Characterization of an Autoreduction Pathway for the [Fe4S4]3+ Cluster of Mutant Chromatium vinosum High-Potential Iron Proteins. Site-Directed Mutagenesis Studies To Probe the Role of Phenylalanine 66 in Defining the Stability of the [Fe4S4] Center Provide Evidence for Oxidative Degradation via a [Fe3S4] Cluster

Bian, Shumin; Hemann, Craig; Hille, Russ; Cowan, J. A.

doi:10.1021/bi961658l

Cited by 25 publications

(32 citation statements)

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“…The oxidized Trx-Fe 4 S 4 cluster is only transiently stable, as evidenced by rapid bleaching of the visible absorbance upon ferricyanide addition, and the disappearance of the g avg Ͼ 2 EPR signal upon prolonged incubation at 4°C. Such cluster degradation has been reported for HiPIPs and is believed to produce reducing equivalents (Fe 2ϩ , S 2Ϫ , and CysSH) (41). While these factors complicate the determination of a redox potential, our observation of complete oxidation upon addition of Ͼ7 equivalents of potassium ferricyanide, and incomplete oxidation at lower concentrations allows us to place a lower bound of ϩ300 mV vs. NHE on the redox potential of Trx-Fe 4 4 S 4 ] cluster in bacterial Fds is Cys-X 2 -Cys-X 2 -Cys with a more remote Cys residue supplying the fourth ligating sulfur center.…”

Section: Discussionmentioning

confidence: 52%

The rational design and construction of a cuboidal iron–sulfur protein

Coldren

Hellinga²,

Caradonna³

1997

Proc. Natl. Acad. Sci. U.S.A.

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Metalloproteins offer a particularly interesting target for the design of function, because the biological chemistry of metals is extraordinarily rich. Cuboidal [Fe 4 S 4 ] clusters are among the most common electron-transfer centers found in biology (1). These clusters act as either simple soluble electron-transfer agents, membrane-bound components of electron-transfer chains, or parts of the electron reservoir found in complex metalloenzymes in plants, animals, and bacteria. In addition to being intimately involved in electron-transport systems and in the metabolism of carbon, oxygen, hydrogen, sulfur, and nitrogen, studies have suggested that these centers also exhibit gene regulatory, catalytic, and structural functions, and have been found as part of a morphogenetic protein (1).The successful construction of a functional metal center requires that three sets of factors are taken into account: (i) the construction of a correctly folded protein, (ii) the coordination requirements of the metal, (iii) the modulation of the properties of the metal center and protein matrix to achieve the required control of reactivity. Thus far, metalloprotein designs have focused primarily on the first two factors (2-8). The redox properties of [Fe 4 S 4 ] centers, which are strongly dependent on the protein environment, offer a good system to explore the modulation of the metal center by the protein (1, 9). Cuboidal [Fe 4 S 4 ] proteins, which contain structurally equivalent metal clusters, are grouped in two classes, the ferredoxins (Fds) and the high-potential iron-sulfur proteins (HiPIPs couple (E o ϭ ϩ100 mV to ϩ450 mV). Although several high-resolution x-ray crystallographic structures of HiPIP systems as well as 4Fe-, 7Fe-, and 8Fe-Fds are available (1), the identification of those factors that determine the redox couple are just beginning to emerge. Conjectures have been presented that cluster-protein (amide NOH to cluster S 2Ϫ or cysteinyl S) hydrogen bonding (10), solvent accessibility of cluster, and electrostatic field gradients arising from the presence of acidic, basic, and hydrophobic residues in the vicinity of the [Fe 4 S 4 ] cluster, are important in determining the redox potential (9). Spectroscopic studies (11), ab inito calculations (12), and computer simulations (13, 14) support these factors as determinants of cluster redox potential. Studies of model systems (15), mutant iron-sulfur proteins (16,17), and interspecies variants (18) have also indicated the importance of the protein matrix and solvent. In addition, recent quantitative modeling simulations of the redox potentials of iron-sulfur proteins using the protein dipoles Langevin dipoles model have implicated the Coulombic interaction of the cluster with the protein atom charges as a major determinant of the variations in measured redox potentials (9). Specifically, the proximity and orientation of protein matrix dipoles arising from the parallel alignment of trans amide NOH and CAO bonds, which depends on the intrinsic structure of the polyp...

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Section: Discussionmentioning

confidence: 52%

The rational design and construction of a cuboidal iron–sulfur protein

Coldren

Hellinga²,

Caradonna³

1997

Proc. Natl. Acad. Sci. U.S.A.

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Section: The Reduction Potential (E 0 ) Of the [4fe-4s]mentioning

confidence: 99%

“…3ϩ/2ϩ, 2ϩ/ϩ, or ϩ/0) or by modulation of the reduction potential (E 0 Ј) of a particular redox couple (7-19). Thus, high potential iron proteins have 3ϩ/2ϩ E 0 Ј ranging from 90 to 450 mV (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)20), while ferredoxins that contain structurally indistinguishable 2ϩ/ϩ clusters have E 0 Ј ranging from Ϫ280 to Ϫ715 mV in different native proteins (10, 21).Both experimental and theoretical research has been directed toward understanding how the polypeptide surrounding the cluster controls the reduction potential. Factors that have been proposed as being important include (a) solvent exposure of the cluster, (b) specific hydrogen bonding networks especially NH-S bonds, (c) the proximity and orientation of protein backbone and side chain dipoles, and/or (d) the proximity of charged residues to the cluster (7,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).…”

mentioning

confidence: 99%

“…[4Fe-4S] clusters with four cysteine ligands), proteins can control reactivity further by stabilizing a particular redox couple (e.g. 3ϩ/2ϩ, 2ϩ/ϩ, or ϩ/0) or by modulation of the reduction potential (E 0 Ј) of a particular redox couple (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Thus, high potential iron proteins have 3ϩ/2ϩ E 0 Ј ranging from 90 to 450 mV (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)20), while ferredoxins that contain structurally indistinguishable [4Fe-4S] 2ϩ/ϩ clusters have E 0 Ј ranging from Ϫ280 to Ϫ715 mV in different native proteins (10,21).…”

