Neutral complexes as oxidants for the reduced form of parsley (Petroselinum crispum) [2Fe--2S] ferredoxin. Evidence for partial blocking by redox-inactive Cr(III) complexes.
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ABSTRACT:
SUBMITTER: Adzamli IK
PROVIDER: S-EPMC1158282 | biostudies-other | 1982 Jun
REPOSITORIES: biostudies-other
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Project description:Knowing the manner of protein-protein interactions is vital for understanding biological events. The plant-type [2Fe-2S] ferredoxin (Fd), a well-known small iron-sulfur protein with low redox potential, partitions electrons to a variety of Fd-dependent enzymes via specific protein-protein interactions. Here we have refined the crystal structure of a recombinant plant-type Fd I from the blue green alga Aphanothece sacrum (AsFd-I) at 1.46 Å resolution on the basis of the synchrotron radiation data. Incorporating the revised amino-acid sequence, our analysis corrects the 3D structure previously reported; we identified the short α-helix (67-71) near the active center, which is conserved in other plant-type [2Fe-2S] Fds. Although the 3D structures of the four molecules in the asymmetric unit are similar to each other, detailed comparison of the four structures revealed the segments whose conformations are variable. Structural comparison between the Fds from different sources showed that the distribution of the variable segments in AsFd-I is highly conserved in other Fds, suggesting the presence of intrinsically flexible regions in the plant-type [2Fe-2S] Fd. A few structures of the complexes with Fd-dependent enzymes clearly demonstrate that the protein-protein interactions are achieved through these variable regions in Fd. The results described here will provide a guide for interpreting the biochemical and mutational studies that aim at the manner of interactions with Fd-dependent enzymes.
| S-EPMC3132287 | biostudies-literature
Project description:Human glutaredoxin 3 (Glrx3) is an essential [2Fe-2S]-binding protein with ill-defined roles in immune cell response, embryogenesis, cancer cell growth, and regulation of cardiac hypertrophy. Similar to other members of the CGFS monothiol glutaredoxin (Grx) family, human Glrx3 forms homodimers bridged by two [2Fe-2S] clusters that are ligated by the conserved CGFS motifs and glutathione (GSH). We recently demonstrated that the yeast homologues of human Glrx3 and the yeast BolA-like protein Fra2 form [2Fe-2S]-bridged heterodimers that play a key role in signaling intracellular iron availability. Herein, we provide biophysical and biochemical evidence that the two tandem Grx-like domains in human Glrx3 form similar [2Fe-2S]-bridged complexes with human BolA2. UV-visible absorption and circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic analyses of recombinant [2Fe-2S] Glrx3 homodimers and [2Fe-2S] Glrx3-BolA2 complexes indicate that the Fe-S coordination environments in these complexes are virtually identical to those of the analogous complexes in yeast. Furthermore, we demonstrate that apo BolA2 binds to each Grx domain in the [2Fe-2S] Glrx3 homodimer forming a [2Fe-2S] BolA2-Glrx3 heterotrimer. Taken together, these results suggest that the unusual [2Fe-2S]-bridging Grx-BolA interaction is conserved in higher eukaryotes and may play a role in signaling cellular iron status in humans.
| S-EPMC3331715 | biostudies-literature
Project description:Kinetic results are presented for the reaction of reduced [2Fe-2S] ferredoxin from the blue-green alga Spirulina platensis with Co(NH3)6(3+), Co(edta)- and Co(acac)3 as oxidants at pH 8.0 at I0.10 (NaCl). The aim is to compare results obtained with those previously reported for the [2Fe-2S] ferredoxin from parsley, where the two ferredoxins under consideration are in evolutionary terms widely divergent (35% amino acid variations). The three oxidants chosen have different ligand sets and different charges, and are the complexes that in previous studies have given greatest diversity in behaviour. With Co(NH3)6(3+) first-order rate constants (oxidant in large excess) tend to a limiting value with increasing concentration of oxidant. With Co(edta)- and Co(acac)3 there is no similar tendency to limiting behaviour and a first-order dependence on oxidant is observed. The temperature-dependence of the Co(NH3)6(3+) reaction was investigated, and values were obtained for delta H0 [19.8kJ X mol-1 (4.7kcal X mol-1)] and delta S0 [129.3J X K-1 X mol-1 (30.9 cal X K-1 X mol-1)] for the association step that occurs before electron transfer. Whereas redox-inactive Cr(NH3)6(3+) displays competitive inhibition in the reaction of Co(NH3)6(3+), it accelerates the reaction of Co(edta)-, and only partially blocks the reaction with Co(acac)3. Results obtained are similar to those previously reported for parsley (and spinach) ferredoxin. It is concluded that electrostatics play a dominant role and that a negatively charged functional site on the protein common to all three ferredoxins is influential. Conserved negative patches at positions 67-69 and 94-96 within 1.0 nm (10A) of an Fe atom of the active site, as well as the exposed S atoms of cysteine residues 41 and 46, which are a part of the Fe2S*2(SR)4(3-) cluster, are the most likely possibilities. The various effects of Cr(NH3)6(3+) provide a means of testing for utilization of the same site in reactions of the ferredoxins with physiological partners.
