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Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase.Biophysical Journal May 2022Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing...
Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR's efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP's flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. Here, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR's higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR's dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR's higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.
Topics: NADP; Neutrons; Oxidation-Reduction; Oxidoreductases; Sulfite Reductase (NADPH); Sulfur
PubMed: 35443926
DOI: 10.1016/j.bpj.2022.04.021 -
Essays in Biochemistry Aug 2023Life sustains itself using energy generated by thermodynamic disequilibria, commonly existing as redox disequilibria. Metals are significant players in controlling redox...
Life sustains itself using energy generated by thermodynamic disequilibria, commonly existing as redox disequilibria. Metals are significant players in controlling redox reactions, as they are essential components of the engine that life uses to tap into the thermodynamic disequilibria necessary for metabolism. The number of proteins that evolved to catalyze redox reactions is extraordinary, as is the diversification level of metal cofactors and catalytic domain structures involved. Notwithstanding the importance of the topic, the relationship between metals and the redox reactions they are involved in has been poorly explored. This work reviews the structure and function of different prokaryotic organometallic-protein complexes, highlighting their pivotal role in controlling biogeochemistry. We focus on a specific subset of metal-containing oxidoreductases (EC1 or EC7.1), which are directly involved in biogeochemical cycles, i.e., at least one substrate or product is a small inorganic molecule that is or can be exchanged with the environment. Based on these inclusion criteria, we select and report 59 metalloenzymes, describing the organometallic structure of their active sites, the redox reactions in which they are involved, and their biogeochemical roles.
Topics: Oxidoreductases; Metals; Oxidation-Reduction; Metalloproteins; Catalytic Domain
PubMed: 37503682
DOI: 10.1042/EBC20230012 -
Cell Reports May 2019The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between the force and the velocity of its constituent...
The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between the force and the velocity of its constituent molecular systems. While the mechanisms of force generation have been studied at the single molecular motor level, there is little understanding of the magnitude of power that can be generated by folding proteins. Here, we use single-molecule force spectroscopy techniques to measure the force-velocity relation of folding titin domains that contain single internal disulfide bonds, a common feature throughout the titin I-band. We find that formation of the disulfide regulates the peak power output of protein folding in an all-or-none manner, providing at 6.0 pN, for example, a boost from 0 to 6,000 zW upon oxidation. This mechanism of power generation from protein folding is of great importance for muscle, where titin domains may unfold and refold with each extension and contraction of the sarcomere.
Topics: Biomechanical Phenomena; Connectin; Disulfides; Models, Biological; Molecular Chaperones; Oxidation-Reduction; Oxidoreductases; Peptides; Protein Disulfide-Isomerases; Protein Domains; Protein Folding
PubMed: 31067467
DOI: 10.1016/j.celrep.2019.04.046 -
Antioxidants & Redox Signaling May 2022The presence of a large number of thioredoxin superfamily members suggests a complex mechanism of redox-based regulation in mammalian cells. However, whether these... (Review)
Review
The presence of a large number of thioredoxin superfamily members suggests a complex mechanism of redox-based regulation in mammalian cells. However, whether these members are functionally redundant or play separate and distinct roles in each cellular compartment remains to be elucidated. In the mammalian endoplasmic reticulum (ER), ∼20 thioredoxin-like proteins have been identified. Most ER oxidoreductases are soluble proteins located in the luminal compartment, whereas a small family of five thioredoxin-related transmembrane proteins (TMX) also reside in the ER membrane and play crucial roles with specialized functions. In addition to the predicted function of ER protein quality control, several independent studies have suggested the diverse roles of TMX family proteins in the regulation of cellular processes, including calcium homeostasis, bioenergetics, and thiol-disulfide exchange in the extracellular space. Moreover, recent studies have provided evidence of their involvement in the pathogenesis of various diseases. Extensive research is required to unravel the physiological roles of TMX family proteins. Given that membrane-associated proteins are prime targets for drug discovery in a variety of human diseases, expanding our knowledge on the mechanistic details of TMX action on the cell membrane will provide the molecular basis for developing novel diagnostic and therapeutic approaches as a potent molecular target in a clinical setting. 36, 984-1000.
