Metallomics

We recently produced three web-based calculators that predict in vivo metal occupancies of proteins, based on the metal affinities of a protein of interest along with estimates of the availabilities of the labile buffered pools of metals inside a cell. Metal availabilities were calculated from the calibrated responses of DNA-binding, metal-sensing, transcriptional regulators. The availability of intracellular Fe(II) was estimated to be similar in E. coli grown under anaerobic conditions compared to cells grown aerobically in LB medium. The purpose of this article is to archive the background data that underpins the release of a new calculator for hyperaerated cells grown in flasks with baffles, with relatively low culture volumes plus high shaking speeds to give elevated oxygenation. The intracellular availability of Fe(II) calculated from the responses of the intracellular Fe(II) sensor Fur was estimated to be significantly lower in these hyperaerated cells than either of the previous values determined for anaerobic or aerobic cultures. The total number of atoms of Mn(II) per cell increased in hyperaerated cells albeit with only modest change in intracellular Mn(II) availability as estimated from the responses of the Mn(II) sensor MntR. Accurate determination of intracellular Ni(II) availability will require further calibration of the magnitude of the responses of the Ni(II) sensor NikR in hyperaerated cells to take account of the state of Fnr. The hyperaerated metalation calculator is made available online and as a spreadsheet, for use by others.

In an effort to gain insight into cellular systems impacted by neurotrophic trans-banglene (t-BG), global proteomic profiling and Western blot analyses were employed. Expression level changes in response to t-BG treatment were compared to those observed with nerve growth factor (NGF), a natural neurotrophic protein and functional analog to t-BG. Findings from these studies did not point to direct interception of NGF/TrkA signaling by t-BG. Instead, significant alterations in iron-binding and iron-regulating proteins were observed. Intracellular iron measurements by FerroOrange indicate lower ferrous (Fe2+) iron levels in t-BG treated cells but not in NGF treated cells. These results highlight a potential connection between iron regulation and neurotrophic activity.

The concentration-dependent decrease in viable cells is a well-documented phenomenon in cytotoxicity assays for most toxic substances. We report that arsenite (As3+), a widely recognized oxidative toxicant, exhibited lower cytotoxic effects at 300 {micro}M compared to 100 {micro}M As3+ in CHO-K1 and Jurkat cells. Formation of stress granules (SGs), which appear in the cytoplasm shortly after exposure to hypertonicity, heat shock, and high concentrations of As3+ is considered as a pro-survival cellular event. We hypothesized that unusual cytotoxicity profile of As3+ could be attributed to SG formation. In both CHO-K1 and Jurkat cells stably expressing GFP-tagged G3BP1, SGs were more rapidly and distinctly induced by 300 {micro}M As3+ than 100 {micro}M As3+. Other toxic metals and a metalloid such as Cd2+, Cu2+, Ag+, and Se4+ did not clearly induce SG formation and instead reduced the viability in a concentration-dependent manner. Exposure to As3+ led to phosphorylation of eIF2, a key regulator of polysome stability and a hallmark of SG formation. Depletion of intracellular glutathione (GSH) increased the susceptibility of cells to As3+, highlighting its role in cellular defense mechanisms. Exposure to As3+ activated small ubiquitin-like modifier (SUMO) which is implicated in phase separation. However, neither depletion of GSH nor overexpression of SUMO contributed As3+-induced SG formation. Consistently, THP-1 and HL60 cells exposed to As3+ also exhibited non-canonical cytotoxic features, albeit at higher concentrations (1 mM). These findings underscore the need for further mechanistic investigations into As3+-induced SG formation, given that As3+ is a promising anti-cancer agent, and resistance of tumor cells to As3+ is a critical issue.

