Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

OCTDL: Optical Coherence Tomography Dataset for Image-Based Deep Learning Methods.

Optical coherence tomography (OCT) is a non-invasive imaging technique with extensive clinical applications in ophthalmology. OCT enables the visualization of the retinal layers, playing a vital role in the early detection and monitoring of retinal diseases. OCT uses the principle of light wave interference to create detailed images of the retinal microstructures, making it a valuable tool for diagnosing ocular conditions. This work presents an open-access OCT dataset (OCTDL) comprising over 2000 OCT images labeled according to disease group and retinal pathology. The dataset consists of OCT records of patients with Age-related Macular Degeneration (AMD), Diabetic Macular Edema (DME), Epiretinal Membrane (ERM), Retinal Artery Occlusion (RAO), Retinal Vein Occlusion (RVO), and Vitreomacular Interface Disease (VID). The images were acquired with an Optovue Avanti RTVue XR using raster scanning protocols with dynamic scan length and image resolution. Each retinal b-scan was acquired by centering on the fovea and interpreted and cataloged by an experienced retinal specialist. In this work, we applied Deep Learning classification techniques to this new open-access dataset.

Mechanism of Ion Translocation by Na+-Rhodopsin.

This review provides a brief description of the structure and transport function of the recently discovered family of retinal-containing Na+-translocating rhodopsins. The main emphasis is put on the kinetics of generation of electric potential difference in the membrane during a single transporter turnover. According to the proposed transport mechanism of Na+-rhodopsin, the driving force for the Na+ translocation from the cytoplasm is the local electric field created by the H+ movement from the Schiff base.

Pre-steady-state kinetics and solvent isotope effects support the "billiard-type" transport mechanism in Na+ -translocating pyrophosphatase.

Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H+ pump that transports the H+ ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na+ via a hypothetical "billiard-type" mechanism, also involving the hydrolysis-generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid-quench and pulse-chase measurements with [32 P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate-limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme-substrate complex. Na+ , whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H2 O with D2 O decreased the rates of pyrophosphate hydrolysis by both Na+ - and H+ -transporting bacterial mPPases, the effect being more significant than with a non-transporting soluble pyrophosphatase. We also show that the Na+ -pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis-generated proton both in H+ -pumping and Na+ -pumping by mPPases.

The Mechanism of Energy Coupling in H+/Na+-Pumping Membrane Pyrophosphatase-Possibilities and Probabilities.

Membrane pyrophosphatases (mPPases) found in plant vacuoles and some prokaryotes and protists are ancient cation pumps that couple pyrophosphate hydrolysis with the H+ and/or Na+ transport out of the cytoplasm. Because this function is reversible, mPPases play a role in maintaining the level of cytoplasmic pyrophosphate, a known regulator of numerous metabolic reactions. mPPases arouse interest because they are among the simplest membrane transporters and have no homologs among known ion pumps. Detailed phylogenetic studies have revealed various subtypes of mPPases and suggested their roles in the evolution of the "sodium" and "proton" bioenergetics. This treatise focuses on the mechanistic aspects of the transport reaction, namely, the coupling step, the role of the chemically produced proton, subunit cooperation, and the relationship between the proton and sodium ion transport. The available data identify H+-PPases as the first non-oxidoreductase pump with a "direct-coupling" mechanism, i.e., the transported proton is produced in the coupled chemical reaction. They also support a "billiard" hypothesis, which unifies the H+ and Na+ transport mechanisms in mPPase and, probably, other transporters.

A Novel, NADH-Dependent Acrylate Reductase in Vibrio harveyi.

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the FAD_binding_2 domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities. Here, we produced and characterized one such protein encoded in the Vibrio harveyi genome (GenBank ID: AIV07243) and found it to be a specific NADH:acrylate oxidoreductase (ARD). This previously unknown enzyme is formed by the OYE-like, FMN_bind, and FAD_binding_2 domains and contains covalently bound flavin mononucleotide (FMN) and noncovalently bound flavin adenine dinucleotide (FAD) and FMN in a ratio of 1:1:1. The covalently bound FMN is absolutely required for activity and is attached by the specific flavin transferase, ApbE, to the FMN_bind domain. Quantitative reverse transcription PCR (RT-qPCR) and activity measurements indicated dramatic stimulation of ARD biosynthesis by acrylate in the V. harveyi cells grown aerobically. In contrast, the ard gene expression in the cells grown anaerobically without acrylate was higher than that in aerobic cultures and increased only 2-fold in the presence of acrylate. These findings suggest that the principal role of ARD in Vibrio is energy-saving detoxification of acrylate coming from the environment. IMPORTANCE The benefits of the massive genomic information accumulated in recent years for biological sciences have been limited by the lack of data on the function of most gene products. Approximately half of the known prokaryotic genes are annotated as "proteins with unknown functions," and many other genes are annotated incorrectly. Thus, the functional and structural characterization of the products of such genes, including identification of all existing enzymatic activities, is a pressing issue in modern biochemistry. In this work, we have shown that the product of the V. harveyi ard gene exhibits a yet-undescribed NADH:acrylate oxidoreductase activity. This activity may allow acrylate detoxification and its use as a terminal electron acceptor in anaerobic or substrate in aerobic respiration of marine and other bacteria.

