Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme composed of two sepa- rately purifiable component proteins encoded by the structural genes nifH, nifD, and nifK. Deletion of the Azotobacter vine- landii nijS gene lowers the activities of both nitrogenase com- ponent proteins. Because both nitrogenase component proteins have metallocluster prosthetic groups that are composed of iron- and sulfur-containing cores, this result indicated that the nifS gene product could be involved in the mobilization of the iron or sulfur required for metallocluster formation. In the present work, it is shown that NIFS is a pyridoxal phosphate- containing homodimer that catalyzes the formation of L-alanine and elemental sulfur by using L-cysteine as substrate. NIFS activity is extremely sensitive to thiol-specific alkylating re- agents, which indicates the participation of a cysteinyl thiolate at the active site. Based on these results we propose that an enzyme-bound cysteinyl persulfide that requires the release of the sulfur from the substrate L-cysteine for its formation ultimately provides the inorganic sulfide required for nitroge- nase metallocluster formation. The recent discovery of nifS- like genes in non-nitrogen-fixing organisms also raises the possibility that the reaction catalyzed by NIFS represents a universal mechanism that involves pyridoxal phosphate chem- istry, in the mobilization of the sulfur required for metallo- cluster formation.

Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis (nitrogenase/FeS cores/pyridoxal phosphate/sulfur mobilization/cysteinyl persulfide)

The role of mitochondria in cellular iron–sulfur protein biogenesis and iron metabolism ☆

Mammalian iron metabolism and its control by iron regulatory proteins.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors composed of iron and inorganic sulfur. They are required for the function of proteins involved in a wide range of activities, including electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. The proteins involved in the biogenesis of Fe-S clusters are evolutionarily conserved from bacteria to humans, and many insights into the process of Fe-S cluster biogenesis have come from studies of model organisms, including bacteria, fungi and plants. It is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of Fe-S cluster biogenesis. Although these diseases –which include Friedreich’s ataxia (FRDA), ISCU myopathy, a rare form of sideroblastic anemia, an encephalomyopathy caused by dysfunction of respiratory chain complex I and multiple mitochondrial dysfunctions syndrome – affect different tissues, a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in Fe-S cluster biogenesis. This Commentary outlines the basic steps of Fe-S cluster biogenesis as they have been defined in model organisms. In addition, it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of Fe-S cluster biogenesis proteins in some eukaryotes, including mammals. Finally, it outlines several important unresolved questions in the field that, once addressed, should offer important clues into how mitochondrial iron homeostasis is regulated, and how dysfunction in Fe-S cluster biogenesis can contribute to disease.

/pdf/biogenesis-of-iron-sulfur-clusters-in-mammalian-cells-new-3qybgte5tr.pdf

Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease

Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity.

Cellular depletion of the human protein frataxin is correlated with the neurodegenerative disease Friedreich’s ataxia and results in the inactivation of Fe−S cluster proteins. Most researchers agree that frataxin functions in the biogenesis of Fe−S clusters, but its precise role in this process is unclear. Here we provide in vitro evidence that human frataxin binds to a Nfs1, Isd11, and Isu2 complex to generate the four-component core machinery for Fe−S cluster biosynthesis. Frataxin binding dramatically changes the KM for cysteine from 0.59 to 0.011 mM and the catalytic efficiency (kcat/KM) of the cysteine desulfurase from 25 to 7900 M−1 s−1. Oxidizing conditions diminish the levels of both complex formation and frataxin-based activation, whereas ferrous iron further stimulates cysteine desulfurase activity. Together, these results indicate human frataxin functions with Fe2+ as an allosteric activator that triggers sulfur delivery and Fe−S cluster assembly. We propose a model in which cellular frataxin l...

Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex.

Iron–sulfur clusters are ubiquitous protein cofactors with critical cellular functions The mitochondrial Fe–S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe–S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe–S clusters The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich’s ataxia Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe–S cluster biosynthesis activities of the Fe–S assembly complex Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe–S clusters Additional mutagenesis, enzyme kinetic, UV–visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe–S cluster formation on the assembly complex These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe–S cluster biosynthesis, and further support an allosteric regulator role for FXN Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe–S cluster biosynthesis

https://pubs.acs.org/doi/pdf/10.1021/bi500532e

Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ∼15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ∼ I154F > W155F > W155A ∼ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.

/pdf/friedreich-s-ataxia-variants-i154f-and-w155r-diminish-4l8d0z5rnj.pdf

Friedreich's Ataxia Variants I154F and W155R Diminish Frataxin-Based Activation of the Iron-Sulfur Cluster Assembly Complex.

His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q ...

/pdf/mechanistic-and-structural-studies-of-h373q-flavocytochrome-1n6v9cfyva.pdf

Chi Lin Tsai

Papers

Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex.

Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry.

Friedreich's Ataxia Variants I154F and W155R Diminish Frataxin-Based Activation of the Iron-Sulfur Cluster Assembly Complex.

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.