Four-iron Cluster Research Articles

Differential Protonation at the Catalytic Six-Iron Cofactor of [FeFe]-Hydrogenases Revealed by 57Fe Nuclear Resonance X-ray Scattering and Quantum Mechanics/Molecular Mechanics Analyses.

[FeFe]-hydrogenases are efficient biological hydrogen conversion catalysts and blueprints for technological fuel production. The relations between substrate interactions and electron/proton transfer events at their unique six-iron cofactor (H-cluster) need to be elucidated. The H-cluster comprises a four-iron cluster, [4Fe4S], linked to a diiron complex, [FeFe]. We combined 57Fe-specific X-ray nuclear resonance scattering experiments (NFS, nuclear forward scattering; NRVS, nuclear resonance vibrational spectroscopy) with quantum-mechanics/molecular-mechanics computations to study the [FeFe]-hydrogenase HYDA1 from a green alga. Selective 57Fe labeling at only [4Fe4S] or [FeFe], or at both subcomplexes was achieved by protein expression with a 57Fe salt and in vitro maturation with a synthetic diiron site precursor containing 57Fe. H-cluster states were populated under infrared spectroscopy control. NRVS spectral analyses facilitated assignment of the vibrational modes of the cofactor species. This approach revealed the H-cluster structure of the oxidized state (Hox) with a bridging carbon monoxide at [FeFe] and ligand rearrangement in the CO-inhibited state (Hox-CO). Protonation at a cysteine ligand of [4Fe4S] in the oxidized state occurring at low pH (HoxH) was indicated, in contrast to bridging hydride binding at [FeFe] in a one-electron reduced state (Hred). These findings are direct evidence for differential protonation either at the four-iron or diiron subcomplex of the H-cluster. NFS time-traces provided Mössbauer parameters such as the quadrupole splitting energy, which differ among cofactor states, thereby supporting selective protonation at either subcomplex. In combination with data for reduced states showing similar [4Fe4S] protonation as HoxH without (Hred') or with (Hhyd) a terminal hydride at [FeFe], our results imply that coordination geometry dynamics at the diiron site and proton-coupled electron transfer to either the four-iron or the diiron subcomplex discriminate catalytic and regulatory functions of [FeFe]-hydrogenases. We support a reaction cycle avoiding diiron site geometry changes during rapid H2 turnover.

Paramagnetic NMR Analysis of the Seven‐Iron Ferredoxin from the Hyperthermoacidophilic Archaeon Desulfurolobus ambivalens Reveals Structural Similarity to other Dicluster Ferredoxins

The seven-iron ferredoxin from the hyperthermophilic archaeon Desulfurolobus ambivalens has been investigated by one-dimensional and two-dimensional 1H-NMR in its oxidized and dithionite-reduced states. All iron atoms of both the three-iron and the four-iron cluster are bound to cysteine residues whose hyperfine-shifted resonances were characterized. The pattern of these resonances is similar to those from three-iron, four-iron and eight-iron ferredoxins previously described in the literature, but the four-iron cluster has a shift pattern different from that in other seven-iron proteins. A second set of hyperfine-shifted resonances clearly indicates sample heterogeneity, which possibly involves the four-iron cluster. The observation of interresidue NOEs between two different cysteine residues proves the existence of close spatial proximity of the two clusters in D. ambivalens ferredoxin and therefore indicates structural homology to other dicluster ferredoxins. Moreover, this feature is crucial for the sequence-specific assignment of the hyperfine-shifted resonances. The C alpha-C beta-S-Fe dihedral angles of the cysteine residues coordinating the four-iron cluster could be estimated, and the electronic structure of the three-iron cluster is discussed.

1H NMR investigation of the electronic and molecular structure of the four-iron cluster ferredoxin from the hyperthermophile Pyrococcus furiosus. Identification of Asp 14 as a cluster ligand in each of the four redox states.

