Fe—S蛋白(MoFe、Fe蛋白)中酸不稳定硫的测定

一、原理 Fe—S蛋白中酸不稳定硫的测定是在碱性条件下,使Fe—S蛋白与Zn(AC),作用所形成的ZnS,在酸性条件下,用对-氨基二甲基苯胺偶联并与FeCl_3作用生成次甲基兰。在670nm处测定光密度。保险粉的干扰可借助于取蛋白样品体积少,浓度高或将蛋白样品见氧等办法来消除。反应式:     

Proton magnetic resonance studies of 7 Fe ferredoxins: Lower potential of the [4 Fe–4 S] redox center than the [3 Fe–3 S] cluster

Two types of iron-sulfur clusters, [3 Fe–3 S] and [4 Fe–4 S], were identified by ¹H-NMR in ferredoxins from Thermus thermophilus, Mycobacterium smegmatis and Pseudomonas ovalis. The [4 Fe–4 S] clusters always showed the redox couples which had potentials lower than that of the [3 Fe–3 S] clusters.     

Glutathione-complexed [2Fe-2S] clusters function in Fe–S cluster storage and trafficking

Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS₄) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe–S cluster transfer reactions. UV–vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS₄) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS₄) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe–S cluster proteins, thereby supporting a possible physiological role for such centers.     

Structure of a [2Fe–2S] ferredoxin from Rhodobacter capsulatus likely involved in Fe–S cluster biogenesis and conformational changes observed upon reduction

FdVI from Rhodobacter capsulatus is structurally related to a group of [2Fe–2S] ferredoxins involved in iron–sulfur cluster biosynthesis. Comparative genomics suggested that FdVI and orthologs found in α-Proteobacteria are involved in this process. Here, the crystal structure of FdVI has been determined for both the oxidized and the reduced protein. The [2Fe–2S] cluster lies 6 Å below the protein surface in a hydrophobic pocket without access to the solvent. This particular cluster environment might explain why the FdVI midpoint redox potential (?306 mV at pH 8.0) did not show temperature or ionic strength dependence. Besides the four cysteines that bind the cluster, FdVI features an extra cysteine which is located close to the S1 atom of the cluster and is oriented in a position such that its thiol group points towards the solvent. Upon reduction, the general fold of the polypeptide chain was almost unchanged. The [2Fe–2S] cluster underwent a conformational change from a planar to a distorted lozenge. In the vicinity of the cluster, the side chain of Met24 was rotated by 180°, bringing its S atom within hydrogen-bonding distance of the S2 atom of the cluster. The reduced molecule also featured a higher content of bound water molecules, and more extensive hydrogen-bonding networks compared with the oxidized molecule. The unique conformational changes observed in FdVI upon reduction are discussed in the light of structural studies performed on related ferredoxins.     

Reactions of thiols and selenols with the FeMoS cluster [Fe(MoS4)2]3−

The known complex [Et₄N]₃[Fe(MoS₄)₂] has been shown by EPR and visible spectral studies to react with both thiophenol and selenophenol. The reaction results in a change in the characteristic S=3/2 EPR spectrum of this species from a complex rhombic pattern to one of a very simple axial appearance. Although this effect is similar to that observed for reaction of these species with the iron- molybdenum cofactor of nitrogenase, a moiety known to consist of a FeMoS cluster species, the large excesses of reagents and the long reaction times required for complete formation of product indicate that these reactions are of questionable direct relevance to the biological system. The reaction corresponding to the EPR spectral change from rhombic to axial in the [Fe(MoS₄)₂]³⁻/PhSeH system has also been partially characterized by product isolation which indicates that attack by selenol of the two terminal MoS₂ moieties in the starting material has occurred.     

