Manganese-induced toxicity has been linked to mitochondrial dysfunction and an increased generation of reactive oxygen species (ROS). We could recently show in mechanistic studies that Mn2+ ions induce hydrogen peroxide (H2O2) production from the ubiquinone binding site of mitochondrial complex II (IIQ) and generally enhance H2O2 formation by accelerating the rate of superoxide dismutation. The present study with intact mitochondria reveals that manganese additionally enhances H2O2 emission by inducing mitochondrial permeability transition (mPT). In mitochondria fed by NADH-generating substrates, the combination of Mn2+ and different respiratory chain inhibitors led to a dynamically increasing H2O2emission which was sensitive to the mPT inhibitor cyclosporine A (CsA) as well as Ru-360, an inhibitor of the mitochondrial calcium uniporter (MCU). Under these conditions, flavin-containing enzymes of the mitochondrial matrix, e.g. the mitochondrial 2-oxoglutaratedehydrogenase (OGDH), were major sources of ROS. With succinate as substrate, Mn2+ stimulated ROS production mainly at complex II, whereby the applied succinate concentration had a marked effect on the tendency for mPT. Also Ca2+ increased the rate of H2O2 emission by mPT, while no direct effect on ROS-production of complex II was observed. The present study reveals a complex scenario through which manganese affects mitochondrial H2O2 emission: stimulating its production from distinct sites (e.g. site IIQ), accelerating superoxide dismutation and enhancing the emission via mPT which also leads to the loss of soluble components of the mitochondrial antioxidant systems and favors the ROS production from flavin-containing oxidoreductases of the Krebs cycle.

Febuxostat, a drug recently approved in the US, European Union and Japan for treatment of gout, inhibits xanthine oxidoreductase (XOR)-mediated generation of uric acid during purine catabolism. It inhibits bovine milk XOR with a K(i) in the picomolar-order, but we found that it is a much weaker inhibitor of Rhodobacter capsulatus XOR, even though the substrate-binding pockets of mammalian and bacterial XOR are well-conserved as regards to catalytically important residues and three-dimensional structure, and both permit the inhibitor to be accommodated in the active site, as indicated by computational docking studies. To clarify the reason for the difference of inhibitory potency towards the two XORs, we performed molecular dynamics simulations. The results indicate that differences in mobility of hydrophobic residues that do not directly interact with the substrate account for the difference in inhibitory potency.

Light-dependent protochlorophyllide oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase are evolutionary and structurally distinct enzymes that are essential for the synthesis of (bacterio)chlorophyll, the primary pigment needed for both anoxygenic and oxygenic photosynthesis. In contrast to the long-held hypothesis that LPORs are only present in oxygenic phototrophs, we recently identified a functional LPOR in the aerobic anoxygenic phototrophic bacterium (AAPB) Dinoroseobacter shibae and attributed its presence to a single horizontal gene transfer event from cyanobacteria. Here, we provide evidence for the more widespread presence of genuine LPOR enzymes in AAPBs. An exhaustive bioinformatics search identified 36 putative LPORs outside of oxygenic phototrophic bacteria (cyanobacteria) with the majority being AAPBs. Using in vitro and in vivo assays, we show that the large majority of the tested AAPB enzymes are genuine LPORs. Solution structural analyses, performed for two of the AAPB LPORs, revealed a globally conserved structure when compared with a well-characterized cyanobacterial LPOR. Phylogenetic analyses suggest that LPORs were transferred not only from cyanobacteria but also subsequently between proteobacteria and from proteobacteria to Gemmatimonadetes. Our study thus provides another interesting example for the complex evolutionary processes that govern the evolution of bacteria, involving multiple horizontal gene transfer events that likely occurred at different time points and involved different donors.

