Ferredoxin 1 is essential for embryonic development and lipid homeostasis.

Mammalian ferredoxin 1 and 2 (FDX1/2) belong to an evolutionary conserved family of iron-sulfur cluster containing proteins and act as electron shutters between ferredoxin reductase (FDXR) and numerous proteins involved in critical biological pathways. FDX1 is involved in biogenesis of steroids and bile acids, Vitamin A/D metabolism, and lipoylation of tricarboxylic acid (TCA) cycle enzymes. FDX1 has been extensively characterized biochemically but its role in physiology and lipid metabolism has not been explored. In this study, we generated Fdx1-deficient mice and showed that knockout of both alleles of the Fdx1 gene led to embryonic lethality. We also showed that like Fdxr+/-+/-, Fdx1+/-+/- had a shorter life span and were prone to steatohepatitis. However, unlike Fdxr+/-+/-, Fdx1+/-+/- were not prone to spontaneous tumors. Additionally, we showed that FDX1 deficiency led to lipid droplet accumulation possibly via the ABCA1-SREBP1/2 pathway. Specifically, untargeted lipidomic analysis showed that FDX1 deficiency led to alterations in several classes of lipids, including cholesterol, triacylglycerides, acylcarnitines, ceramides, phospholipids and lysophospholipids. Taken together, our data indicate that FDX1 is essential for mammalian embryonic development and lipid homeostasis at both cellular and organismal levels.

A Versatile Aldehyde: Ferredoxin Oxidoreductase from the Organic Acid Reducing Thermoanaerobacter sp. Strain X514.

Aldehyde:ferredoxin oxidoreductases (AORs) have been isolated and biochemically-characterized from a handful of anaerobic or facultative aerobic archaea and bacteria. They catalyze the ferredoxin (Fd)-dependent oxidation of aldehydes to acids. Recently, the involvement of AOR in the reduction of organic acids to alcohols with electrons derived from sugar or synthesis gas was demonstrated, with alcohol dehydrogenases (ADHs) carrying out the reduction of the aldehyde to the alcohol (AOR-ADH pathway). Here, we describe the biochemical characterization of an AOR of the thermophilic fermentative bacterium Thermoanaerobacter sp. strain X514 (AORX514). The putative aor gene (Teth514_1380) including a 6x-His-tag was introduced into the genome of the genetically-accessible, related species Thermoanaerobacter kivui. The protein was purified to apparent homogeneity, and indeed revealed AOR activity, as measured by acetaldehyde-dependent ferredoxin reduction. AORX514 was active over a wide temperature (10 to 95 °C) and pH (5.5 to 11.5) range, utilized a wide variety of aldehydes (short and branched-chained, aliphatic, aromatic) and resembles archaeal sensu stricto AORs, as the protein is active in a homodimeric form. The successful, recombinant production of AORX514 in a related, well-characterized and likewise strict anaerobe paves the road towards structure-function analyses of this enzyme and possibly similar oxygen-sensitive or W/Mo-dependent proteins in the future.

Overexpressing plant ferredoxin-like protein enhances photosynthetic efficiency and carbohydrates accumulation in Phalaenopsis.

Crassulacean acid metabolism (CAM) is one of three major models of carbon dioxide assimilation pathway with better water-use efficiency and slower photosynthetic efficiency in photosynthesis. Previous studies indicated that the gene of sweet pepper plant ferredoxin-like protein (PFLP) shows high homology to the ferredoxin-1(Fd-1) family that belongs to photosynthetic type Fd and involves in photosystem I. It is speculated that overexpressing pflp in the transgenic plant may enhance photosynthetic efficiency through the electron transport chain (ETC). To reveal the function of PFLP in photosynthetic efficiency, pflp transgenic Phalaenopsis, a CAM plant, was generated to analyze photosynthetic markers. Transgenic plants exhibited 1.2-folds of electron transport rate than that of wild type (WT), and higher CO2 assimilation rates up to 1.6 and 1.5-folds samples at 4 pm and 10 pm respectively. Enzyme activity of phosphoenolpyruvate carboxylase (PEPC) was increased to 5.9-folds in Phase III, and NAD+-linked malic enzyme (NAD+-ME) activity increased 1.4-folds in Phase IV in transgenic plants. The photosynthesis products were analyzed between transgenic plants and WT. Soluble sugars contents such as glucose, fructose, and sucrose were found to significantly increase to 1.2, 1.8, and 1.3-folds higher in transgenic plants. The starch grains were also accumulated up to 1.4-folds in transgenic plants than that of WT. These results indicated that overexpressing pflp in transgenic plants increases carbohydrates accumulation by enhancing electron transport flow during photosynthesis. This is the first evidence for the PFLP function in CAM plants. Taken altogether, we suggest that pflp is an applicable gene for agriculture application that enhances electron transport chain efficiency during photosynthesis.