HdrC2 from Acidithiobacillus ferrooxidans Owns Two Iron–Sulfur Binding Motifs But Binds Only One Variable Cluster Between [4Fe–4S] and [3Fe–4S]

The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was suggested to own novel features that act in reverse to convert the sulfane sulfur of GS _n H species (n > 1) into sulfite in sulfur oxidation. The HdrC subunit is potentially encoded by two different highly upregulated genes sharing only 29 % identity in A. ferrooxidans grown in sulfur-containing medium, which were named as HdrC1 and HdrC2, respectively and had been confirmed to contain iron–sulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe–4S] cluster by mutations. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here, we report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans. This HdrC2 had two identical motifs (Cx₂Cx₂Cx₃C) containing total of eight cysteine residues potentially for iron–sulfur cluster binding. This purified HdrC2 was exhibited to contain one variable cluster converted between [4Fe–4S] and [3Fe–4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of these eight residues further confirmed that the HdrC2 in reduction with Fe²⁺ condition loaded only one [4Fe–4S]⁺ with spin S = 1/2 ligated by the residues of Cys73, Cys109, Cys112, and Cys115; the HdrC2 in natural aeration condition lost the Fe atom ligated by the residue of Cys73 and loaded only one [3Fe–4S]⁰ with spin S = 0; the HdrC2 in oxidation condition loaded only one [3Fe–4S]⁺ with spin S = 1/2. Molecular modeling results were also in line with the experiment results.     

Preliminary X-ray diffraction studies on a [4Fe4S] ferredoxin from Bacillus thermoproteolyticus

A [4Fe4S] ferredoxin from Bacillus thermoproteolyticus has been crystallized. The space group is P1 with two molecules in the unit cell, with the dimensions $\text{a = 32.96}\text{A}\text{?}\text{, b = 37.83}\text{A}\text{?}\text{, c = 39.82}\text{A}\text{?}\text{, α = 118.1 °, β = 104.2 °}\text{and}\text{γ = 89.7 °}$. The Bijvoet-difference Patterson map of the native crystal shows up a prominent peak of [4Fe4S] cluster.     

Conformational Change of the Rieske [2Fe–2S] Protein inCytochrome bc 1 Complex

Structures of mitochondrial bc ₁ complex have been reported based on four different crystalforms by three different groups. In these structures, the extrinsic domain of the Rieske [2Fe–2S]protein, surprisingly, appeared at three different positions: the c ₁ position, where the [2Fe–2S]cluster exists in close proximity to the heme c ₁; the b position, where the [2Fe–2S] clusterexist in close proximity to the cytochrome b; and the intermediate position where the[2Fe–2S] cluster exists in between c ₁ and b positions. The conformational changes betweenthese three positions can be explained by a combination of two rotations; (1) a rotation of theentire extrinsic domain and (2) a relative rotation between the cluster-binding fold and thebase fold within the extrinsic domain. The hydroquinone oxidation and the electron bifurcationmechanism at the Q_P binding pocket of the bc ₁ complex is well explained using theseconformational changes of the Rieske [2Fe–2S] protein.     

Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe–4S] cluster

Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe₃S₄ cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdII_int) with four ¹H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdII_ox and FdII_int were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same ¹H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state (DgFdII_ox) was used here for the assignment of the same three ligands in the intermediate state (DgFdII_int). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm⁻¹ and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdII_int. This study, together with the spin delocalization mechanism proposed here for DgFdII_int, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdII_ox and DgFdII_int.     

EPR analysis of multiple forms of [4Fe–4S]3+ clusters in HiPIPs

The electron paramagnetic resonance (EPR) spectrum from the [4Fe–4S]³⁺ cluster in several high-potential iron–sulfur proteins (HiPIPs) is complex: it is not the pattern of a single, isolated S=1/2 system. Multifrequency EPR from 9 to 130 GHz reveals that the apparent peak positions (g values) are frequency-independent: the spectrum is dominated by the Zeeman interaction plus g-strain broadening. The spectra taken at frequencies above the X-band are increasingly sensitive to rapid-passage effects; therefore, the X-band data, which are slightly additionally broadened by dipolar interaction, were used for quantitative spectral analysis. For a single geometrical [4Fe–4S]³⁺ structure the (Fe–Fe)⁵⁺ mixed-valence dimer can be assigned in six different ways to a pair of iron ions, and this defines six valence isomers. Systematic multicomponent g-strain simulation shows that the [4Fe–4S]³⁺ paramagnets in seven HiPIPs from different bacteria each consist of three to four discernible species, and these are assigned to valence isomers of the clusters. This interpretation builds on previous EPR analyzes of [4Fe–4S]³⁺ model compounds, and it constitutes a high-resolution extension of the current literature model, proposed from paramagnetic NMR studies.     

