protein of Desulfovibrio vulgaris, in its isolated, its one-electron-reduced and its oxidized states, was the subject of a detailed Mössbauer investigation. Measurements were recorded in the range 0.295Ϫ77 K and in the field range 0Ϫ6.2 T (parallel and perpendicular to the γ beam). The paramagnetic parts of the magnetically split Mössbauer spectra were analyzed with the spin-Hamiltonian formalism, including the nuclear Hamiltonian ; the diamagnetic parts result from the nuclear Hamiltonian only. The field-dependent spectra at 295 mK and 4.2 K indicate that the paramagnetic part of the isolated protein represents a spin-coupled 4Fe unit with the spin of one Fe site (5/2) oriented antiparallel to the spins of the other three Fe sites (5/2, 5/2 and 2), yielding a total cluster spin, S tot of 9/2. The Mössbauer parameters of the individual Fe sites indicated that this unit represents a 4Fe cluster with an unusual mixture of bridging and terminal ligands and metal coordinations (hybrid cluster). The diamagnetic part of the isolated protein represents an additional 4Fe unit, which, according to its Mössbauer parameters, is a [4Fe 2.5ϩ -4S] cubane. The parameter changes upon one-electron oxidation or reduction and the magnetic properties of the two clusters in the three oxidation states of the protein investigated here reveal that the redox behavior of the prismane protein is exclusively related with the hybrid cluster. . However, they are in full agreement with the crystal structure of the isolated protein, which, concurrent with our Mössbauer investigation, has been solved.Keywords : prismane; hybrid cluster; Desulfovibrio; Mössbauer.Hagen et al. discovered the prismane protein accidentally graphic result and the originally proposed structure is extensively discussed elsewhere. Simultaneously with the crystalwhile purifying the soluble hydrogenase of the sulphate-reducing anaerobic bacterium Desulfovibrio vulgaris [1]. Their EPR structure investigation the prismane protein has been evaluated spectroscopically in several laboratories. This complementary spectroscopic analysis led to the hypothesis that the Fe-S prosthetic group of this protein is unusual in its structure and its approach has provided a consistent picture of the structure of the two 4Fe clusters. The present paper is intended to describe redox chemistry : a [6Fe-6S] prismane core with four stable oxidation states. Moura et al. purified the analogous protein from the detailed Mössbauer investigation of the prismane protein in three oxidation states, i.e. as isolated, one-electron oxidized, and Desulfovibrio desulfuricans and proposed it to contain two 6Fe clusters [2]. Recently the crystal structure of the isolated one-electron reduced. prismane protein from D. vulgaris has been solved to 1.7-Å resolution [3]. Surprisingly, it does not show any 6Fe cluster; instead, it reveals two 4Fe clusters, a [4Fe-4S] cubane and a 4Fe MATERIALS AND METHODS cluster with an unusual mixture of bridging ligands and coordiSample preparation. The prismane pro...
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