Neurotoxin {beta}-N-methylamino-L-alanine (BMAA) has been deemed a pathogenic factor for human neurodegenerative diseases. It is an important issue to disclose the biosynthesis mechanism of BMAA in marine diatoms. In the present study, the iron (Fe) limitation (1/3 x Fe) was found to suppress the growth of diatoms but stimulate the production of BMAA-containing proteins, maximum 7.7 fold in Thalassiosira minima. Transcriptome analysis showed that energy metabolism, protein biosynthesis and carbon fixation functions were mainly affected by the Fe limitation in the diatom. Analysis of subcellular distribution of BMAA showed that BMAA-containing proteins were mainly detected in the endoplasmic reticulum and the Golgi apparatus. Combination results of the responses of the diatom to Fe deficiency and co-culture with cyanobacteria in our previous study, we speculate that cysteine embedded in peptide chains and methylamine produced by the diatom itself are possibly catalyzed by the cysteine synthase (cysK) to form the BMAA structure in situ. Spiked methylamine in culture media significantly stimulated the production of BMAA, and BMAA amounts were correlated with the expression of cysK gene in different diatoms. The reduced ubiquitination-mediated proteolysis and vesicle trafficking precision through the COPII system would aggravate the accumulation of BMAA-containing proteins in the diatom. Significance StatementWith the detection of neurotoxin BMAA in diverse marine diatoms, the pathogenic risk of BMAA has been further concerned to human neurodegenerative diseases such as Alzheimers disease. Interestingly, BMAA-containing proteins are the dominant forms of this neurotoxin in diatoms. It is a keystone issue to disclose the biosynthesis mechanism of BMAA in marine diatoms. We found Fe-limitation could stimulate the production of BMAA-containing proteins in diatoms and explored its biosynthesis using transcriptomics in this study. Results suggested that cysteine embedded in peptides and methylamine in cytoplasm were catalyzed by the cysteine synthase (cysK) to form BMAA. This study hints that the biosynthesis of BMAA would be improved by the worldwide prevalence of iron deficiency in the coastal waters.

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

The role of the cytosolic O-acetylserine-(thiol) lyase A (OASTLA), chloroplastic OASTLB and mitochondrion OASTLC in plant resistance/sensitivity to selenate was studied in Arabidopsis plants. Impairment in OASTLA and B resulted in reduced biomass, chlorophyll and soluble protein levels compared with impaired OASTL C and Wild-Type treated with selenate. The lower organic-Se and protein-Se levels followed by decreased organic-S, S in proteins and total glutathione in oastlA and oastlB compared to Wild-Type and oastlC are indicative that Se accumulation is not the main cause for the stress symptoms, but rather the interference of Se with the S-reduction pathway. The increase in sulfite oxidase, adenosine 5'-phosphosulfate reductase, sulfite reductase and OASTL activity levels, followed by enhanced sulfite and sulfide, indicate a futile anabolic S-starvation response to selenate-induced organic-S catabolism in oastlA and oastlB compared to Wild-Type and oastlC. Additionally, the catabolic pathway of L-cysteine degradation was enhanced by selenate, and similar to L-cysteine producing activity, oastlA and B exhibited a significant decrease in L-cysteine desulfhydrase (DES) activity, compared with WT, indicating a major role of OASTLs in L-cysteine degradation. This notion was further evidenced by sulfide dependent DES in-gel activity, immunoblotting, immunoprecipitation with specific antibodies and identification of unique peptides in activity bands generated by OASTLA, B and C. Similar responses of the OASTLs in Seleno-Cysteine degradation was demonstrated in selenate stressed plants. Notably, no L-cysteine and L-Seleno-Cysteine DES activity bands but those related to OASTLs were evident. These results indicate the significance of OASTLs in degrading L-cysteine and L-SelenoCysteine in Arabidopsis. SummaryThe cytosolic OASTLA and chloroplastic OASTLB have significantly higher desulfhydrase activity rates than the cytosolic DES1 and are able to degrade L-Cys and L-SeCys to sulfide and selenide, respectively in Arabidopsis.

Cadmium (Cd) is a toxic pollutant in soil and water affecting plants, animals, and humans. Autophagy, a cellular recycling process, is crucial for different biotic and abiotic plant stress responses. This study explores the autophagy role in Arabidopsis under Cd stress, using wild-type, autophagy-deficient mutants (atg5, and atg7) and overexpressing lines (35S:ATG5, 35S:ATG7). Cd exposure induced autophagy, as evidenced by ATG8a and ATG8a-PE accumulation, GFP-ATG8a fluorescence, and upregulation of ATG genes and proteins. Responses differed between Col-0 and Ws backgrounds, with Ws showing higher Cd tolerance. atg5 mutants were more sensitive to Cd, indicating the autophagy protective role, whereas ATG5/ATG7 overexpression did not significantly enhance Cd tolerance. Although oxidative stress may activate autophagy, ATG5/ATG7 overexpression did not significantly change the oxidative stress response. Notably, atg5 mutants displayed marked disruptions in metal/ion homeostasis under control and Cd conditions, reinforcing autophagys role inion homeostasis. In contrast, atg7 showed no significant differences from its WT (Ws), suggesting genotype-specific effects. Transcriptional analysis of metal transporters and ion flux analyses indicates that autophagy can regulate metal/ion accumulation through transcriptional control and post-translational modifications (e.g., ROS) with a differential response being observed between Ws and Col-0 plants.