The molecular and electronic structure of the four-iron cluster of the ferredoxin (Fd) from the hyperthermophilic archaeon, Pyrococcus furiosus, Pf (which has only three Cys in the cluster binding consensus sequence), has been investigated by 1H NMR in order to determine the identity of the noncysteinyl cluster ligand in each of the four redox states [Gorst, C. M., Zhou, Z. H., Ma, K., Teng, Q., Howard, J. B., Adams, M. W., & La Mar, G. N. (1995) Biochemistry 34, 8788-8795], and to characterize the electron spin ground state for the reduced cluster which at 10 K exhibits an unusual predominant S = 3/2 ground state [Conover, R. C., Kowal, A. T., Fu, W., Park, J. -B., Aono, S., Adams, M. W. W., & Johnson, M. K. (1990) J. Biol. Chem. 265, 8533-8541]. It is demonstrated that a combination of 1D and 2D NMR tailored to relaxed resonances allows the location of four hyperfine shifted and paramagnetically relaxed spin systems which dictates that all four cluster ligands are amino acid side chains, rather than a solvent water/hydroxide at the unique non-Cys ligation site. Three of the ligands could be sequence-specifically assigned to the three Cys residues (positions 11, 17, and 56) in the consensus sequence for cluster binding, hence identifying the fourth ligand as Asp 14. It is concluded that the identification of Asp ligation to a 4Fe cluster is readily achieved in the reduced, but not in the oxidized cluster of Fd. Analysis of the relaxation properties and pattern of the hyperfine shifts in Pf Fd reveals very strong similarities to other Fds with S = 1/2 ground states, leading to the conclusion that the S = 3/2 ground state is not detected in solution at ambient temperatures, and this in independent of the redox state of the two remaining Cys residues in the protein (positions 21 and 48). However, the electron self-exchange rate for 4Fe Pf Fd is significantly slower than for other 4Fe Fd with complete Cys ligation. Changes in the pattern of hyperfine shifts between oxidized and reduced clusters for the four ligands in Pf Fd reveal that the most significant variation occurs for the Asp 14 orientation, suggesting that the altered Asp orientation may "gate" the electron transfer.

Two-dimensional NMR investigation of iron-sulfur cluster electronic and molecular structure of oxidized Clostridium pasteurianum ferredoxin. Interpretability of contact shifts in terms of cysteine orientation.

A two-dimensional NMR study has been carried out on the four-iron clusters of a bacterial oxidized ferredoxin for the purpose of investigating the relationship between contact shift patterns and the orientation of the individual coordinated cysteines. The ferredoxin from Clostridium pasteurianum, CpFdox, was selected because of its extensive sequence homology, and likely close structural similarity, to the crystallographically characterized ferredoxin from Peptococcus aerogenes, Pa Fdox (Adman, E.T., Sieker, L.C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996). Rapid data collection rates with minimal but adequate acquisition time allowed the detection of numerous CpFdox cross-peaks from the contact-shifted and strongly relaxed coordinated cysteinyl C beta H protons in the resolved 10-20 ppm window. Relatively strong magnitude COSY cross peaks from the resolved eight cysteinyl C beta H resonance unambiguously locate the geminal C beta H partner for each residue; weaker cross-peaks locate the C alpha Hs from three of the residues. The geminal nature of the magnitude-COSY detected partners to the resolved C beta H peaks is confirmed by strong NOESY cross-peaks. The NOESY spectra, moreover, assign an additional two cysteinyl C alpha H resonances. The present results confirm some previous one-dimensional NOE assignments, revise others, and locate resonances previously undetected (Bertini, I., Briganti, F., Luchinat, C., and Scozzafara, A. (1990) Inorg. Chem. 29, 1874-1880). A striking pairwise pseudo-symmetry in cysteinyl contact shift patterns is observed which is attributed to the previously recognized pseudo-symmetry in the crystal of PaFdox. A detailed analysis of the structural/electronic determinants of the coordinated cysteine C beta H contact shift pattern is made, and the NMR data necessary for unique interpretation are identified. It is shown that analysis of the relaxation properties of cysteine beta-methylene protons provides the stereospecific assignments necessary for comparison of shift ratios with crystallographic structural data. The available structural data on PaFdox (Backes, G., Mino, Y., Loehr, T., Meyer, T., Cusanovich, M., Sweeney, W., Adman, E., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 13, 2055-2064) are qualitatively but not quantitatively consistent with the observed cysteinyl contact shift pattern, with the NMR data reflecting more asymmetry than previous studies. A tentative assignment of a single pair of symmetry-related cysteines is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)

ChemInform Abstract: Systematics of the Formation of Π‐CO Ligands in Four‐Iron Clusters.

AbstractDie Lewissäure‐Liganden [AuPR3]+, [CuL]+ und [HgMe]+ reagieren mit dem tetraedrischen Fe‐Clusterion [Fe4(CO),3]2′.

Solution equilibria between carbon-bonded carbonyl and .PI.-carbonyl ligands in four-iron clusters and cleavage of the .PI.-carbonyl ligand

Solution equilibria between carbon-bonded carbonyl and .PI.-carbonyl ligands in four-iron clusters and cleavage of the .PI.-carbonyl ligand

A study of one of the iron-sulphur clusters in oxidized hydrogenase from Megasphaera elsdenii by magnetic-circular-dichroism spectroscopy.

