Spectroscopic evidence for an all-ferrous [4Fe–4S]0 cluster in the superreduced activator of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans

The key enzyme of the fermentation of glutamate by Acidaminococcus fermentans, 2-hydroxyglutarylcoenzyme A dehydratase, catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-coenzyme A, resulting in (E)-glutaconylcoenzyme A. The dehydratase system consists of two oxygen-sensitive protein components, the activator (HgdC) and the actual dehydratase (HgdAB). Previous biochemical and spectroscopic studies revealed that the reduced [4Fe–4S]+ cluster containing activator transfers one electron to the dehydratase driven by ATP hydrolysis, which activates the enzyme. With a tenfold excess of titanium(III) citrate at pH 8.0 the activator can be further reduced, yielding about 50% of a superreduced [4Fe–4S]0 cluster in the all-ferrous state. This is inferred from the appearance of a new Mössbauer spectrum with parameters δ = 0.65 mm/s and ΔEQ = 1.51–2.19 mm/s at 140 K, which are typical of Fe(II)S4 sites. Parallel-mode electron paramagnetic resonance (EPR) spectroscopy performed at temperatures between 3 and 20 K showed two sharp signals at g = 16 and 12, indicating an integer-spin system. The X-band EPR spectra and magnetic Mössbauer spectra could be consistently simulated by adopting a total spin St = 4 for the all-ferrous cluster with weak zero-field splitting parameters D = −0.66 cm−1 and E/D = 0.17. The superreduced cluster has apparent spectroscopic similarities with the corresponding [4Fe–4S]0 cluster described for the nitrogenase Fe-protein, but in detail their properties differ. While the all-ferrous Fe-protein is capable of transferring electrons to the MoFe-protein for dinitrogen reduction, a similar physiological role is elusive for the superreduced activator. This finding supports our model that only one-electron transfer steps are involved in dehydratase catalysis. Nevertheless we discuss a common basic mechanism of the two diverse systems, which are so far the only described examples of the all-ferrous [4Fe–4S]0 cluster found in biology

The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a [4Fe–4S] cluster

In type E succinate:quinone reductase (SQR), subunit SdhE (formerly SdhC) is thought to function as monotopic membrane anchor of the enzyme. SdhE contains two copies of a cysteine-rich sequence motif (CXnCCGXmCXXC), designated as the CCG domain in the Pfam database and conserved in many proteins. On the basis of the spectroscopic characterization of heterologously produced SdhE from Sulfolobus tokodaii, the protein was proposed in a previous study to contain a labile [2Fe–2S] cluster ligated by cysteine residues of the CCG domains. Using UV/vis, electron paramagnetic resonance (EPR), 57Fe electron–nuclear double resonance (ENDOR) and Mössbauer spectroscopies, we show that after an in vitro cluster reconstitution, SdhE from S. solfataricus P2 contains a [4Fe–4S] cluster in reduced (2+) and oxidized (3+) states. The reduced form of the [4Fe–4S]2+ cluster is diamagnetic. The individual iron sites of the reduced cluster are noticeably heterogeneous and show partial valence localization, which is particularly strong for one unique ferrous site. In contrast, the paramagnetic form of the cluster exhibits a characteristic rhombic EPR signal with gzyx = 2.015, 2.008, and 1.947. This EPR signal is reminiscent of a signal observed previously in intact SQR from S. tokodaii with gzyx = 2.016, 2.00, and 1.957. In addition, zinc K-edge X-ray absorption spectroscopy indicated the presence of an isolated zinc site with an S3(O/N)1 coordination in reconstituted SdhE. Since cysteine residues in SdhE are restricted to the two CCG domains, we conclude that these domains provide the ligands to both the iron–sulfur cluster and the zinc site

METAL-METAL INTERACTIONS IN COMPLEXES WITH A DOUBLE DIMETHYLSILANDIYL-BRIDGED DICYCLOPENTADIENYL LIGAND

SIEMELING U, Jutzi P, BILL E, TRAUTWEIN AX. METAL-METAL INTERACTIONS IN COMPLEXES WITH A DOUBLE DIMETHYLSILANDIYL-BRIDGED DICYCLOPENTADIENYL LIGAND. JOURNAL OF ORGANOMETALLIC CHEMISTRY. 1993;463(1-2):151-154.The metal-metal interaction in the mixed-valence ferrocenium salt [trans-Cp*FeLFeCp*]+PF6- ([5(+)]PF6-) (Cp* = C(5)Me(5), L = [C(5)H(3)SiMe(2)](2)) was investigated by cyclic voltammetry, electron spectroscopy, EPR and temperature dependent Mossbauer spectroscopy. The compound is best described as a valence-localised system (class I according to Robin and Day)