Dihydroorotate:quinone oxidoreductases (DHOQOs) are membrane bound enzymes responsible for oxidizing dihydroorotate (DHO) to orotate with concomitant reduction of quinone to quinol. They have FMN as prosthetic group and are part of the monotopic quinone reductase superfamily. These enzymes are also members of the dihydroorotate dehydrogenases (DHODHs) family, which besides membrane bound DHOQOs, class 2, includes soluble enzymes which reduce either NAD+ or fumarate, class 1. As key enzymes in both the de novo pyrimidine biosynthetic pathway as well as in the energetic metabolism, inhibitors of DHOQOs have been investigated as leads for therapeutics in cancer, immunological disorders and bacterial/viral infections. This work is a thorough bioinformatic approach on the structural conservation and taxonomic distribution of DHOQOs. We explored previously established structural/functional hallmarks of these enzymes, while searching for uncharacterized common elements. We also discuss the cellular role of DHOQOs and organize the identified protein sequences within six sub-classes 2A to 2F, according to their taxonomic origin and sequence traits. We concluded that DHOQOs are present in Archaea, Eukarya and Bacteria, including the first recognition in Gram-positive organisms. DHOQOs can be the single dihydroorotate dehydrogenase encoded in the genome of a species, or they can coexist with other DHODHs, as the NAD+ or fumarate reducing enzymes. Furthermore, we show that the type of catalytic base present in the active site is not an absolute criterium to distinguish between class 1 and class 2 enzymes. We propose the existence of a quinone binding motif ("ExAH") adjacent to a hydrophobic cavity present in the membrane interacting N-terminal domain.

Last step of electron transport from ferredoxin to NADP+ in photosynthesis light reactions catalyses ferredoxin: NADP+ oxidoreductase (FNR). FNR is present as soluble protein in stroma, but also bound to the protein complexes on the membrane with thylakoid rhodanase-like protein (TROL) and translocon on the inner envelope chloroplast membrane (Tic62), which have identical C terminal FNR binding domain [1,2]. During the electron transport, FNR anchored by TROL protein transfers electrons on NADP+ and forms NADPH which is then used in Calvin cycle as reducing agent. TROL is an integral membrane protein [3] with an inactive rhodanase-like domain (RHO) facing stroma which, as proposed earlier [4], could bind a small ligand leading to releasing or binding of FNR. FNR-TROL protein complex is necessary for optimal photosynthetic electron flow [1]. It has been shown that C4 plant maize FNR isomers have different N-terminal structure which determines binding affinity to protein complexes and different ratios of bound and unbound FNR in bundle sheath and mesophyll cells, depending on preferable photosynthetic electron transport [5]. Mutant Arabidopsis plant that contain maize FNR1 protein showed influence on dynamic association of FNR and change in excitation balance between photosystems which then influenced photo induced electron transport and finally photosynthesis [5]. In order to determine the influence of maize FNR1 on photosynthesis in C3 plants and difference in interaction strength with TROL, we preformed Yeast two hybrid screening, x-alpha-gal assay and β-galactosidase assay.

Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases.

Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.

Cellular form and function - and thus normal development and physiology - are specified via proteins that control the organization and dynamic properties of the actin cytoskeleton. Using the Drosophila model, we have recently identified an unusual actin regulatory enzyme, Mical, which is directly activated by F-actin to selectively post-translationally oxidize and destabilize filaments - regulating numerous cellular behaviors. Mical proteins are also present in mammals, but their actin regulatory properties, including comparisons among different family members, remain poorly defined. We now find that each human MICAL family member, MICAL-1, MICAL-2, and MICAL-3, directly induces F-actin dismantling and controls F-actin-mediated cellular remodeling. Specifically, each human MICAL selectively associates with F-actin, which directly induces MICALs catalytic activity. We also find that each human MICAL uses an NADPH-dependent Redox activity to post-translationally oxidize actin's methionine (M) M44/M47 residues, directly dismantling filaments and limiting new polymerization. Genetic experiments also demonstrate that each human MICAL drives F-actin disassembly in vivo, reshaping cells and their membranous extensions. Our results go on to reveal that MsrB/SelR reductase enzymes counteract each MICAL's effect on F-actin in vitro and in vivo. Collectively, our results therefore define the MICALs as an important phylogenetically-conserved family of catalytically-acting F-actin disassembly factors.