The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii.

Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin), acting as an electron donor for nitrogenase. However, the relative contribution of Rnf to nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely unknown. We constructed A. vinelandii cells with impaired activities of FixABCX and/or putative flavin transferase ApbE. The ApbE-deficient mutant could not produce covalently flavinylated membrane proteins and demonstrated markedly decreased flavodoxin:NAD+ oxidoreductase activity and significant growth defects under diazotrophic conditions. The double ΔFix/ΔApbE mutation abolished the flavodoxin:NAD+ oxidoreductase activity and the ability of A. vinelandii to grow in the absence of a fixed nitrogen source. ApbE flavinylated a truncated RnfG subunit of Rnf1 by forming a phosphoester bond between flavin mononucleotide and a threonine residue. These findings indicate that Rnf (presumably its Rnf1 form) is the major ferredoxin-reducing enzyme in the nitrogen fixation system and that the activity of Rnf depends on its covalent flavinylation by the flavin transferase ApbE.

Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs.

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H+ and Na+ transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a "direct coupling" mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na+ transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K+-dependent H+-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PPi analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PPi hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg2+ (essential cofactor). The replacement of K+, the second metal cofactor, by Na+ increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PPi hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell's direct coupling with conformational coupling to catalyze cation transport across the membrane.

A new water-soluble bacterial NADH: fumarate oxidoreductase.

The cytoplasmic fumarate reductase of Klebsiella pneumoniae (FRD) is a monomeric protein which contains three prosthetic groups: noncovalently bound FMN and FAD plus a covalently bound FMN. In the present work, NADH is revealed to be an inherent electron donor for this enzyme. We found that the fumarate reductase activity of FRD significantly exceeds its NADH dehydrogenase activity. During the catalysis of NADH:fumarate oxidoreductase reaction, FRD turnover is limited by a very low rate (∼10/s) of electron transfer between the noncovalently and covalently bound FMN moieties. Induction of FRD synthesis in K. pneumoniae cells was observed only under anaerobic conditions in the presence of fumarate or malate. Enzymes with the FRD-like domain architecture are widely distributed among various bacteria and apparently comprise a new type of water-soluble NADH:fumarate oxidoreductases.

A simple strategy to differentiate between H+- and Na+-transporting NADH:quinone oxidoreductases.

We describe here a simple strategy to characterize transport specificity of NADH:quinone oxidoreductases, using Na+-translocating (NQR) and H+-translocating (NDH-1) enzymes of the soil bacterium Azotobactervinelandii as the models. Submillimolar concentrations of Na+ and Li+ increased the rate of deaminoNADH oxidation by the inverted membrane vesicles prepared from the NDH-1-deficient strain. The vesicles generated carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-resistant electric potential difference and CCCP-stimulated pH difference (alkalinization inside) in the presence of Na+. These findings testified a primary Na+-pump function of A. vinelandii NQR. Furthermore, ΔpH measurements with fluorescent probes (acridine orange and pyranine) demonstrated that A. vinelandii NQR cannot transport H+ under various conditions. The opposite results obtained in similar measurements with the vesicles prepared from the NQR-deficient strain indicated a primary H+-pump function of NDH-1. Based on our findings, we propose a package of simple experiments that are necessary and sufficient to unequivocally identify the pumping specificity of a bacterial Na+ or H+ transporter. The NQR-deficient strain, but not the NDH-1-deficient one, exhibited impaired growth characteristics under diazotrophic condition, suggesting a role for the Na+ transport in nitrogen fixation by A. vinelandii.

Mutational analysis of the flavinylation and binding motifs in two protein targets of the flavin transferase ApbE.

Many flavoproteins belonging to three domain types contain an FMN residue linked through a phosphoester bond to a threonine or serine residue found in a conserved seven-residue motif. The flavinylation reaction is catalyzed by a specific enzyme, ApbE, which uses FAD as a substrate. To determine the structural requirements of the flavinylation reaction, we examined the effects of single substitutions in the flavinylation motif of Klebsiella pneumoniae cytoplasmic fumarate reductase on its modification by its own ApbE in recombinant Escherichia coli cells. The replacement of the flavin acceptor threonine with alanine completely abolished the modification reaction, whereas the replacements of conserved aspartate and serine had only minor effects. Effects of other substitutions, including replacing the acceptor threonine with serine, (a 10-55% decrease in the flavinylation degree) pinpointed important glycine and alanine residues and suggested an excessive capacity of the ApbE-based flavinylation system in vivo. Consistent with this deduction, drastic replacements of conserved leucine and threonine residues in the binding pocket that accommodates FMN residue still allowed appreciable flavinylation of the NqrC subunit of Vibrio harveyi Na+-translocating NADH:quinone oxidoreductase, despite a profound weakening of the isoalloxazine ring binding and an increase in its exposure to solvent.