Topics: Animals; Endoplasmic Reticulum; Humans; Mammals; Membrane Proteins; Oxidation-Reduction; Oxidoreductases; Thioredoxins
PubMed: 34465218
DOI: 10.1089/ars.2021.0187 -
International Journal of Molecular... Jan 2017Epigenetic mechanisms influence molecular patterns important for the bone-metastatic process, and here we highlight the role of WW-domain containing oxidoreductase... (Comparative Study)
Comparative Study Review
Epigenetic mechanisms influence molecular patterns important for the bone-metastatic process, and here we highlight the role of WW-domain containing oxidoreductase (Wwox). The tumor-suppressor Wwox lacks in almost all cancer types; the variable expression in osteosarcomas is related to lung-metastasis formation, and exogenous Wwox destabilizes HIF-1α (subunit of Hypoxia inducible Factor-1, HIF-1) affecting aerobic glycolysis. Our recent studies show critical functions of Wwox present in 1833-osteotropic clone, in the corresponding xenograft model, and in human bone metastasis from breast carcinoma. In hypoxic-bone metastatic cells, Wwox enhances HIF-1α stabilization, phosphorylation, and nuclear translocation. Consistently, in bone-metastasis specimens Wwox localizes in cytosolic/perinuclear area, while TAZ (transcriptional co-activator with PDZ-binding motif) and HIF-1α co-localize in nuclei, playing specific regulatory mechanisms: TAZ is a co-factor of HIF-1, and Wwox regulates HIF-1 activity by controlling HIF-1α. In vitro, DNA methylation affects Wwox-protein synthesis; hypoxia decreases Wwox-protein level; hepatocyte growth factor (HGF) phosphorylates Wwox driving its nuclear shuttle, and counteracting a Twist program important for the epithelial phenotype and metastasis colonization. In agreement, in 1833-xenograft mice under DNA-methyltransferase blockade with decitabine, Wwox increases in nuclei/cytosol counteracting bone metastasis with prolongation of the survival. However, Wwox seems relevant for the autophagic process which sustains metastasis, enhancing more Beclin-1 than p62 protein levels, and p62 accumulates under decitabine consistent with adaptability of metastasis to therapy. In conclusion, Wwox methylation as a bone-metastasis therapeutic target would depend on autophagy conditions, and epigenetic mechanisms regulating Wwox may influence the phenotype of bone metastasis.
Topics: Animals; Bone Neoplasms; Bone and Bones; Breast; Breast Neoplasms; Carcinogenesis; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Hippo Signaling Pathway; Humans; Oxidoreductases; Protein Serine-Threonine Kinases; Signal Transduction; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase
PubMed: 28045433
DOI: 10.3390/ijms18010075 -
The Journal of Biological Chemistry Dec 2015WW domain-containing oxidoreductase (WWOX), originally marked as a likely tumor suppressor gene, has over the years become recognized for its role in a much wider range... (Review)
Review
WW domain-containing oxidoreductase (WWOX), originally marked as a likely tumor suppressor gene, has over the years become recognized for its role in a much wider range of cellular activities. Phenotypic effects displayed in animal studies, along with resolution of WWOX's architecture, fold, and binding partners, point to the protein's multifaceted biological functions. Results from a series of complementary experiments seem to indicate WWOX's involvement in metabolic regulation. More recently, clinical studies involving cases of severe encephalopathy suggest that WWOX also plays a part in controlling CNS development, further expanding our understanding of the breadth and complexity of WWOX behavior. Here we present a short overview of the various approaches taken to study this dynamic gene, emphasizing the most recent findings regarding WWOX's metabolic- and CNS-associated functions and their underlying molecular basis.
Topics: Animals; Central Nervous System; Humans; Neoplasms; Oxidoreductases; Protein Structure, Tertiary; Tumor Suppressor Proteins
PubMed: 26499798
DOI: 10.1074/jbc.R115.676346 -
Experimental Biology and Medicine... Mar 2015Glioblastoma multiforme (GBM) is the most aggressive and malignant brain tumor. Delicate microenvironment and lineage heterogeneity of GBM cells including infiltration,... (Review)
Review
Glioblastoma multiforme (GBM) is the most aggressive and malignant brain tumor. Delicate microenvironment and lineage heterogeneity of GBM cells including infiltration, hypoxia, angiogenesis, and stemness make them highly resistant to current conventional therapies, with an average life expectancy for GBM patients of less than 15 months. Poor response to cytotoxic agents of GBM cells remains the major challenge of GBM treatment. Resistance of GBM to clinical treatment is a result of genomic alternation and deregulated signaling pathways, such as p53 mutation and apoptosis signaling blockage, providing cancer cells more opportunities for survival rather than cell death. WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene, commonly downregulated in various types of tumors, including GBM. It has been found that the reintroduction of WWOX induced p53-mutant GBM cells to undergo apoptosis, but not in p53 wild-type GBM cells, indicating WWOX is likely to reopen apoptosis pathways in a p53-independent manner in GBM. Identifying the crucial target modulated by WWOX deficiency provides a potential therapeutic target for GBM treatment. Here, we have reviewed the literatures about the role of WWOX in development, signaling pathway, prognosis, and treatment response in malignant glioma.
