NifS-Mediated Assembly of [4Fe−4S] Clusters in the N- and C-Terminal Domains of the NifU Scaffold Protein

The specialized yeast mitochondrial chaperone system, composed of the Hsp70 Ssq1p, its co-chaperone J-protein Jac1p, and the nucleotide release factor Mge1p, perform a critical function in the biogenesis of iron-sulfur (Fe/S) proteins. Using a spectroscopic assay, we have analyzed the potential role of the chaperones in Fe/S cluster assembly on the scaffold protein Isu1p in vitro in the presence of the cysteine desulfurase Nfs1p. In the absence of chaperones, the kinetics of Fe/S cluster formation on Isu1p were compatible with a chemical reconstitution pathway with Nfs1p functioning as a sulfide donor. Addition of Ssq1p improved the rates of Fe/S cluster assembly 3-fold. However, this stimulatory effect of Ssq1p required neither ATP nor Jac1p and could be fully attributed to the activation of the Nfs1p desulfurase activity by Ssq1p. Furthermore, chaperone-stimulated Fe/S cluster assembly did not involve the specific interaction between Isu1p and Ssq1p, since the effect was observed with Isu1p mutant proteins defective in this interaction, suggesting that nonspecific binding of Ssq1p to Nfs1p helped to prevent its unfolding. Consistent with this idea, these Isu1p mutants were capable of binding an Fe/S cluster in vivo but failed to restore the growth and Fe/S cluster assembly defects of a Isu1p/ Isu2p-deficient yeast strain. Taken together, these data suggest that Ssq1p/Jac1p/Mge1p are not important for Fe/S cluster synthesis on Isu1p. Hence, consistent with previous in vivo data, these chaperones likely function in steps subsequent to the de novo synthesis of the Fe/S cluster on Isu1p.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The Hsp70 Chaperone Ssq1p Is Dispensable for Iron-Sulfur Cluster Formation on the Scaffold Protein Isu1p

Dutkiewicz

Marszałek

Schilke

et al. 2006

“…The variants IscU D39A and Nfu-1 D39A were shown to be compromised in [Fe-S] cluster transfer to apo-aconitase and apo-dinitrogen reductase, respectively (64,65). These protein variants have been useful in dissecting the mechanism of [Fe-S] cluster biosynthesis and transfer (36, 66 -69).…”

Section: Variant Growth Atp Hydrolysismentioning

confidence: 99%

Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function

Boyd

Sondelski

Downs

2009

Analysis of the metabolic network anchored to thiamine biosynthesis in S. enterica identified lesions in three non-isc or -suf loci that compromise Fe-S metabolism as follows: apbC, apbE, and rseC (17-21). This metabolic system was subsequently used to dissect a role for cyaY and gshA in [Fe-S] cluster metabolism (6,22,23). Of these, the apbC (mrp in E. coli) locus was identified as the predominant site of lesions that altered thiamine synthesis by disrupting [Fe-S] cluster metabolism (17,18).ApbC is a member of the ParA subfamily of proteins that have a wide array of functions, including electron transfer (24), initiation of cell division (25), and DNA segregation (26,27). Importantly, ATP hydrolysis is required for function of all well characterized members of this subfamily, and all members contain a "deviant" Walker A motif, which contains two lysine residues instead of one (GKXXXGK(S/T)) (28). ApbC has been shown to hydrolyze ATP (17).Recently, five proteins with a high degree of identity to ApbC have been shown to be involved in [Fe-S] cluster metabolism in eukaryotes. The sequence alignments of the central portion of these proteins and bacterial ApbC are shown in Fig. 1. HCF101 was demonstrated to be involved in chloroplast [Fe-S] cluster metabolism (29, 30). The CFD1, Npb35, and huNbp35 (formally Nubp1) proteins were demonstrated to be involved in cytoplasmic [Fe-S] cluster metabolism (31,32). Ind1 was demonstrated to be involved in the maturation of [Fe-S] clusters in the mitochondrial enzyme NADH:ubiquinone oxidoreductase (33). There is currently no report of any of these proteins hydrolyzing ATP.Biochemical analysis of ApbC indicated that it could bind and transfer [Fe-S] clusters to Saccharomyces cerevisiae apo-isopropylmalate isomerase (34). Additional genetic studies indicated that ApbC has a degree of functional redundancy with IscU, a known [Fe-S] cluster scaffolding protein (35,36).In this study we investigate the correlation between the biochemical properties of ApbC (i.e. ATPase activity, [Fe-S] cluster binding, and [Fe-S] cluster transfer rates) and the in vivo function of this protein. This is the first detailed kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of deviant Walker A proteins and the first functional analysis of a member of the ever expanding family of ApbC/Nbp35 proteins. Data presented indicate that noncomplementing variants have distinct biochemical properties that place them in three distinct classes.