Zinc is an essential transition metal that participates in many biological processes. In C. elegans, excess zinc is stored in lysosomes in intestinal cells; this process involves increasing the expression of the zinc transporter CDF-2 and remodeling of lysosomes characterized by an increase in the volume of the expansion compartment. To determine if this is a more general property, we investigated other metals. Here we report that lysosomes are remodeled in response to excess copper, manganese, and cadmium, with each metal causing an increase in the volume of the expansion compartment. Mutants with a reduced number of lysosomes were hypersensitive to growth retardation caused by excess copper and manganese, suggesting metal toxicity is prevented by metal sequestration in lysosomes. Using a novel method to analyze isolated lysosomes by X-ray Fluorescence Microscopy we demonstrated that zinc, copper and manganese are detectable in the lumen of lysosomes. To further analyze copper, we examined localization of CUA-1.1, a copper transporter that moves copper into the lumen of lysosomes. Like the zinc transporter CDF-2, CUA-1.1 localizes to both the acidified and expansion compartments in excess copper. These results indicate that the same intestinal lysosomes store zinc, copper and manganese. Lysosome remodeling characterized by an increase in volume of the expansion compartment is not specific to zinc but is a more general phenomenon during metal storage in lysosomes.

Promyelocytic leukemia-nuclear bodies (PML-NBs) are dot-like protein assemblies and implicated in the pathogenesis of leukemia and viral infection. PML is the scaffold protein of PML-NB and its client proteins such as SUMO, DAXX, and Sp100 reside in PML-NBs. It is known that a short exposure to trivalent arsenic (As3+) induces the solubility change and the subsequent SUMOylation of PML, and the SUMO interacting motif (SIM) is not necessary for these biochemical changes. However, it has not been well studied how As3+ initiates or enhances the association of SUMO with PML and the other PML-NB client proteins. Here, we report that As3+ enhanced non-covalent association of PML with SUMO via the SUMO-SIM interaction which is dispensable for the solubility change and SUMOylation of PML. We also report that the As3+-induced solubility change of PML was not affected by ML792, a SUMO E1 enzyme inhibitor, even though the nuclear localization of SUMO2/3 and protein SUMOylation were halted by ML792. As3+ did not change the solubility of DAXX and SUMOylation enzymes such as SAE1, UBA2, and UBC9. In contrast, As3+ induced SUMOylation of Sp100 with a concomitant loss of its solubility like PML in human leukemia cell lines. Our current results indicate that both covalent and non-covalent associations of SUMO with PML are increased in As3+-exposed cells, and Sp100 may play a role in the maintenance of PML-NBs.

Hydrogen sulfide (H2S) has traditionally been considered as an environmental toxin for animal lineages; yet, it plays a signaling role in various processes at low concentrations. Mechanisms controlling H2S in animals, especially in sulfide-rich environments, are not fully understood. The main detoxification pathway involves the conversion of H2S into less harmful forms, through a mitochondrial oxidation pathway. The first step of this pathway oxidizes sulfide and reduces ubiquinone (UQ) through sulfide-quinone oxidoreductase (SQRD/SQOR). Because H2S inhibits cytochrome oxidase and hence UQ regeneration, this pathway becomes compromised at high H2S concentrations. The free-living nematode C. elegans feeds on bacteria and can face high sulfide concentrations in its natural environment. This organism has an alternative ETC that uses rhodoquinone (RQ) as the lipidic electron transporter and fumarate as the final electron acceptor. In this study, we demonstrate that RQ is essential for survival in sulfide. RQ-less animals (kynu-1 and coq-2e KO) cannot survive high H2S concentrations, while UQ-less animals (clk-1 and coq-2a KO) exhibit recovery, even when provided with a UQ-deficient diet. Our findings highlight that sqrd-1 uses both benzoquinones and that RQ-dependent ETC confers a key advantage (RQ regeneration) over UQ in sulfide conditions. C. elegans also faces cyanide, another cytochrome oxidase inhibitor, whose detoxification leads to H2S production, via cysl-2. Our study reveals that RQ delays killing by the HCN-producing bacteria Pseudomonas aeruginosa PAO1. These results underscore the fundamental role that RQ-dependent ETC serves as a biochemical adaptation to H2S environments, and to pathogenic bacteria producing cyanide and H2S toxins.