Topics: Apoptosis; Brain Neoplasms; Down-Regulation; Glioblastoma; Humans; Mutation; Oxidoreductases; Prognosis; Signal Transduction; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase
PubMed: 25432984
DOI: 10.1177/1535370214561588 -
Nature Microbiology Sep 2021Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the...
Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex.
Topics: Bacteria; Bacterial Proteins; Catalytic Domain; Cryoelectron Microscopy; Crystallography, X-Ray; Kinetics; Multiprotein Complexes; Nitrates; Nitrites; Oxidation-Reduction; Oxidoreductases
PubMed: 34267357
DOI: 10.1038/s41564-021-00934-8 -
The Journal of Physical Chemistry. B Oct 2022Oxidoreductases have evolved over millions of years to perform a variety of metabolic tasks crucial for life. Understanding how these tasks are engineered relies on...
Oxidoreductases have evolved over millions of years to perform a variety of metabolic tasks crucial for life. Understanding how these tasks are engineered relies on delivering external electron donors or acceptors to initiate electron transfer reactions. This is a challenge. Small-molecule redox reagents can act indiscriminately, poisoning the cell. Natural redox proteins are more selective, but finding the right partner can be difficult due to the limited number of redox potentials and difficulty tuning them. proteins offer an alternative path. They are robust and can withstand mutations that allow for tailorable changes. They are also devoid of evolutionary artifacts and readily bind redox cofactors. However, no reliable set of engineering principles have been developed that allow for these proteins to be fine-tuned so their redox midpoint potential () can form donor/acceptor pairs with any natural oxidoreductase. This work dissects protein-cofactor interactions that can be tuned to modulate redox potentials of acceptors and donors using a mutable designed tetrahelical protein platform with iron tetrapyrrole cofactors as a test case. We show a series of engineered heme -binding proteins and quantify their resulting effect on . By focusing on the surface charge and buried charges, as well as cofactor placement, chemical modification, and ligation of cofactors, we are able to achieve a broad range of values spanning a range of 330 mV. We anticipate this work will guide the design of proteinaceous tools that can interface with natural oxidoreductases inside and outside the cell while shedding light on how natural proteins modulate values of bound cofactors.
Topics: Oxidation-Reduction; Heme; Proteins; Oxidoreductases; Tetrapyrroles; Iron
PubMed: 36219580
DOI: 10.1021/acs.jpcb.2c05119 -
Biochimica Et Biophysica Acta.... Nov 2022The heme‑copper oxidoreductase (HCO) superfamily is a large superfamily of terminal respiratory enzymes that are widely distributed across the three domains of life....
The heme‑copper oxidoreductase (HCO) superfamily is a large superfamily of terminal respiratory enzymes that are widely distributed across the three domains of life. The superfamily includes biochemically diverse oxygen reductases and nitric oxide reductases that are pivotal in the pathways of aerobic respiration and denitrification. The adaptation of HCOs to use quinol as the electron donor instead of cytochrome c has significant implication for the respiratory flexibility and energetic efficiency of the respiratory chains that include them. In this work, we explore the adaptation of this scaffold to two different electron donors, cytochromes c and quinols, with extensive sequence analysis of these enzymes from publicly available datasets. Our work shows that quinol oxidation evolved independently within the HCO superfamily at least seven times. Enzymes from only two of these independently evolved clades have been biochemically well-characterized. Combining structural modeling with sequence analysis, we identify putative quinol binding sites in each of the novel quinol oxidases. Our analysis of experimental and modeling data suggests that the quinol binding site appears to have evolved at the same structural position within the scaffold more than once.
Topics: Copper; Cytochromes c; Heme; Hydroquinones; Nitric Oxide; Oxidoreductases; Oxygen
PubMed: 35944661
DOI: 10.1016/j.bbabio.2022.148907