FDX1 Is Required for the Biogenesis of Mitochondrial Cytochrome c Oxidase in Mammalian Cells.

Ferredoxins (FDXs) are evolutionarily conserved iron-sulfur (Fe-S) proteins that function as electron transfer proteins in diverse metabolic pathways. Mammalian mitochondria contain two ferredoxins, FDX1 and FDX2, which share a high degree of structural similarity but exhibit different functionalities. Previous studies have established the unique role of FDX2 in the biogenesis of Fe-S clusters; however, FDX1 seems to have multiple targets in vivo, some of which are only recently emerging. Using CRISPR-Cas9-based loss-of-function studies in rat cardiomyocyte cell line, we demonstrate an essential requirement of FDX1 in mitochondrial respiration and energy production. We attribute reduced mitochondrial respiration to a specific decrease in the abundance and assembly of cytochrome c oxidase (CcO), a mitochondrial heme-copper oxidase and the terminal enzyme of the mitochondrial respiratory chain. FDX1 knockout cells have reduced levels of copper and heme a/a3, factors that are essential for the maturation of the CcO enzyme complex. Copper supplementation failed to rescue CcO biogenesis, but overexpression of heme a synthase, COX15, partially rescued COX1 abundance in FDX1 knockout cells. This finding links FDX1 function to heme a biosynthesis, and places it upstream of COX15 in CcO biogenesis like its ancestral yeast homolog. Taken together, our work has identified FDX1 as a critical CcO biogenesis factor in mammalian cells.

Α rare case of myopathy, lactic acidosis, and severe rhabdomyolysis, due to a homozygous mutation of the ferredoxin-2 (FDX2) gene.

Mitochondrial myopathy is a severe metabolic myopathy related to nuclear or mitochondrial DNA dysfunction. We present a rare case of mitochondrial myopathy, presented with multiple episodes of proximal muscle weakness, lactic acidosis, and severe rhabdomyolysis (CPK 319,990 U/L, lactic acid 22.31 mmol/L, and GFR 3.82 mL/min/1.73m2 ). She was hospitalized in the pediatric intensive care unit due to acute kidney injury, elevated blood pressure, and deterioration of respiratory and cardiac function. Investigation for inherited metabolic disorders showed elevated levels of ammonia, lactic acid to pyruvic acid ratio, and urine ketone bodies. Exome sequencing detected a homozygous pathogenic variant in FDX2 (ENST00000541276:p.Met4Leu/c.10A > T) and a heterozygous variant of uncertain significance in MSTO1 (ENST00000538143:p.Leu137Pro/c.410 T > C). After Sanger sequencing, the p.Met4Leu pathogenic variant in FDX2 (ENST00000541276:p.Met4Leu/c.10A > T) was identified in a heterozygous state in both her parents and sister. Recently, pathogenic variants in the FDX2 gene have been associated with mitochondrial myopathy, lactic acidosis, optic atrophy, and leukoencephalopathy. Only four reports of FDX2-related rhabdomyolysis have been described before, but none of the previous patients had hyperammonemia. This is a rare case of severe mitochondrial myopathy in a pediatric patient related to a pathogenic FDX2 variant, suggesting the need for genetic analysis of the FDX2 gene in cases of suspicion of mitochondrial myopathies.

Lipoylation is dependent on the ferredoxin FDX1 and dispensable under hypoxia in human cells.