“…The central domain contains a stable redoxactive [2Fe-2S] cluster with an as-yet-unknown function (7). In vitro and in vivo experiments have established that labile [Fe-S] clusters can be assembled on both the N-terminal and C-terminal domains of NifU, and such cluster-loaded forms of NifU can be used for activation of the nitrogenase Fe-protein (8,9). Thus, NifU contains two different sites upon which labile [Fe-S] clusters can be assembled in vitro, but the functional relationship between these sites is not yet known.…”

mentioning

confidence: 99%

A Proposed Role for the Azotobacter vinelandii NfuA Protein as an Intermediate Iron-Sulfur Cluster Carrier

Bandyopadhyay

Naik

O’Carroll

et al. 2008

Self Cite

129

Iron-sulfur clusters ([Fe-The in vivo maturation of simple [Fe-S] proteins is proposed to require preassembly of [Fe-S] species on molecular scaffolds. The first [Fe-S] cluster assembly system to be described is the NIF 2 system from Azotobacter vinelandii. This system consists of a cysteine desulfurase, encoded by nifS, which supplies the S for [Fe-S] cluster formation, and a proposed scaffold protein, encoded by nifU (1). The NIF system is specialized for the maturation of [Fe-S] proteins involved in nitrogen fixation.A. vinelandii also contains a second [Fe-S] protein maturation system designated ISC. The ISC system is required for the general maturation of cellular [Fe-S] proteins involved in intermediary metabolism, such as aconitase (2). The ISC system is more complicated than the NIF system as it includes the products of eight contiguous genes : iscR, iscS, iscU, iscA, hscB, hscA, fdx, and iscX (3). Although the NIF and ISC systems exhibit physiological target specificity, each can partially replace the function of the other, when expressed at high levels (4, 5).Even though the NIF and ISC systems are differentiated by their apparent target specificities, they share a number of common structural and functional features. For example, NifS and IscS have similar sequences, and they both exhibit cysteine desulfurase activity (2). IscU also shares considerable sequence identity when compared with the N-terminal domain of NifU, including conservation of three cysteine residues that are likely to provide the nucleation site(s) for [Fe-S] cluster assembly (2, 6).NifU is a modular protein that contains three distinct domains (Fig. 1). The central domain contains a stable redoxactive [2Fe-2S] cluster with an as-yet-unknown function (7). In vitro and in vivo experiments have established that labile [Fe-S] clusters can be assembled on both the N-terminal and C-terminal domains of NifU, and such cluster-loaded forms of NifU can be used for activation of the nitrogenase 9). Thus, NifU contains two different sites upon which labile [Fe-S] clusters can be assembled in vitro, but the functional relationship between these sites is not yet known.There are no genes within the ISC transcriptional unit that encode proteins with sequence similarity to the C-terminal domain of NifU. However, located elsewhere on the A. vinelandii genome is a gene, designated nfuA, whose product encodes a protein having a C-terminal sequence similar to the C-terminal domain of NifU (Fig. 1). The sequence conservation between NifU and NfuA includes two cysteine residues that are required for the in vitro assembly of [Fe-S] clusters within the NifU C-terminal domain. Like NifU, NfuA also appears to be a modular protein because the amino acid sequence within its N-terminal region shares some sequence similarity with another protein involved in [Fe-S] protein maturation designated IscA (Fig. 1). IscA is a nonessential protein encoded within the ISC transcriptional unit, and it has been proposed to serve as an alternative [Fe-S] cluster ass...