EPR AND MOSSBAUER SPECTROSCOPIC STUDIES ON THE TETRAMERIC, NAD-LINKED HYDROGENASE OF NOCARDIA-OPACA-1B AND ITS 2 DIMERS .1. THE BETA-DELTA-DIMER - A PROTOTYPE OF A SIMPLE HYDROGENASE

ZABOROSCH C, KOSTER M, BILL E, Schneider K, SCHLEGEL HG, TRAUTWEIN AX. EPR AND MOSSBAUER SPECTROSCOPIC STUDIES ON THE TETRAMERIC, NAD-LINKED HYDROGENASE OF NOCARDIA-OPACA-1B AND ITS 2 DIMERS .1. THE BETA-DELTA-DIMER - A PROTOTYPE OF A SIMPLE HYDROGENASE. BIOMETALS. 1995;8(2):149-162.The cytoplasmic, tetrameric NAD-linked hydrogenase from Nocardia opaca Ib can be separated in two dimeric substructures, an ay-dimer with NADH:electron acceptor oxidoreductase (diaphorase) activity and a PG-dimer which displays hydrogenase activity with artificial electron carriers, These two dimers were preparatively isolated by a FPLC Mono Q procedure in the absence of nickel and at alkaline pH values, The hydrogenase-active PS-dimer contained, as analyzed by inductively coupled plasma mass spectrometry (TCP-MS), 3.5-3.9 iron atoms and 1.3-1.7 nickel atoms per dimer molecule, EPR and Mossbauer spectra indicated the presence of a [4Fe-4S] cluster, This center turned out to be extremely labile towards oxidants, Oxidation led to irreversible convertion into a [4Fe-4S] form, thus representing an artifact and not a regulatory state of the cluster, The midpoint redox potential of the [4Fe-4S] cluster was determined to be -385 mV, Very weak EPR Ni signals of the PG-dimer were detectable in the oxidized as well as in the reduced state, The diaphorase-active ay-dimer was free of nickel and the iron content corresponded to 11.2-12.8 Fe atoms per dimer molecule, From EPR and Mossbauer measurements it was concluded that this dimer contained two [4Fe-4S] clusters, one [2Fe-2S] and one [3Fe-4S] cluster, In accordance with the results obtained for the diner proteins, for the whole enzyme an iron content of 15.8-16.2 atoms per enzyme molecule have been determined, EPR spectra and spectrum simulations of the native hydrogenase corroborate the cluster assignments of the two dimers: in total the enzyme contains one [2Fe-2S] cluster, one [3Fe-4S] cluster and three [4Fe-4S] clusters

Multiple K-edge XAS for the structural analysis of thiophenolate bridged heterotrinuclear complexes

Meyer-Klaucke W, Glaser T, Froba M, Tiemann M, Wong J, Trautwein AX. Multiple K-edge XAS for the structural analysis of thiophenolate bridged heterotrinuclear complexes. Journal of Synchrotron Radiation. 1999;6(3):397-399.A multiple K-edge X-ray absorption spectroscopic (XAS) approach to determine the structural parameters of an isostructural series of heterotrinuclear thiophenolate-bridged metal-complexes of the general formula [LFeCrFeL]nt (with n=1,2,3) is presented. The analysis focuses on the variation of the metal-metal distances and the metal-sulfur distances with the cluster charge n. XAS measurements on the Fe, Cr, and S-Kedges were performed to allow a fit of these distances from these constituent elements. Results show that 3 3 atoms and 3 N atoms coordinate each of the terminal Fe atoms, whereas 6 S atoms coordinate the central Cr atom. This symmetry allows extracting structural information with high accuracy especially when the spectra obtained at different edges are refined at the same time. A decrease of the metal-metal distance with increasing cluster charge is also derived from this multiple-edge analysis