Iron-sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environmental sensing, and catalysis. Amongst the most ancient ISC-containing proteins are the ferredoxin (FDX) family of electron carriers. Humans have two FDXs- FDX1 and FDX2, both of which are localized to mitochondria, and the latter of which is itself important for ISC synthesis. We have previously shown that hypoxia can eliminate the requirement for some components of the ISC biosynthetic pathway, but FDXs were not included in that study. Here, we report that FDX1, but not FDX2, is dispensable under 1% O2 in cultured human cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC-containing enzyme lipoyl synthase. While hypoxia can rescue the growth phenotype of either FDX1 or lipoyl synthase KO cells, lipoylation in these same cells is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work reveals the divergent roles of FDX1 and FDX2 in mitochondria, identifies a role for FDX1 in lipoate synthesis, and suggests that loss of lipoic acid can be tolerated under low oxygen tensions in cell culture.

FDX1 regulates cellular protein lipoylation through direct binding to LIAS.

Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D, and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore-induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post-translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 directly regulates protein lipoylation by binding the lipoyl synthase (LIAS) enzyme promoting its functional binding to the lipoyl carrier protein GCSH and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss of function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling established that FDX1 loss-of-function results in the induction of both compensatory metabolism-related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-function is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting its role in cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.

Ferredoxin C2 is required for chlorophyll biosynthesis and accumulation of photosynthetic antennae in Arabidopsis.

Ferredoxins (Fd) are small iron-sulphur proteins, with sub-types that have evolved for specific redox functions. Ferredoxin C2 (FdC2) proteins are essential Fd homologues conserved in all photosynthetic organisms and a number of different FdC2 functions have been proposed in angiosperms. Here we use RNAi silencing in Arabidopsis thaliana to generate a viable fdC2 mutant line with near-depleted FdC2 protein levels. Mutant leaves have ~50% less chlorophyll a and b, and chloroplasts have poorly developed thylakoid membrane structure. Transcriptomics indicates upregulation of genes involved in stress responses. Although fdC2 antisense plants show increased damage at photosystem II (PSII) when exposed to high light, PSII recovers at the same rate as wild type in the dark. This contradicts literature proposing that FdC2 regulates translation of the D1 subunit of PSII, by binding to psbA transcript. Measurement of chlorophyll biosynthesis intermediates revealed a build-up of Mg-protoporphyrin IX, the substrate of the aerobic cyclase. We localise FdC2 to the inner chloroplast envelope and show that the FdC2 RNAi line has a disproportionately lower protein abundance of antennae proteins, which are nuclear-encoded and must be refolded at the envelope after import.

One-Pot De Novo Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions.

Fe-S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe-S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis.

BLZ8 activates a plastidial peroxiredoxin and a ferredoxin to protect Chlamydomonas reinhardtii against oxidative stress.

Reactive oxygen species (ROS) cause damage to various cellular processes in almost all organisms, in particular photosynthetic organisms that depend on the electron transfer chain for CO2 fixation. However, the detoxifying process to mitigate ROS damage has not been studied intensively in microalgae. Here, we characterized the ROS detoxifying role of a bZIP transcription factor, BLZ8, in Chlamydomonas reinhardtii. To identify downstream targets of BLZ8, we carried out comparative genome-wide transcriptomic profiling of BLZ8 OX and its parental CC-4533 under oxidative stress conditions. Luciferase reporter activity assays and RT-qPCR were performed to test whether BLZ8 regulates downstream genes. We performed an in silico functional gene network analysis and an in vivo immunoprecipitation assay to identify the interaction between downstream targets of BLZ8. Comparative transcriptomic analysis and RT-qPCR revealed that overexpression of BLZ8 increased the expression levels of plastid peroxiredoxin1 (PRX1) and ferredoxin-5 (FDX5) under oxidative stress conditions. BLZ8 alone could activate the transcriptional activity of FDX5 and required bZIP2 to activate transcriptional activity of PRX1. Functional gene network analysis using FDX5 and PRX1 orthologs in A. thaliana suggested that these two genes were functionally associated. Indeed, our immunoprecipitation assay revealed the physical interaction between PRX1 and FDX5. Furthermore, the complemented strain, fdx5 (FDX5), recovered growth retardation of the fdx5 mutant under oxidative stress conditions, indicating that FDX5 contributes to oxidative stress tolerance. These results suggest that BLZ8 activates PRX1 and FDX5 expression, resulting in the detoxification of ROS to confer oxidative stress tolerance in microalgae.