Huntington's disease (HD) is caused by a trinucleotide CAG repeat in the huntingtin gene (HTT) that results in expression of a polyglutamine-expanded mutant huntingtin protein (mHTT). N-terminal fragments of mHTT accumulate in brain neurons and glia as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD.

NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.

The nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation toward the emergence of novel cofactor regeneration routes has not been previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotrophic strain. We continuously cultivated this strain under selective conditions. After 500 to 1,100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD+-dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wild-type variant with NAD+. In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP+, thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve toward reducing NADP+, which we attribute to several biochemical constraints, including unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context toward changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries. IMPORTANCE In the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism. Correspondingly, the ratio of the reduced to the oxidized form differs for NAD+ (low) and NADP+ (high), reflecting their distinct roles. We challenged the flexibility of E. coli's central metabolism in multiple adaptive evolution experiments using an NADPH-auxotrophic strain. We found several mutations in two enzymes, changing the cofactor preference of malic enzyme and dihydrolipoamide dehydrogenase. Upon deletion of their corresponding genes we performed additional evolution experiments which did not lead to the emergence of any additional mutants. We attribute this restricted number of mutational targets to intrinsic thermodynamic barriers; the high ratio of NADPH to NADP+ limits metabolic redox reactions that can regenerate NADPH, mainly by mass action constraints.

Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.

The biochemical and genetic search for a bc(1) complex in Rhodothermus marinus was always fruitless; however, a functional equivalent, i.e. having quinol:cytochrome c oxidoreductase activity was characterized. Now, with the sequencing of R. marinus genome, it was possible to assign the N-terminal sequences of several proteins of this complex to its coding genes. The alternative complex III from R. marinus has the same genomic organization of the so-called MFIcc complexes, proposed to be oxidoreductases of the respiratory and photosynthetic electron transfer chains. In this report, we establish undoubtedly the existence of an alternative complex III, a functional substitute of the bc(1) complex, by its identification at both the biochemical and genomic level.

Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. Thiol-disulphide oxidoreductases are enzymes that catalyse the formation of disulphide bonds. The bacterium Streptomyces lividans is a well-known host for the efficient secretion of overproduced homologous and heterologous secretory proteins of industrial application. Therefore, the correct conformation of these extracellular proteins is of great importance when engineering that overproduction.

Flavoprotein oxidoreductases are members of a large protein family of specialized dehydrogenases, which include type II NADH dehydrogenase, pyridine nucleotide-disulphide oxidoreductases, ferredoxin-NAD+ reductases, NADH oxidases, and NADH peroxidases, playing a crucial role in the metabolism of several prokaryotes and eukaryotes. Although several studies have been performed on single members or protein subgroups of flavoprotein oxidoreductases, a comprehensive analysis on structure-function relationships among the different members and subgroups of this great dehydrogenase family is still missing. Here, we present a structural comparative analysis showing that the investigated flavoprotein oxidoreductases have a highly similar overall structure, although the investigated dehydrogenases are quite different in functional annotations and global amino acid composition. The different functional annotation is ascribed to their participation in species-specific metabolic pathways based on the same biochemical reaction, i.e., the oxidation of specific cofactors, like NADH and FADH2. Notably, the performed comparative analysis sheds light on conserved sequence features that reflect very similar oxidation mechanisms, conserved among flavoprotein oxidoreductases belonging to phylogenetically distant species, as the bacterial type II NADH dehydrogenases and the mammalian apoptosis-inducing factor protein, until now retained as unique protein entities in Bacteria/Fungi or Animals, respectively. Furthermore, the presented computational analyses will allow consideration of FAD/NADH oxidoreductases as a possible target of new small molecules to be used as modulators of mitochondrial respiration for patients affected by rare diseases or cancer showing mitochondrial dysfunction, or antibiotics for treating bacterial/fungal/protista infections.

