Bacterial Approaches for Assembling Iron-Sulfur Proteins

Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins.

The long-standing relationship between paramagnetic NMR and iron–sulfur proteins: the mitoNEET example. An old method for new stories or the other way around?

Paramagnetic NMR spectroscopy and iron–sulfur (Fe–S) proteins have maintained a synergic relationship for decades. Indeed, the hyperfine shifts with their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues have been extensively used as a fingerprint of the type and of the oxidation state of the Fe–S cluster within the protein frame. The identification of NMR signals from residues surrounding the metal cofactor is crucial for understanding the structure–function relationship in Fe–S proteins, but it is generally impaired in standard NMR experiments by paramagnetic relaxation enhancement due to the presence of the paramagnetic cluster(s). On the other hand, the availability of systems of different sizes and stabilities has, over the years, stimulated NMR spectroscopists to exploit iron–sulfur proteins as paradigmatic cases to develop experiments, models, and protocols. Here, the cluster-binding properties of human mitoNEET have been investigated by 1D and 2D 1H diamagnetic and paramagnetic NMR, in its oxidized and reduced states. The NMR spectra of both oxidation states of mitoNEET appeared to be significantly different from those reported for previously investigated [Fe2S2]2+/+ proteins. The protocol we have developed in this work conjugates spectroscopic information arising from “classical” paramagnetic NMR with an extended mapping of the signals of residues around the cluster which can be taken, even before the sequence-specific assignment is accomplished, as a fingerprint of the protein region constituting the functional site of the protein. We show how the combined use of 1D NOE experiments, 13C direct-detected experiments, and double- and triple-resonance experiments tailored using R1- and/or R2-based filters significantly reduces the “blind” sphere of the protein around the paramagnetic cluster. This approach provided a detailed description of the unique electronic properties of mitoNEET, which are responsible for its biological function. Indeed, the NMR properties suggested that the specific electronic structure of the cluster possibly drives the functional properties of different [Fe2S2] proteins.

Structural basis of RICs iron donation for iron-sulfur cluster biogenesis

Escherichia coli YtfE is a di-iron protein, of the widespread RIC family, with capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. Herein we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron centre to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.ImportanceThe ubiquitous iron-sulfur proteins require specialized cellular machineries to promote the assembly of the cofactor. These systems include proteins that provide sulfur and iron, and scaffold proteins where the cluster is formed. Although largely studied the nature of the iron donor remains to be fully clarified. In this work, we show that Escherichia coli YtfE, which belongs to the RIC protein family, establishes protein-protein interactions with two of the major proteins of the ISC system, and we reveal the structural characteristics necessary for the exit of iron ions from YtfE. Altogether our results prove that RICs can be considered a family of iron donor proteins involved in the biogenesis of iron-sulfur containing proteins.

The long-standing relationship between Paramagnetic NMR and Iron-Sulfur proteins: the mitoNEET example. An old method for new stories or the other way around?

Paramagnetic NMR spectroscopy and iron-sulfur (Fe–S) proteins have maintained a synergic relationship for decades. Indeed, the hyperfine shifts with their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues have been extensively used as a fingerprint of the type and of the oxidation state of the Fe–S cluster within the protein frame. The identification of NMR signals from residues surrounding the metal cofactor is crucial for understanding the structure-function relationship in Fe–S proteins, but it is generally impaired in standard NMR experiments by paramagnetic relaxation enhancement due to the presence of the paramagnetic cluster(s). On the other hand, the availability of systems of different size and stability has, over the years, stimulated NMR spectroscopists to exploit iron-sulfur proteins as paradigmatic cases to develop experiments, models and protocols. Here, the cluster binding properties of human mitoNEET have been investigated by one-dimensional and two-dimensional 1H diamagnetic and paramagnetic NMR, in its oxidized and reduced states. The NMR spectra of both oxidation states of mitoNEET appeared to be significantly different from those reported for previously investigated [Fe2S2]2+/+ proteins. We show how the use of 1D NOE experiments, 13C direct-detected experiments, and the optimization of NMR experiments for paramagnetic systems significantly reduce the blind sphere of the protein around the paramagnetic cluster. The application of this approach provided a detailed description of the unique electronic properties of mitoNEET, that are responsible for its biological function. Indeed, the NMR properties suggested that the specific electronic structure of the cluster possibly drives the functional properties of different [Fe2S2] proteins.

Treatment-resistant schizophrenia: focus on the transsulfuration pathway

Treatment-resistant schizophrenia (TRS) is a severe form of schizophrenia. The severity of illness is positively related to homocysteine levels, with high homocysteine levels due to the low activity of the transsulfuration pathway, which metabolizes homocysteine in synthesizing L-cysteine. Glutathione levels are low in schizophrenia, which indicates shortages of L-cysteine and low activity of the transsulfuration pathway. Hydrogen sulfide (H2S) levels are low in schizophrenia. H2S is synthesized by cystathionine β-synthase and cystathionine γ-lyase, which are the two enzymes in the transsulfuration pathway. Iron-sulfur proteins obtain sulfur from L-cysteine. The oxidative phosphorylation (OXPHOS) pathway has various iron-sulfur proteins. With low levels of L-cysteine, iron-sulfur cluster formation will be dysregulated leading to deficits in OXPHOS in schizophrenia. Molybdenum cofactor (MoCo) synthesis requires sulfur, which is obtained from L-cysteine. With low levels of MoCo synthesis, molybdenum-dependent sulfite oxidase (SUOX) will not be synthesized at appropriate levels. SUOX detoxifies sulfite from sulfur-containing amino acids. If sulfites are not detoxified, there can be sulfite toxicity. The transsulfuration pathway metabolizes selenomethionine, whereby selenium from selenomethionine can be used for selenoprotein synthesis. The low activity of the transsulfuration pathway decreases selenoprotein synthesis. Glutathione peroxidase (GPX), with various GPXs being selenoprotein, is low in schizophrenia. The dysregulations of selenoproteins would lead to oxidant stress, which would increase the methylation of genes and histones leading to epigenetic changes in TRS. An add-on treatment to mainline antipsychotics is proposed for TRS that targets the dysregulations of the transsulfuration pathway and the dysregulations of other pathways stemming from the transsulfuration pathway being dysregulated.