| S-EPMC1154345 | biostudies-other
Project description:The [2Fe-2S] cluster-bridged complex of BOLA3 with GLRX5 has been implicated in cluster trafficking, but cluster exchange involving this heterocomplex has not been reported. Herein we describe an investigation of the cluster exchange reactivity of holo BOLA3-GLRX complexes using two different monothiol glutaredoxins, H.s. GLRX5 and S.c. Grx3, which share significant identity. We observe that a 1?:?1 mixture of apo BOLA3 and glutaredoxin protein is able to accept a cluster from donors such as ISCU and a [2Fe-2S](GS)4 complex, with preferential formation of the cluster-bridged heterodimer over the plausible holo homodimeric glutaredoxin. Holo BOLA3-GLRX5 transfers clusters to apo acceptors at rates comparable to other Fe-S cluster trafficking proteins. Isothermal titration calorimetry experiments with apo proteins demonstrated a strong binding of BOLA3 with both GLRX5 and Grx3, while binding with an alternative mitochondrial partner, NFU1, was weak. Cluster exchange and calorimetry experiments resulted in a very similar behavior for yeast Grx3 (cytosolic) and human GLRX5 (mitochondrial), indicating conservation across the monothiol glutaredoxin family for interactions with BOLA3 and supporting a functional role for the BOLA3-GLRX5 heterocomplex relative to the previously proposed BOLA3-NFU1 interaction. The results also demonstrate rapid formation of the heterocomplexed holo cluster via delivery from a glutathione-complexed cluster, again indicative of the physiological relevance of the [2Fe-2S](GS)4 complex in the cellular labile iron pool.
| S-EPMC6146392 | biostudies-literature
Project description:The physiological function of Rhodobacter capsulatus FdVI, a [2Fe-2S] ferredoxin, was investigated by the cloning, sequence analysis, and mutagenesis of its structural gene, called fdxE. The DNA region surrounding fdxE was mapped, and the nucleotide sequence of a 4.2-kb fragment was determined. fdxE is preceded by a sequence that is very similar to a sigma54 recognition site and is followed by a putative transcription stop signal, suggesting that fdxE forms a separate cistron. Two open reading frames were identified upstream and downstream of fdxE and were named ORFE0 and ORFE1, respectively. The former may encode a polypeptide having 34% similarity with HtrA, a serine protease found in enteric bacteria. ORFE1 is homologous to purU, a gene involved in purine biosynthesis. Interposon mutagenesis of fdxE was unsuccessful when attempted on the wild-type strain B10. Disruption of fdxE could be achieved only in strains harboring an additional copy of fdxE on a plasmid. Mutants obtained in this way and carrying a plasmid-borne copy of fdxE under the control of the nifH promoter grew only in N-free medium, thus demonstrating that fdxE expression is required for growth. Nevertheless, such mutants were found to spontaneously revert at a frequency of 5 x 10(-6) to an apparent wild-type phenotype, although they contained no detectable amount of FdVI. Taken together, the results indicate that FdVI is required for an essential metabolic function in R. capsulatus and that this FdVI dependence could be relieved by a single-mutation event. In accordance, FdVI biosynthesis was found to be constitutive in R. capsulatus.
| S-EPMC179111 | biostudies-other
Project description:The essential process of iron-sulfur (Fe/S) cluster assembly (ISC) in mitochondria occurs in three major phases. First, [2Fe-2S] clusters are synthesized on the scaffold protein ISCU2; second, these clusters are transferred to the monothiol glutaredoxin GLRX5 by an Hsp70 system followed by insertion into [2Fe-2S] apoproteins; third, [4Fe-4S] clusters are formed involving the ISC proteins ISCA1-ISCA2-IBA57 followed by target-specific apoprotein insertion. The third phase is poorly characterized biochemically, because previous in vitro assembly reactions involved artificial reductants and lacked at least one of the in vivo-identified ISC components. Here, we reconstituted the maturation of mitochondrial [4Fe-4S] aconitase without artificial reductants and verified the [2Fe-2S]-containing GLRX5 as cluster donor. The process required all components known from in vivo studies (i.e., ISCA1-ISCA2-IBA57), yet surprisingly also depended on mitochondrial ferredoxin FDX2 and its NADPH-coupled reductase FDXR. Electrons from FDX2 catalyze the reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 in an IBA57-dependent fashion. This previously unidentified electron transfer was occluded during previous in vivo studies due to the earlier FDX2 requirement for [2Fe-2S] cluster synthesis on ISCU2. The FDX2 function is specific, because neither FDX1, a mitochondrial ferredoxin involved in steroid production, nor other cellular reducing systems, supported maturation. In contrast to ISC factor-assisted [4Fe-4S] protein assembly, [2Fe-2S] cluster transfer from GLRX5 to [2Fe-2S] apoproteins occurred spontaneously within seconds, clearly distinguishing the mechanisms of [2Fe-2S] and [4Fe-4S] protein maturation. Our study defines the physiologically relevant mechanistic action of late-acting ISC factors in mitochondrial [4Fe-4S] cluster synthesis, trafficking, and apoprotein insertion.