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Azotobacter vinelandii Ferredoxin I

Chen¹,

Jung²,

Bonagura³

et al. 2002

Journal of Biological Chemistry

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The reduction potential (E 0 ) of the [4Fe-4S]2؉/؉ cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is ϳ200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences in E 0 do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the Most iron-sulfur ([Fe-S]) proteins are intimately involved in essential electron transfer reactions, including membranebound electron transport systems; their functions, however, are not restricted to electron transfer and include direct catalysis of hydration/dehydration reactions, O 2 and iron sensing, regulation of gene expression, iron storage, metal cluster assembly, and the generation and stabilization of radical intermediates (for reviews, see Refs. 1-6). To meet these diverse functions, individual proteins have increased the variety of [Fe-S] cluster structures by adding or subtracting iron and sulfide atoms to vary the cluster type, or by introducing noncysteine ligands. Even after adopting a specific cluster type (e.g. [4Fe-4S] clusters with four cysteine ligands), proteins can control reactivity further by stabilizing a particular redox couple (e.g. 3ϩ/2ϩ, 2ϩ/ϩ, or ϩ/0) or by modulation of the reduction potential (E 0 Ј) of a particular redox couple (7-19). Thus, high potential iron proteins have 3ϩ/2ϩ E 0 Ј ranging from 90 to 450 mV (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)20), while ferredoxins that contain structurally indistinguishable 2ϩ/ϩ clusters have E 0 Ј ranging from Ϫ280 to Ϫ715 mV in different native proteins (10, 21).Both experimental and theoretical research has been directed toward understanding how the polypeptide surrounding the cluster controls the reduction potential. Factors that have been proposed as being important include (a) solvent exposure of the cluster, (b) specific hydrogen bonding networks especially NH-S bonds, (c) the proximity and orientation of protein backbone and side chain dipoles, and/or (d) the proximity of charged residues to the cluster (7,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). This study concerns protein control of the reduction potential of a 2ϩ/ϩ cluster that is ligated via a typical CXXCXXC motif with one remote Cys ligand.Previous studies in this area have focused on comparing the environments of the clusters in structurally characterized proteins. 2ϩ/ϩ cluster of Azotobacter vinelandii ferredoxin I (AvFdI) 1 has a low E 0 Ј of about Ϫ620 mV (pH 7.0) (36), whereas the analogous clusters in Peptostreptococcus asaccharolyticus ferredoxin (PaFd) and Clostridium acidiurici ferredoxin have E 0 Ј of about Ϫ430 mV (not pH-dependent) (37). Comparison of the structures of these proteins showed that the peptide folding around the analogous clusters is highly conserved with respect to the location of the four Cys ligands, the Cys dihedral angles, and the eight amide groups hydr...

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“…39,40 However, these studies could not differentiate between IscU functioning as an iron or an iron-sulfur cluster delivery protein. Given our long-standing interest in Fe-S biochemistry and mechanisms of cluster assembly and disassembly, [41][42][43] we initiated a program to compare and contrast the properties of IscU from a variety of sources, including human (Hs ISU), yeast (Schizosaccharomyces pombe, Sp ISU), and a hyperthermophilic bacterium (Thermotoga maritima, Tm IscU). In agreement with Dean and colleagues, 27 we found that IscU coordinates one reductively labile [2Fe-2S] 2+ cluster per monomeric subunit that is stabilized by substitution of a highly conserved aspartate (D37 for Hs ISU and Sp ISU, and D40 for Tm IscU), as judged by Mössbauer, EXAFS, EPR, and UV-vis spectroscopies.…”

Section: Cast Of Characters In Iron-sulfur Cluster Biosynthesismentioning

confidence: 99%

Iron Sulfur Cluster Biosynthesis

Cowan

2009

ACS Symposium Series

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Iron-sulfur clusters are among the most complex metal-containing prosthetic centers in biology. Most if not all of the proteins involved in the biosynthesis of "simple" Fe-S clusters have been identified. The structural and functional chemistry of these proteins has been the subject of intense research efforts, and many of the key details are now understood in structural and mechanistic detail. The fact that Fe-S cluster-binding proteins can be reconstituted in vitro with no accessory proteins provides an important indicator of the intracellular roles for many proteins on the Fe-S cluster assembly pathway. Indeed, such proteins are more correctly viewed as carrier proteins, rather than as catalysts for the reaction, that both avoid the toxicity associated with free iron and sulfide and allow delivery at lower intracellular concentrations of these species. The IscU (or ISU) family of proteins serves a key role as scaffolding proteins on which [2Fe-2S] building blocks are assembled prior to transfer to final apo target proteins. IscU in particular exhibits highly unusual conformational flexibility that appears critical to its function.

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Cited by 25 publications

References 55 publications

The rational design and construction of a cuboidal iron–sulfur protein

The rational design and construction of a cuboidal iron–sulfur protein

Azotobacter vinelandii Ferredoxin I

Iron Sulfur Cluster Biosynthesis

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