In this paper, a novel electron mediator, 1-methoxy-5-ethyl phenazinium ethyl sulfate (mPES), was introduced as a versatile mediator for disposable enzyme sensor strips, employing representative flavin oxidoreductases, lactate oxidase (LOx), glucose dehydrogenase (GDH), and fructosyl peptide oxidase (FPOx). A disposable lactate enzyme sensor with oxygen insensitive Aerococcus viridans-derived engineered LOx (AvLOx), with A96L mutant as the enzyme, was constructed. The constructed lactate sensor exhibited a high sensitivity (0.73 ± 0.12 μA/mM) and wide linear range (0-50 mM lactate), showings that mPES functions as an effective mediator for AvLOx. Employing mPES as mediator allowed this amperometric lactate sensor to be operated at a relatively low potential of +0.2 V to 0 V vs. Ag/AgCl, thus avoiding interference from uric acid and acetaminophen. The lactate sensors were adequately stable for at least 48 days of storage at 25 °C. These results indicated that mPES can be replaced with 1-methoxy-5-methyl phenazinium methyl sulfate (mPMS), which we previously reported as the best mediator for AvLOx-based lactate sensors. Furthermore, this study revealed that mPES can be used as an effective electron mediator for the enzyme sensors employing representative flavin oxidoreductases, GDH-based glucose sensors, and FPOx-based hemoglobin A1c (HbA1c) sensors.

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.

The formation of disulfides in proteins entering the secretory pathway is catalysed by the protein disulfide isomerase (PDI) family of enzymes. These enzymes catalyse the introduction, reduction and isomerization of disulfides. To function continuously they require an oxidase to reform the disulfide at their active site. To determine how each family member can be recycled to catalyse disulfide exchange, we have studied whether disulfides are transferred between individual PDI family members. We studied disulfide exchange either between purified proteins or by identifying mixed disulfide formation within cells grown in culture. We show that disulfide exchange occurs efficiently and reversibly between specific PDIs. These results have allowed us to define a hierarchy for members of the PDI family, in terms of ability to act as electron acceptors or donors during thiol-disulfide exchange reactions and indicate that there is no kinetic barrier to the exchange of disulfides between several PDI proteins. Such promiscuous disulfide exchange negates the necessity for each enzyme to be oxidized by Ero1 (ER oxidoreductin 1) or reduced by a reductive system. The lack of kinetic separation of the oxidative and reductive pathways in mammalian cells contrasts sharply with the equivalent systems for native disulfide formation within the bacterial periplasm.

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.

The alarming presence of hazardous halo-organic pollutants in wastewater and soils generated by industrial growth, pharmaceutical and agricultural activities is a major environmental concern that has drawn the attention of scientists. Unfortunately, the application of conventional technologies within hazardous materials remediation processes has radically failed due to their high cost and ineffectiveness. Consequently, the design of innovative and sustainable techniques to remove halo-organic contaminants from wastewater and soils is crucial. Altogether, these aspects have led to the search for safe and efficient alternatives for the treatment of contaminated matrices. In fact, over the last decades, the efficacy of immobilized oxidoreductases has been explored to achieve the removal of halo-organic pollutants from diverse tainted media. Several reports have indicated that these enzymatic constructs possess unique properties, such as high removal rates, improved stability, and excellent reusability, making them promising candidates for green remediation processes. Hence, in this current review, we present an insight of green remediation approaches based on the use of immobilized constructs of phenoloxidases (e.g., laccase and tyrosinase) and peroxidases (e.g., horseradish peroxidase, chloroperoxidase, and manganese peroxidase) for sustainable decontamination of wastewater and soil matrices from halo-organic pollutants, including 2,4-dichlorophenol, 4-chlorophenol, diclofenac, 2-chlorophenol, 2,4,6-trichlorophenol, among others.