The m.c.d. spectrum of the oxidized state of hydrogenase from Megasphaera elsdenii has been measured at liquid-helium temperatures. This oxidation state of the enzyme displays a characteristic rhombic e.p.r. signal with g-values of 2.101, 2.052 and 2.005 assigned previously to a [4Fe-4S]3+ cluster as in oxidized HiPIP (high-potential iron-sulphur protein) [Van Dijk, Grande, Mayhew & Veeger (1980) Eur. J. Biochem. 107, 251-261]. The low-temperature m.c.d. spectrum shows no features attributable to an oxidized four-iron cluster of the HiPIP type, but does reveal broad, positive peaks at 460 and 730 nm, which magnetize in a manner untypical of a spin S = 1/2 cluster with g-values close to 2. The m.c.d. spectrum is most closely similar to that of dye-oxidized P-clusters known in the enzyme nitrogenase. It is therefore proposed that the rhombic e.p.r. spectrum at a g-value close to 2 arises from an m.c.d.-silent radical species that may be related chemically to the cysteine persulphide species, RS-S., recently found in the hexacyanoferrate-oxidized seven-iron ferredoxin of Azotobacter vinelandii [Morgan, Stephens, Devlin, Stout, Melis & Burgess (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1931-1935].

Intermediates in the proton-induced reduction of CO in iron clusters

After an induction period, solutions of HFe4(CH)(CO)12 in HSO3CF3 slowly produce methane at room temperature. The predominant iron-containing species throughout the initial stages of the reaction is H2Fe4-(η2-CH)(CO)12+, for which NMR studies indicate diprotonation of the iron framework and very slow exchange between one of the cluster protons and the CH proton. Although three-iron clusters such as Fe3(CH)(CO)10− can be recovered from the reaction mixture, this compound and a two-iron methylene compound, Fe2(CH2)(CO)8, do not yield significant quantities of methane under these reaction conditions. On the other hand, reducing agents eliminate the induction period and in some cases increase the rate of conversion of HFe4(CH)(CO)12 to methane. We conclude that reducing equivalents for the conversion of CH in HFe4(CH)(CO)12 to methane are likely to be produced by intermolecular electron transfer rather than successive oxidative cleavage of the four-iron cluster.

Nucleophilic attack and structural rearrangements in some iron carbonyl cluster carbides. Syntheses, structures, and reactions of the tetrahedral four-iron clusters [Fe4(CO)12(.mu.3-COMe)]- and [Fe4(CO)12(CMe)

Nucleophilic attack and structural rearrangements in some iron carbonyl cluster carbides. Syntheses, structures, and reactions of the tetrahedral four-iron clusters [Fe4(CO)12(.mu.3-COMe)]- and [Fe4(CO)12(CMe)

Mechanism of formation, spectrum and reactivity of half-reduced eight-iron Clostridium pasteurianum ferredoxin in pulse-radiolysis studies and the non-co-operativity of the four-iron clusters.

Reduction of fully oxidized Clostridium pasteurianum 8-Feox.,ox. ferredoxin by using pulse-radiolysis techniques yields the half-reduced species 8-Feox.,red. ferredoxin. The subsequent oxidation of 8-Feox.,red. ferredoxin with Co(NH3)5Cl2+ was studied. From a comparison with stopped-flow studies on the 2:1 Co(NH3)5Cl2+ oxidation of 8-Fered.,red. ferredoxin to the 8-Feox.,ox. form it is concluded that there is no redox co-operativity between the two 4-Fe centres in these reactions.

On the Nature of the Spin Coupling between the Iron-Sulfur Clusters in the Eight-Iron Ferredoxins

Abstract Evidence is presented to show that the two four-iron clusters in several 8-iron ferredoxins each have spin S = 1/2 in the reduced state and are spin-coupled to yield the complex EPR signals previously observed in the fully reduced states of these 8-iron ferredoxins.

“Super-reduction” of [formula omitted] high-potential iron-sulphur protein in the presence of dimethyl sulphoxide

In solutions containing more than 70% dimethyl sulphoxide (DMSO) by volume, the normal reduced state of Chromatium HIPIP can be further reduced by dithionite to give a form with a decreased absorption around 400 nm and an EPR signal similar to those of the reduced ferredoxins. These changes are reversible and the native HIPIP is recovered on removal of DMSO. These observations favour the hypothesis that the four-iron cluster in ferredoxins and HIPIP can exist in three oxidation states.