Redox properties of the iron‐sulfur clusters in activated Fe‐hydrogenase from <i>Desulfovibrio vulgaris</i> (Hildenborough)

Pierik, Antonio J.; Hagen, Wilfred R.; Redeker, Jan S.; Wolbert, R.B.G.; Boersma, Marelle G.; Verhagen, Marc F. J. M.; Grande, Hans J.; Veeger, C.; Mutsaers, P.H.A.; Sands, Richard H.; Dunham, William

doi:10.1111/j.1432-1033.1992.tb17261.x

Cited by 133 publications

(125 citation statements)

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“…This is especially critical for hydrogenase (proton reduction) and photosystem II (water oxidation), since their substrate is inseparable from the solvent. 157,378 Figure 39 illustrates the first method that can be used with PFV to access this invaluable information on the redox properties of the active site in the presence of substrate. 56 It is based on the examination of noncatalytic data recorded in the presence of substrate at fast scan rate; the second method below will deal with the interpretation of catalytic data.…”

Section: Active Site Chemistrymentioning

confidence: 99%

“…This is exemplified by DesulfoVribrio africanus pyruvate-ferredoxin oxidoreductase 413,414 and DesulfoVibrio Vulgaris Hildenborough FeFe-hydrogenase. 377,378 Other examples of redox-poise controlling catalytic efficiency include diheme peroxydases and galactose oxidase. Pre-steady-state measurements in PFV can easily resolve these potential-dependent processes.…”

Section: Redox-dependent Slow or Irreversible (In)activation Processesmentioning

confidence: 99%

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Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

2008

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Section: Active Site Chemistrymentioning

confidence: 99%

Section: Redox-dependent Slow or Irreversible (In)activation Processesmentioning

confidence: 99%

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

2008

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“…The following published methods were used: manipulation of DNA and PCR (34); transformation of yeast cells (35); enzyme activities of isopropylmalate isomerase and alcohol dehydrogenase (8,36); quantitative analysis of iron by the colorimetric chelator ferene (37) and of sulfur in purified Nar1 proteins (33); and radiolabeling and cell lysis of the Fe/S reporter proteins of interest (38,39). All experiments were repeated at least three times.…”

Section: Methodsmentioning

confidence: 99%

“…Purified, desalted hexahistidinyl-tagged wild-type Nar1 or mutant Nar1 proteins were used for EPR spectroscopy. Low-temperature X-band EPR spectra were recorded after anaerobic reduction of these proteins using sodium dithionite or 5 0 -deazaflavin and light with a Bruker ESP 300E cw spectrometer, equipped with an ESR910 helium flow cryostat (Oxford Instruments) (22,33).…”

Section: Methodsmentioning

confidence: 99%

Crucial Role of Conserved Cysteine Residues in the Assembly of Two Iron−Sulfur Clusters on the CIA Protein Nar1

et al. 2009

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Iron-sulfur (Fe/S) protein maturation in the eukaryotic cytosol and nucleus requires conserved components of the essential CIA machinery. The CIA protein Nar1 performs a specific function in transferring an Fe/S cluster that is assembled de novo on the Cfd1-Nbp35 scaffold to apoproteins. Here, we used systematic site-directed mutagenesis and a combination of in vitro and in vivo studies to show that Nar1 holds two Fe/S clusters at conserved N-and C-terminal cysteine motifs. A wealth of biochemical studies suggests that the assembly of these Fe/S clusters on Nar1 cannot be studied in Escherichia coli, as the recombinant protein does not contain the native Fe/S clusters. We therefore followed Fe/S cluster incorporation directly in yeast by a 55 Fe radiolabeling method in vivo, and we measured the functional consequences of Nar1 mutations in the assembly of cytosolic Fe/S proteins. We find that both Fe/S clusters are essential for Nar1 function and cell viability. Molecular modeling using a structurally but not functionally related bacterial iron-only hydrogenase as a template provided compelling structural explanations for our mutational data. The C-terminal Fe/S cluster is stably buried within Nar1, whereas the N-terminal one is exposed at the protein surface and hence may be more easily lost. Insertion of an Fe/S cluster into the C-terminal location depends on the N-terminal motif, suggesting the participation of the latter motif in the assembly process of the C-terminal cluster. The vicinity of the two Fe/S centers suggests a close functional cooperation during cytosolic Fe/S protein maturation.Iron-sulfur (Fe/S) 1 clusters are inorganic cofactors of many proteins found in nearly all prokaryotic and eukaryotic organisms. Fe/S proteins play important roles in many cellular processes, including electron transport, enzyme catalysis, and regulation of gene expression. Cells have developed dedicated systems for synthesizing Fe/S clusters and insert them into apoproteins. In Saccharomyces cerevisiae, three different machineries encompassing more than 20 proteins were identified with mature Fe/S proteins located in mitochondria, cytosol, and nucleus (1, 2). The mitochondrial ISC (Fe/S cluster) assembly machinery is needed for the biosynthesis of all cellular Fe/S proteins, whereas the ISC export and CIA machineries are specifically required for the biogenesis of cytosolic and nuclear Fe/S proteins. A central component of the ISC assembly system is the cysteine desulfurase complex Nfs1-Isd11 (3-5) which converts cysteine to alanine and transfers the sulfur to Isu1 and Isu2 which serve as scaffold proteins for Fe/S cluster assembly (6). Thereafter, a dedicated chaperone system is needed for the transfer of the Fe/S cluster from Isu1 and Isu2 to apoproteins. This system includes a mitochondrial Hsp70 protein (Ssq1), its cochaperone Jac1, and nucleotide exchange factor Mge1 (7). The mitochondrial ISC assembly machinery is also required for biogenesis of extra-mitochondrial Fe/S proteins. According to a working ...

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“…However, they offer information on the midpoint potential and the identity of the cofactor. 5 Furthermore in a single titration multiple cofactors in a protein can be monitored.…”

Section: Introductionmentioning

confidence: 99%

EPR Monitored Redox Titration of the Cofactors of <em>Saccharomyces cerevisiae </em>Nar1

Hagedoorn

Weel

Hagen

2014

JoVE

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Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor. Video LinkThe video component of this article can be found at

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Redox properties of the iron‐sulfur clusters in activated Fe‐hydrogenase from Desulfovibrio vulgaris (Hildenborough)

Cited by 133 publications

References 46 publications

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

Crucial Role of Conserved Cysteine Residues in the Assembly of Two Iron−Sulfur Clusters on the CIA Protein Nar1

EPR Monitored Redox Titration of the Cofactors of <em>Saccharomyces cerevisiae </em>Nar1

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