REDOX PROPERTIES OF THE METAL CENTERS IN THE MEMBRANE-BOUND HYDROGENASE FROM ALCALIGENES-EUTROPHUS CH34

KNUTTEL K, Schneider K, ERKENS A, et al. REDOX PROPERTIES OF THE METAL CENTERS IN THE MEMBRANE-BOUND HYDROGENASE FROM ALCALIGENES-EUTROPHUS CH34. BULLETIN OF THE POLISH ACADEMY OF SCIENCES-CHEMISTRY. 1994;42(4):495-511.For highly active preparations of the membrane-bound, respiratory chain-linked hydrogenase from Alcaligenes eutrophus strains H16 and CH34, 0.7 moles of nickel and approximately 10 moles of iron per mole of enzyme were determined. The hydrogenase isolated from A. eutrophus CH34, displayed the highest specific activity (540 U/mg protein) and the most intense Ni signals and was therefore used to study the redox properties of the metal centres present in this type of hydrogenase. At 80 K two well-resolved Ni spectra were detected in the course of a redox titration (at pH 7.0): one displayed a Ni-B type signal at g = 2.30, 2.17 and 2.01 representing the Ni(III) centre in the ''ready state'' of the enzyme (''active'' protein conformation, oxidized catalytic centre), the other one consisted of a Ni-C type signal at g = 2.20, 2.16 and 2.01 apparently representing monovalent nickel in the ''active state'' of the enzyme (''active'' protein conformation, reduced catalytic centre) and a minor proportion of a second Ni-C signal (g = 2.31, 2.12, 2.05) induced by the influence of normal daylight. The midpoint potentials determined are -30 mV for Ni(III), -260 mV (appearance of the Ni-C signal) and -340 mV (disappearance of the Ni-C signal) for apparent Ni(I). The midpoint potentials calculated for the FeS clusters are: + 100 mV ([3Fe-4S]1+/0), + 50 m V ([4Fe-4S]2+/1+ cluster I) and -80 mV ([4Fe-4S]2+/1+ cluster II)

Is the corrolate macrocycle innocent or noninnocent? Magnetic susceptibility, Mossbauer, H-1 NMR, and DFT investigations of chloro- and phenyliron corrolates

an attempt to determine the electron configuration of (anion)iron corrolates, i.e., whether they are S = 1 Fe(IV)-corrolate(3(-)) or S = (3)/(2) Fe(III)-corrolate(2(-.)), with antiferromagnetic coupling between the iron and macrocycle electrons to yield overall S = 1, two axial ligand complexes of an iron octaalkylcorrolate have been studied by temperature-dependent magnetic susceptibility, magnetic Mossbauer, and H-1 NMR spectroscopy, and the results have been compared to those determined on the basis of spin-unrestricted DFT calculations. Magnetic susceptibility measurements indicate the presence of a noninnocent macrocycle (corrolate (2-(.))) for the chloroiron corrolate, with strong antiferromagnetic coupling to the S = (3)/(2) Fe(III) center, while those for the phenyliron corrolate are not conclusive as to the electron configuration. Temperature- and field-dependent Mossbauer spectroscopic investigations of these two complexes yielded spectra that could be simulated with either electron configuration, except that the isomer shift of the phenyliron complex is -0.10 mm/s while that of the chloroiron complex is +0.21 mm/s, suggesting that the iron in the former is Fe(IV) while in the latter it is Fe(III). 1H NMR spectroscopic studies of both axial ligand complexes show large negative spin density at the meso carbons, with those of the chloroiron complex (Cal, S.; Walker, F. A.; Licoccia, S. Inorg. Chem. 2000, 39, 3466) being roughly four times larger than those of the phenyliron complex. The temperature dependence of the proton chemical shifts of the phenyliron complex is strictly linear. DFT calculations are consistent with the chloroiron complex being formulated as S-1 = (3)/(2) Fe(III)-corrolate (2(-.)) S-2 = (1)/(2), with negative spin density at all nitrogens and meso carbons, and a net spin density of -0.79 on the corrolate ring and positive spin density (+0.17) on the chloride ion and +2.58 on the iron. In contrast, the phenyliron complex is best formulated as S = I Fe(IV)-corrolate (3-), but again with negative spin density at all nitrogens and meso carbons of the macrocycle, yet with the net spin density on the corrolate ring being virtually zero; the phenyl carbanion carbon has relatively large negative spin density of -0.15 and the iron +2.05. On the basis of all of the results, we conclude that in both the chloroiron and phenyliron complexes the corrolate ring is noninnocent, in the chloroiron complex to a much larger extent than in the phenyliron complex