| S-EPMC7456137 | biostudies-literature
Project description:The iron-sulfur (Fe-S) cluster-biosynthesis (ISC) system of the gamma-proteobacterium Pseudomonas putida JCM 20004 contains a constitutively expressed vertebrate-type [2Fe-2S] ferredoxin, FdxB, which lacks the conserved free cysteine residue near the Fe-S cluster site that has been proposed to function in the catalysis of biological Fe-S cluster assembly in other bacterial homologues. Recombinant FdxB was heterologously overproduced in Escherichia coli, purified and crystallized in its oxidized form by the hanging-drop vapour-diffusion and streak-seeding methods using 1.6 M trisodium citrate dihydrate pH 6.5. The thin needle-shaped crystals diffract to 1.90 A resolution and belong to the hexagonal space group P6(1)22, with unit-cell parameters a = 87.58, c = 73.14 A. The asymmetric unit contains one protein molecule.
| S-EPMC2344098 | biostudies-literature
Project description:We have used X-band ESEEM to study the reduced [2Fe-2S] cluster in adrenodoxin and Arthrospira platensis ferredoxin. By use of a 2D approach (HYSCORE), we have shown that the cluster is involved in weak magnetic interactions with several nitrogens in each protein. Despite substantial differences in the shape and orientational dependence of individual cross-peaks, the major spectral features in both proteins are attributable to two peptide nitrogens (N1 and N2) with similar hyperfine couplings approximately 1.1 and approximately 0.70 MHz. The couplings determined represent a small fraction (0.0003-0.0005) of the unpaired spin density of the reduced cluster transferred to these nitrogens over H-bond bridges or the covalent bonds of cysteine ligands. Simulation of the HYSCORE spectra has allowed us to estimate the orientation of the nuclear quadrupole tensors of N1 and N2 in the g-tensor coordinate system. The most likely candidates for the role of N1 and N2 have been identified in the protein environment by comparing magnetic-resonance data with crystallographic structures of the oxidized proteins. A possible influence of redox-linked structural changes on ESEEM data is analyzed using available structures for related proteins in two redox states.
| S-EPMC2773023 | biostudies-literature
Project description:Human cytosolic monothiol glutaredoxin-3 (GLRX3) is a protein essential for the maturation of cytosolic [4Fe-4S] proteins. We show here that dimeric cluster-bridged GLRX3 transfers its [2Fe-2S]2+ clusters to the human P-loop NTPase NUBP1, an essential early component of the cytosolic iron-sulfur assembly (CIA) machinery. Specifically, we observed that [2Fe-2S]2+ clusters are transferred from GLRX3 to monomeric apo NUBP1 and reductively coupled to form [4Fe-4S]2+ clusters on both N-terminal CX13CX2CX5C and C-terminal CPXC motifs of NUBP1 in the presence of glutathione that acts as a reductant. In this process, cluster binding to the C-terminal motif of NUBP1 promotes protein dimerization, while cluster binding to the N-terminal motif does not affect the quaternary structure of NUBP1. The cluster transfer/assembly process is not complete on both N- and C-terminal motifs and indeed requires a reductant stronger than GSH to increase its efficiency. We also showed that the [4Fe-4S]2+ cluster formed at the N-terminal motif of NUBP1 is tightly bound, while the [4Fe-4S]2+ cluster bound at the C-terminal motif is labile. Our findings provide the first evidence for GLRX3 acting as a [2Fe-2S] cluster chaperone in the early stage of the CIA machinery.
| S-EPMC8007109 | biostudies-literature
Project description:The terminal enzyme of heme biosynthesis, ferrochelatase (EC 4.99.1.1), catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. Prior to the present work, [2Fe-2S] clusters have been identified and characterized in animal ferrochelatases but not in plant or prokaryotic ferrochelatases. Herein we present evidence that ferrochelatases from the bacteria Caulobacter crescentus and Mycobacterium tuberculosis possess [2Fe-2S] clusters. The enzyme from C. crescentus is a homodimeric, membrane-associated protein while the enzyme from M. tuberculosis is monomeric and soluble. The clusters of the C. crescentus and M. tuberculosis ferrochelatases are ligated by four cysteines but possess ligand spacings that are unlike those of any previously characterized [2Fe-2S] cluster-containing protein, including the ferrochelatase of the yeast Schizosaccharomyces pombe. Thus, the microbial ferrochelatases represent a new group of [2Fe-2S] cluster-containing proteins.
| S-EPMC134991 | biostudies-literature