On the evolution of the plant phytochrome chromophore biosynthesis.

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria, and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IXα (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to PФB by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only PФB biosynthesis-related genes (HY2). The HY2 of the streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) has already indirectly been indicated to participate in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in Escherichia coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we confirmed the product and identified intermediates of the reaction. Site-directed mutagenesis revealed 2 aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a PΦB-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of 2 additional members of the HY2 lineage enabled us to define 2 distinct clades, the PCB-HY2 and the PΦB-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui.

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 was determined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.

Cloning, expression, and bioinformatics analysis of heavy metal resistance-related genes fd-I and fd-II from Acidithiobacillus ferrooxidans.

Five heavy metals were introduced into the bacterial heavy metal resistance tests. The results showed that apparent inhibition effects of Cd2+ and Cu2+ on the growth of Acidithiobacillus ferrooxidans BYSW1 occurred at high concentrations (>0.04 mol l-1). Significant differences (P < 0.001) were both noticed in the expression of two ferredoxin-encoding genes (fd-I and fd-II) related to heavy metal resistance in the presence of Cd2+ and Cu2+ . When exposed to 0.06 mol l-1 Cd2+, the relative expression levels of fd-I and fd-II were about 11 and 13 times as much as those of the control, respectively. Similarly, exposure to 0.04 mol l-1 Cu2+ caused approximate 8 and 4 times higher than those of the control, respectively. These two genes were cloned and expressed in Escherichia coli, and the structures, functions of two corresponding target proteins, i.e. Ferredoxin-I (Fd-I) and Ferredoxin-II (Fd-II), were predicted. The recombinant cells inserted by fd-I or fd-II were more resistant to Cd2+ and Cu2+ compared with wild-type cells. This study was the first investigation regarding the contribution of fd-I and fd-II to enhancing heavy metal resistance of this bioleaching bacterium, and laid a foundation for further elucidation of heavy metal resistance mechanisms caused by Fd.

Genetic diversity in the metronidazole metabolism genes nitroreductases and pyruvate ferredoxin oxidoreductases in susceptible and refractory clinical samples of Giardia lamblia.

The effectiveness of metronidazole against the tetraploid intestinal parasite Giardia lamblia is dependent on its activation/inactivation within the cytoplasm. There are several activating enzymes, including pyruvate ferredoxin reductase (PFOR) and nitroreductase (NR) 1 which metabolize metronidazole into toxic forms, while NR2 on the other hand inactivates it. Metronidazole treatment failures have been increasing rapidly over the last decade, indicating genetic resistance mechanisms. Analyzing genetic variation in the PFOR and NR genes in susceptible and refractory Giardia isolates may help identify potential markers of resistance. Full length PFOR1, PFOR2, NR1 and NR2 genes from clinical culturable isolates and non-cultured clinical Giardia assemblage B samples were cloned, sequenced and single nucleotide variants (SNVs) were analyzed to assess genetic diversity and alleles. A similar ratio of amino acid changing SNVs per gene length was found for the NRs; 4.2% for NR1 and 6.4% for NR2, while the PFOR1 and PFOR2 genes had less variability with a ratio of 1.1% and 1.6%, respectively. One of the samples from a refractory case had a nonsense mutation which caused a truncated NR1 gene in one out of six alleles. Further, we found three NR2 alleles with frameshift mutations, possibly causing a truncated protein in two susceptible isolates. One of these isolates was homozygous for the affected NR2 allele. Three nsSNVs with potential for affecting protein function were found in the ferredoxin domain of the PFOR2 gene. The considerable variation and discovery of mutations possibly causing dysfunctional NR proteins in clinical Giardia assemblage B isolates, reveal a potential for genetic link to metronidazole susceptibility and resistance.

Mitochondrial ferredoxin-like is essential for forming complex I-containing supercomplexes in Arabidopsis.