Cadmium (Cd) is a toxic heavy metal that endangers human health through contaminated rice consumption. Reducing Cd uptake in rice is essential for minimizing grain Cd accumulation. However, the transcriptional regulation of Cd uptake in rice remains largely elusive. This study reveals that the transcription factor OsNAC5 in Oryza sativa positively regulates the Cd transporter gene OsNRAMP1, influencing Cd uptake. OsNAC5 is primarily expressed in roots, localized to the nucleus, and upregulated by Cd-induced H2O2. Knocking out OsNAC5 reduces Cd levels in both shoots and roots and increases Cd sensitivity. OsNRAMP1 expression, enhanced by Cd stress, depends on OsNAC5, which binds to "CATGTG" motifs in the OsNRAMP1 promoter activating its expression. Loss of OsNAC5 function decreases Cd accumulation in rice grains. Our findings elucidate the transcriptional regulation of Cd stress response in rice and suggest biotechnological approaches to reduce Cd uptake in crops. Declaration of Competing InterestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Based on available platforms of Saccharomyces cerevisiae mitochondrial proteome and other high throughput studies, we identified the yeast gene DMO2 with a profile of genetic and physical interactions that indicate a putative role in mitochondrial respiration. Dmo2p is a homolog to human DMAC1 with two conserved cysteines in a Cx2C motif. Here, we localized Dmo2p in the mitochondrial inner membrane with the conserved cysteines facing the intermembrane space. The observed phenotypes of the dmo2 null mutant indicate a general function in cellular stress response; the mutant displayed poor growth on non-fermentative media at 37{degrees}C, and in oleate, it is more sensitive to heat and oxidative stress while its overexpression confers resistance to some of the tested stressors. Dmo2p topology and modeled structure suggested a functional redox role for the Cx2C motif sustained by site-directed mutagenesis of both cysteine residues. The respiratory deficiency of dmo2 mutants at 37{degrees}C led to a reduction in cytochrome c oxidase activity (COX) and the formation of bc1-COX supercomplexes; we also observed a rapid turnover of Cox2p, the subunit two of the cytochrome c oxidase complex that harbors the binuclear CuA center. Moreover, Dmo2p co-immunoprecipitated with Cox2p and components of CuA center maturation such as Sco1p and Sco2p; and finally, DMO2 overexpression can suppress cox23 respiratory deficiency, a mutant that has the mitochondrial copper homeostasis impaired. Overall, our data suggest that Dmo2p is required for Cox2p maturation, potentially by aiding proteins involved in copper transport and incorporation into Cox2p.

The red microalga Cyanidioschyzon merolae inhabits extreme environments of high temperature (40-56{degrees}C), high acidity (pH 0.05-4), and the presence of high concentrations of heavy metals and sulphites that are lethal to most other forms of life. However, information is scarce on the precise adaptation mechanisms of this extremophile to such hostile conditions. Gaining such knowledge is important for understanding the evolution of microorganisms in the early stages of life on Earth characterized by such extreme environments. By analyzing the re-programming of the global transcriptome upon long-term (up to 15 days) exposure of C. merolae to extremely high concentrations of nickel (1 and 3 mM), the key adaptive metabolic pathways and associated molecular components were identified. Our work shows that long-term Ni exposure of C. merolae leads to the lagged metabolic switch demonstrated by the transcriptional upregulation of the metabolic pathways critical for cell survival. DNA replication, cell cycle, and protein quality control processes were upregulated while downregulation occurred of energetically costly processes including assembly of the photosynthetic apparatus and lipid biosynthesis. This study paves the way for the multi-omic studies of the molecular mechanisms of abiotic stress adaptation in phototrophs, as well as future development of the rational approaches for bioremediation of contaminated aquatic environments. ImportanceThis study provides the first comprehensive analysis of the global transcriptome re-programming in the extremophilic red microalga Cyanidioschyzon merolae during its long-term adaptation to heavy metals. We show that the lagged metabolic switch, demonstrated by the transcriptional upregulation of the metabolic pathways critical for cell survival, underlies the long-term Ni adaptation of this model extremophile. The transcriptomic results shed light on how life may have adapted to some of the harshest abiotic stresses on Earth including high temperatures, extreme acidity, and high levels heavy metals that are prohibitive to most other organisms. Additionally, the differentially regulated genes identified in this work provide important clues on the rational development of effective bioremediation strategies of removing heavy metals from the heavily contaminated aquatic environments.