Electronic structure of linear thiophenolate-bridged heterotrinuclear complexes [LFeMFeL]n(+) (M = Cr, Co, Fe; n = 1-3): Localized vs delocalized models

Glaser T, Beissel T, Bill E, et al. Electronic structure of linear thiophenolate-bridged heterotrinuclear complexes [LFeMFeL]n(+) (M = Cr, Co, Fe; n = 1-3): Localized vs delocalized models. Journal of the American Chemical Society. 1999;121(10):2193-2208.The reaction of mononuclear [LFeIII] where L represents the trianionic ligand 1,4,7-tris(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclononane with CrSO4. 5H2O, CoCl2. 6H2O, or Fe(BF4)2. 6H2O and subsequent oxidation with ferrocenium hexafluorophosphate or NO(BF4) or reduction with [(tmcn)Mo(CO)3] (tmcn = 1,4,7-trimethyl-1,4,7-triazacyclononane) produced an isostructural series of [LFeMFeL]n+ complexes, the following salts of which were isolated as crystalline solids: (i) [LFeCrFeL](PF6)n with n = 1 (1a), n = 2 (1b), and n = 3 (1c); (ii) [LFeCoFeL]Xn with X = BPh4 and n = 2 (2b) and X = PF6 and n = 3 (2c); (iii) [LFeFeFeL](BPh4)n with n = 2 (3b) and n = 3 (3c). All compounds contain Linear trinuclear cations (face-sharing octahedral) with an N3Fe(mu-SR)3M(mu-SR)3FeN3 core structure. The electron structure of all complexes has been studied by Fe and M K-edge X-ray absorption near edge structure (XANES), UV-vis, and EPR spectroscopy, variable-temperature, variable-field susceptibility measurements, and Mossbauer spectroscopy (in zero and applied field). The following electronic structures have been established: (1a) FeII(ls)CrIIIFeII(ls) (ls = low-spin) with a spin ground state of St = 3/2; (1c) FeIII(ls)CrIIIFeIII(ls) with an St = 1/2 ground state; (2c) FeIII(ls)CoIII(ls)FeIII(ls) with an St = 1 ground state; (3c) FeIII(ls)FeIII(ls)FeIII(ls) with an St = 1/2 ground state. For 1b (St = 2) it is found that the two iron ions are spectroscopically equivalent (Fe2.5) and, therefore, the excess electron is delocalized (class III): [LFe2.5CrIIIFe2.5L]2+. For 2b clearly two different iron sites prevail at low temperatures (4.2 K); at higher temperatures (>200 K) they become equivalent on the Mossbauer time scale. Thus, 2b is class II with temperature-dependent electron hopping between the FeII and FeIII ions. 3b is again fully delocalized (class III) with an St = 1 ground state; the excess electron is delocalized over all three iron sites. The electronic structure of all complexes is discussed in terms of double exchange and superexchange mechanisms

The Fe-only nitrogenase from Rhodobacter capsulatus: identification of the cofactor, an unusual, high-nuclearity iron-sulfur cluster, by FeK-edge EXAFS and Fe-57 Mossbauer spectroscopy

Krahn E, Weiss BJR, Krockel M, et al. The Fe-only nitrogenase from Rhodobacter capsulatus: identification of the cofactor, an unusual, high-nuclearity iron-sulfur cluster, by FeK-edge EXAFS and Fe-57 Mossbauer spectroscopy. JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY. 2002;7(1-2):37-45.Samples of the dithionite-reduced FeFe protein (the dinitrogenase component of the Fe-only nitrogenase) from Rhodobacter capsulatus have been investigated by Fe-57 Mossbauer 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 Angstrom and 2.63 Angstrom, respectively. A significant contribution to the Fe EXAFS must be assigned to an Fe backscatterer shell at 3.68 Angstrom, 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 (FeFe)-Fe-... interactions at 2.92 Angstrom and 4.05 Angstrom clearly indicate that the principal geometry, of the P cluster is also conserved. Mossbauer spectra of Fe-57-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