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of 5 multiprotein complexes (complexes I-V). Analyses of the OXPHOS system by native gel electrophoresis have revealed an organization of OXPHOS complexes into supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mitochondrial ferredoxin-like, mFDX-like). This protein was previously found to be part of the bridge domain linking the matrix and membrane arms of the complex I. Phylogenetic analysis suggested that the Arabidopsis (Arabidopsis thaliana) mFDX-like evolved from classical mitochondrial ferredoxins (mFDXs) but lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster, supposedly essential for the electron transfer function of FDXs. Accordingly, our biochemical study showed that AtmFDX-like does not bind an Fe-S cluster and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9-based strategy. These lines did not show any abnormal phenotype under standard growth conditions. However, the characterization of the OXPHOS system demonstrated that mFDX-like is important for the assembly of complex I and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III2 to form supercomplexes.

High expression of cuproptosis-related gene FDX1 in relation to good prognosis and immune cells infiltration in colon adenocarcinoma (COAD).

BACKGROUND: Cuproptosis induced by FDX1 is a newly discovered mechanism regulating cell death. However, the role of FDX1 in the pathogenesis of colon adenocarcinoma (COAD) remains to be studied. METHODS: FDX1 expression was analyzed with The Cancer Genome Atlas (TCGA) database and Human Protein Atlas (HPA) database. Association between FDX1 expression and COAD prognosis was investigated via the Kaplan-Meier (KM) survival curve. The differentially expressed genes (DEGs) of FDX1 were screened with R packages and the PPI were constructed via STRING database. Cytoscape software was used to detect the most profound modules in the PPIs network. CancerSEA database was used to analyze the effect of FDX1 expression levels on different functional status of COAD cells. The relationship between FDX1 expression and immune infiltration of COAD was analyzed by TIMER2.0 database. The COAD patients with high expression of FDX1 by Western blot, and the levels of immune infiltration were measured by flow cytometry. RESULTS: FDX1 was low expressed in most cancers, such as BRCA, KICH, and COAD. The overall survival (OS) and disease-specific survival (DSS) of COAD with high FDX1 expression were better than that of the low expression group. GO-KEGG enrichment analysis revealed that FDX1 and its co-expressed genes played an important role in the pathogenesis of COAD. Moreover, FDX1 expression in COAD were positively associated with "quiescence" and "inflammation" but negatively correlated with "invasion". FDX1 expression was positively correlated with infiltration levels of CD8+ T cells, NK cells, and neutrophils. Oppositely, FDX1 expression was negatively correlated with that of CD4+ T cells and cancer-associated fibroblasts (CAFs). Finally, 6 COAD patients with high expression of FDX1 were screened, and the proportion of CD8+ T cells in cancer tissues of these patients was significantly higher than that in paracancerous, while the CD4+ T cells presented the opposite pattern. CONCLUSION: FDX1 plays a role in inducing cuproptosis and modulating tumor immunity, which could be considered as potential therapeutic targets in COAD.

The ferredoxin/thioredoxin pathway constitutes an indispensable redox-signaling cascade for light-dependent reduction of chloroplast stromal proteins.

To ensure efficient photosynthesis, chloroplast proteins need to be flexibly regulated under fluctuating light conditions. Thiol-based redox regulation plays a key role in reductively activating several chloroplast proteins in a light-dependent manner. The ferredoxin (Fd)/thioredoxin (Trx) pathway has long been recognized as the machinery that transfers reducing power generated by photosynthetic electron transport reactions to redox-sensitive target proteins; however, its biological importance remains unclear, because the complete disruption of the Fd/Trx pathway in plants has been unsuccessful to date. Especially, recent identifications of multiple redox-related factors in chloroplasts, as represented by the NADPH-Trx reductase C, have raised a controversial proposal that other redox pathways work redundantly with the Fd/Trx pathway. To address these issues directly, we used CRISPR/Cas9 gene editing to create Arabidopsis mutant plants in which the activity of the Fd/Trx pathway was completely defective. The mutants generated showed severe growth inhibition. Importantly, these mutants almost entirely lost the ability to reduce several redox-sensitive proteins in chloroplast stroma, including four Calvin-Benson cycle enzymes, NADP-malate dehydrogenase, and Rubisco activase, under light conditions. These striking phenotypes were further accompanied by abnormally developed chloroplasts and a drastic decline in photosynthetic efficiency. These results indicate that the Fd/Trx pathway is indispensable for the light-responsive activation of diverse stromal proteins and photoautotrophic growth of plants. Our data also suggest that the ATP synthase is exceptionally reduced by other pathways in a redundant manner. This study provides an important insight into how the chloroplast redox-regulatory system operates in vivo.