Comparison and characterization of the [Fe4S4]2+/3+ centre in the wild-type and C77S mutated HiPIPs from Chromatium vinosum monitored by Mössbauer, 57Fe ENDOR and EPR spectroscopies

M?ssbauer, 57Fe ENDOR, CW and pulsed EPR experiments were performed on the reduced and the oxidized high-potential iron proteins (HiPIPs) of the wild type (WT) and the C77S mutant from Chromatium vinosum. The EPR spectra of the oxidized WT and mutant show three species respectively having nearly the same g-values but strongly changed spectral contributions. Relaxation times were estimated for oxidized WT and mutant at T = 5 K with pulsed EPR. A-tensor components of both iron pairs were obtained by 57Fe ENDOR, proving a similar magnetic structure for the WT and the mutant. Electronic relaxation has to be taken into account at T = 5 K in native and mutated oxidized HiPIPs to achieve agreement between M?ssbauer and 57Fe ENDOR spectroscopies. The M?ssbauer spectroscopy shows that the oxidized cluster contains a pure ferric and a mixed-valence iron pair coupled antiparallel. While all cluster irons from reduced C. vinosum WT are indistinguishable in the M?ssbauer spectrum, the reduced C77S mutant shows a non-equivalence between the serine-bound and the three cysteine-ligated iron ions. The M?ssbauer parameters confirm a loss of the covalent character of the iron bond when S is replaced by O and indicate a shift of the cluster's electron cloud towards the serine. M?ssbauer spectra of the oxidized mutant can be simulated with two models: model I introduces a single electronic isomer with the serine always ligated to a ferric iron. Model II assumes two equally populated electronic isomers with the serine ligated to a ferric iron and a mixed-valence iron, respectively. The latter model is in better agreement with EPR and NMR.     

Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the [Fe4S4] cluster in the high-potential iron protein I Ectothiorhodospira halophila

4 S₄]^3 + and the reduced [Fe₄S₄]^2 + clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and ⁵⁷Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe₄S₄]^3 + clusters, the M?ssbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe^3 + atoms couple to the “ferric-ferric” pair, and one Fe^2 + and one Fe^3 + atom give the “ferric-ferrous pair”. For the simulation of the M?ssbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by ⁵⁷Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of M?ssbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical M?ssbauer spectra. Spin-spin and spin-lattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the M?ssbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the M?ssbauer spectrum, indicating a mixed-valence state of Fe^2.5 + for each. Received: 15 February 1999 / Accepted: 31 August 1999     

Stabilities of cubane type [Fe₄S₄(SR)₄](2-) clusters in partially aqueous media

The stability of cubane-type [Fe₄S₄(SR)₄]²⁻ clusters in mixed organic/aqueous solvents was examined as an initial step in the development of stable water-soluble cluster compounds possibly suitable for reconstitution of scaffold proteins in protein biosynthesis. The research involves primarily spectrophotometric assessment of stability in 20-80% Me₂SO/aqueous media (v/v), from which it was found that conventional clusters tend to be stable for up to 12 h in 60% Me₂SO but are much less stable at higher aqueous content. α-Cyclodextrin mono- and dithioesters and thiols were prepared as ligand precursors for cluster binding, which was demonstrated by spectroscopic methods. A potentially bidentate cyclodextrin dithiolate was found to be relatively effective for cluster stabilization in 40% Me₂SO, suggesting (together with earlier results) that other exceptionally large thiolate ligands may promote cluster stability in aqueous media.     

O.S.S浅谈

Oncology Support System是荷兰PHI LIPS公司O.S.S的全称。这是一台在Treatment Planning System(T.P.S治疗计划系统)基础上发展而成、辅助CT诊断、设计三维放射治疗的专用计算机系统。我院放射治疗科于84年完成了SL 75—20医用电子直线加速器、模拟定位机、O.S.S等设备的安装