Human exposure to arsenicals is associated with devastating diseases such as cancer and neurodegeneration. At the same time, arsenic-based drugs are used as therapeutic agents. The ability of arsenic to directly bind to proteins is correlated with its toxic and therapeutic effects highlighting the importance of elucidating arsenic-protein interactions. In this study, we took a proteomic approach and identified 174 proteins that bind to arsenic in Saccharomyces cerevisiae. Proteins involved in nucleocytoplasmic transport were markedly enriched among the arsenic-binding proteins, and we demonstrate that arsenic-binding to nuclear import factors results in their relocation from the nuclear envelope and subsequent aggregation in the cytosol. Similarly, nuclear pore proteins that make up the nuclear pore complex mislocalized and aggregated in arsenic-exposed cells. Consequently, arsenic was shown to inhibit nuclear protein import and export. We propose a model in which arsenic-binding to nuclear transport factors leads to their mislocalization and aggregation, which disrupts nucleocytoplasmic transport and causes arsenic sensitivity.

A hypothetical protein encoded by Synechocystis sp. PCC 6803 open reading frame slr0201shows high sequence similarity to the C subunit of a group of unusual succinate dehydrogenases found in some archaeal species. Slr0201 was originally annotated as HdrB, the B subunit of heterodisulfide reductase, but appears to be SdhC instead. This protein was overexpressed in E. coli by cloning the PCR-derived slr0201 open reading frame into a pET16b-based expression vector. The overproduced Slr0201 accumulated predominantly in inclusion bodies with an apparent molecular mass of 33 kDa. The protein contained at least one [2Fe-2S] cluster based on UV-visible absorbance and CD spectra and EPR spectroscopy, in conjunction with stoichiometric analysis of protein-bound iron and sulfur content. Redox titration showed a midpoint potential (Em) of + 17 mV at pH 7.0, which is consistent with Slr0201 serving a role in transferring electrons between succinate and plastoquinone. Slr0201 was also overproduced in Synechocystis sp. PCC 6803 by introducing an additional, His-tagged slr0201 into the Synechocystis genome replacing psbA3, creating the slr0201+-His overexpression strain. Immunoblot analysis shows that Slr0201 is membrane-associated in the wild type. However, in the Slr0201+-His strain, immunoreaction occurred in both the membrane and soluble fractions, possibly as a consequence of processing near the N-terminus. The results obtained with Slr0201 are discussed in the light of one of the cyanobacterial SdhB subunits, which shares redox commonalities with archaeal SdhB.