Cuproptosis-related gene FDX1 expression correlates with the prognosis and tumor immune microenvironment in clear cell renal cell carcinoma.

Background: Cuproptosis, a newly discovered form of cell death, is regulated by protein lipoylation and is related to mitochondrial metabolism. However, further research is needed to determine how the cuproptosis-related gene ferredoxin 1 (FDX1) affects the tumor immune response and its prognostic significance in clear cell renal cell carcinoma (ccRCC). Methods: The Cancer Genome Atlas was used to screen for FDX1 gene expression in ccRCC and healthy tissue samples. The results were validated using the Gene Expression Omnibus and the Human Protein Atlas. Multivariable analysis and Kaplan-Meier survival curves were used to examine the relationship between FDX1 gene expression, clinicopathological parameters, and overall survival (OS). The protein network containing FDX1 gene interaction was constructed using the online Search Tool for the Retrieval of Interacting Genes/Proteins. The relationship between FDX1 gene expression and immune cell infiltration in ccRCC was examined using Gene Ontology, gene set enrichment analysis (GSEA), and a single-sample GSEA. Using the Gene Expression Profiling Interactive Analysis and Tumor Immune Estimation Resource databases, we investigated the relationship between FDX1 gene expression, the degree of immune cell infiltration, and the corresponding gene marker sets. Results: ccRCC samples had significantly (p < 0.05) lower FDX1 gene expression levels than normal tissue samples. Lower FDX1 gene expression levels were strongly associated with higher cancer grades and more advanced tumor-node-metastasis stages. The findings of multivariate and univariate analyses illustrated that the OS in ccRCC patients with low FDX1 expression is shorter than in patients with high FDX1 expression (p < 0.05). Ferredoxin reductase and CYP11A1 are key proteins interacting with the FDX1 gene, and ccRCC with an FDX1 enzyme defect was associated with a low number of invading immune cells and their corresponding marker. Conclusion: In ccRCC, decreased FDX1 expression was linked to disease progression, an unfavorable prognosis, and dysregulated immune cell infiltration.

Molecular mechanism of negative cooperativity of ferredoxin-NADP + reductase by ferredoxin and NADP(H): role of the ion pair of ferredoxin Arg40 of and FNR Glu154.

Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) and converts NADP+ to NADPH at the end of the photosynthetic electron transfer chain. We previously showed that the interaction between FNR and Fd was weakened by the allosteric binding of NADP(H) on FNR, which was considered as a part of negative cooperativity. In this study, we investigated the molecular mechanism of this phenomenon using maize (Zea mays L.) FNR and Fd, as the 3D structure of this Fd:FNR complex is available. Site-specific mutants of several amino acid residues on the Fd:FNR interface were analysed for the effect on the negative cooperativity, by kinetic analysis of Fd:FNR electron transfer activity and by Fd-affinity chromatography. Mutations of Fd Arg40Gln and FNR Glu154Gln that disrupt one of the salt bridges in the Fd:FNR complex suppressed the negative cooperativity, indicating the involvement of the ion pair of Fd Arg40 and FNR Glu154 in the mechanism of the negative cooperativity. Unexpectedly, either mutation of Fd Arg40Gln or FNR Glu154Gln tends to increase the affinity between Fd and FNR, suggesting the role of this ion pair in the regulation of the Fd:FNR affinity by NADPH, rather than the stabilization of the Fd:FNR complex.

The ferredoxin redox system - an essential electron distributing hub in the apicoplast of Apicomplexa.

The apicoplast, a relict plastid found in most species of the phylum Apicomplexa, harbors the ferredoxin redox system which supplies electrons to enzymes of various metabolic pathways in this organelle. Recent reports in Toxoplasma gondii and Plasmodium falciparum have shown that the iron-sulfur cluster (FeS)-containing ferredoxin is essential in tachyzoite and blood-stage parasites, respectively. Here we review ferredoxin's crucial contribution to isoprenoid and lipoate biosynthesis as well as tRNA modification in the apicoplast, highlighting similarities and differences between the two species. We also discuss ferredoxin's potential role in the initial reductive steps required for FeS synthesis as well as recent evidence that offers an explanation for how NADPH required by the redox system might be generated in Plasmodium spp.