Zrt-/Irt-like proteins (ZIPs), a family of divalent metal transporters, are crucial for maintaining the homeostasis of zinc, an essential trace element involved in numerous biological processes. While extensive research on the prototypical ZIP from Bordetella bronchiseptica (BbZIP) have suggested an elevator transport mechanism, the dynamic conformational changes during the transport cycle have not been thoroughly studied. In this work, we developed a sandwich ELISA using a custom anti-BbZIP monoclonal antibody to investigate the conformational change induced by the metal binding to the transport site. This was achieved by determining the accessibility of a cysteine residue introduced at a position exposed to the solvent only when the transporter adopts an outward-facing conformation. This assay allowed us to report the dissociation constants of BbZIP for Zn2+ and Cd2+ at low and sub-micromolar levels, respectively. Notably, the installation of a positive charge at the M2 site drastically reduced metal binding at the M1 site, consistent with an auxiliary role for the M2 site in metal transport. We also demonstrated that this assay can be used to rapidly screen variants for subsequent structural study. We anticipate that other transporters where substrate binding induces large conformational changes can also be studied using this method.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP ({Delta}ppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and {Delta}ppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while{Delta} ppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of required building blocks. From our dataset, we were particularly interested in Arn and EptA proteins, which were downregulated in {Delta}ppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins, and provide evidence that this mis-regulation in {Delta}ppk cells stems from a failure to induce the BasS/BasR two-component system during starvation. We also show that {Delta}ppk mutants unable to upregulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Rubrerythrins are a group of proteins within the Ferritin-like superfamily that display a defining four-helix bundle domain. They also show multiple structural features that are crucial to their functionality as iron storage proteins and in detoxification and oxidative stress response. Here we investigate rubrerythrin (Rbr) in multiple metalated states, from the pathogen Burkholderia pseudomallei (Bp). We use X-ray crystallography for structure determination of Rbr to probe the capacity and specificity of metal binding. BpRbr lacks the rubredoxin moiety found in canonical Rbrs from anaerobic lineages, and we demonstrate that BpRbr also possesses a domain-swapped dimer, which has functional implications for its putative role in oxidative stress response. We also carry out in crystallo spectroscopic assessment of BpRbr with various metals, using energy dispersive X-ray (EDX) spectroscopy. We observe that samples can contain metals other than those supplied in crystallization conditions, and developed a strategy of utilizing EDX spectroscopy to select those samples with single metal incorporation for downstream diffraction data collection. Our work underscores the importance of spectroscopic probing for definitive metal identification and characterization. HighlightsO_LIPresent structures of apo-, Fe-, Mn-, and Co-bound rubrerythrin from B. pseudomallei C_LIO_LIBpRbr lacks rubredoxin domain and possesses unique domain swap fold in the dimer C_LIO_LIBpRbr features include promiscuous metal binding and pre-formed metal binding sites C_LIO_LIHighlights importance of in crystallo spectroscopy in investigating metalloproteins C_LIO_LIMutant Rbr with abrogated metal binding supports premise of pre-formed binding sites C_LI

A family of cytosolic copper (Cu) storage proteins (the Csps) are widespread in bacteria. The Csps can bind large quantities of Cu(I) via their Cys-lined four-helix bundles, and the majority are cytosolic (Csp3s). This is inconsistent with the current dogma that bacteria, unlike eukaryotes, have evolved not to maintain intracellular pools of Cu due to its potential toxicity. Sporulation in Bacillus subtilis has been used to investigate if a Csp3 can store Cu(I) in the cytosol for a target enzyme. The activity of the Cu-requiring endospore multi-Cu oxidase BsCotA (a laccase) increases under Cu-replete conditions in wild type B. subtilis, but not in the strain lacking BsCsp3. Cuprous ions readily transfer from BsCsp3, but not from the cytosolic copper metallochaperone BsCopZ, to BsCotA in vitro producing active enzyme. Both BsCsp3 and BsCotA are upregulated during late sporulation. The hypothesis we propose is that BsCsp3 acquires and stores Cu(I) in the cytosol for BsCotA.

Iron-sulfur (Fe-S) clusters are ubiquitous metallocofactors found across diverse protein families, where they perform myriad functions including redox chemistry, radical generation, and gene regulation. Monitoring Fe-S cluster occupancy in protein targets directly within native biological systems has been challenging. Commonly utilized spectroscopic methods to detect Fe-S clusters require purification of proteins prior to analysis. Global iron incorporation into the proteome can be monitored using radiolabeled iron, but limitations include the low resolution afforded by gel-based autoradiography. Here, we report the development of a mass spectrometry-based strategy to assess Fe-S cluster binding in a native proteome. This chemoproteomic strategy relies on monitoring changes in the reactivity of Fe-S cluster cysteine ligands upon disruption of Fe-S cluster incorporation. Application to E. coli cells cultured under iron-depleted conditions enabled monitoring of disruptions to Fe-S cluster incorporation broadly across the E. coli Fe-S proteome. Evaluation of E. coli deletion strains of three scaffold proteins within the Isc Fe-S biogenesis pathway enabled the identification of Fe-S clients that are reliant on each individual scaffold protein for proper cluster installation. Lastly, cysteine-reactivity changes characteristic of Fe-S ligands were used to identify previously unannotated Fe-S proteins, including the tRNA hydroxylase, TrhP, and a member of a family of membrane transporter ATPase subunits, DppD. In summary, the chemoproteomic strategy described herein provides a powerful tool to report on Fe-S cluster incorporation directly within a native proteome, to interrogate the role of scaffold and accessory proteins within Fe-S biogenesis pathways, and to identify previously uncharacterized Fe-S proteins.