Flavodoxin with an air-stable flavin semiquinone in a green sulfur bacterium.

Flavodoxins are small proteins with a non-covalently bound FMN that can accept two electrons and accordingly adopt three redox states: oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (quinol). In iron-deficient cyanobacteria and algae, flavodoxin can substitute for ferredoxin as the electron carrier in the photosynthetic electron transport chain. Here, we demonstrate a similar function for flavodoxin from the green sulfur bacterium Chlorobium phaeovibrioides (cp-Fld). The expression of the cp-Fld gene, found in a close proximity with the genes for other proteins associated with iron transport and storage, increased in a low-iron medium. cp-Fld produced in Escherichia coli exhibited the optical, ERP, and electron-nuclear double resonance spectra that were similar to those of known flavodoxins. However, unlike all other flavodoxins, cp-Fld exhibited unprecedented stability of FMN semiquinone to oxidation by air and difference in midpoint redox potentials for the quinone-semiquinone and semiquinone-quinol couples (- 110 and - 530 mV, respectively). cp-Fld could be reduced by pyruvate:ferredoxin oxidoreductase found in the membrane-free extract of Chl. phaeovibrioides cells and photo-reduced by the photosynthetic reaction center found in membrane vesicles from these cells. The green sulfur bacterium Chl. phaeovibrioides appears thus to be a new type of the photosynthetic organisms that can use flavodoxin as an alternative electron carrier to cope with iron deficiency.

Flavin transferase: the maturation factor of flavin-containing oxidoreductases.

Flavins, cofactors of many enzymes, are often covalently linked to these enzymes; for instance, flavin adenine mononucleotide (FMN) can form a covalent bond through either its phosphate or isoalloxazine group. The prevailing view had long been that all types of covalent attachment of flavins occur as autocatalytic reactions; however, in 2013, the first flavin transferase was identified, which catalyzes phosphoester bond formation between FMN and Na+-translocating NADH:quinone oxidoreductase in certain bacteria. Later studies have indicated that this post-translational modification is widespread in prokaryotes and is even found in some eukaryotes. Flavin transferase can occur as a separate ∼40 kDa protein or as a domain within the target protein and recognizes a degenerate DgxtsAT/S motif in various target proteins. The purpose of this review was to summarize the progress already achieved by studies of the structure, mechanism, and specificity of flavin transferase and to encourage future research on this topic. Interestingly, the flavin transferase gene (apbE) is found in many bacteria that have no known target protein, suggesting the presence of yet unknown flavinylation targets.

Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase.

One of the three domains of kinetoplastid NADH:fumarate oxidoreductase (FRD) is homologous to bacterial flavin transferase that catalyzes transfer of FMN residue from FAD to threonine in flavoproteins. Leptomonas pyrrhocoris FRD produced in yeast cells, which lack flavin transferase gene in their proteome, reduces fumarate in the presence of NADH and contains an FMN residue covalently linked to a Ser9 residue. The conserved flavinylation motif of FRD, D3(g/s)x(s/t)(s/g)AS9, is similar to the Dxx(s/t)gAT motif recognized by flavin transferase in prokaryotic proteins. Ser9 replacement abolished the flavinylation and fumarate reductase activity of FRD. These findings suggest that the flavinylation is important for the activity of FRD and that this post-translational modification is carried out by the own flavin transferase domain.

EPR evidence for a fast-relaxing iron center in Na+-translocating NADH:quinone-oxidoreductase.

A paramagnetic Cys4[Fe] center was detected by pulse EPR in Na+-translocating NADH:quinone-oxidoreductase (Na+-NQR) by influence of this center on transverse and longitudinal spin relaxation of Na+-NQR flavin radicals. The oxidation state of the Cys4[Fe] center was Fe3+ in the oxidized and Fe2+ in the reduced Na+-NQR, as deduced from the temperature dependence of spin relaxation rates of different flavin radicals. A high-spin state of iron in the Cys4[Fe] center was assigned to both forms of Na+-NQR.

Identification of the key determinant of the transport promiscuity in Na+-translocating rhodopsins.

Bacterial Na+-transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na+, but some can additionally transport H+. To determine the structural basis of cation promiscuity in Na+-rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na+-rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na+-rhodopsin (Kr2). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na+-rhodopsin (NaR) was indeed found to transport both Na+ and H+ in a light-dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation-capture step by comparison with the wild-type NaR in a Na+-deficient medium. The structural basis for the dependence of the Na+/H+ specificity in Na+-rhodopsin on residue 253 remains to be determined.

Engineering a carotenoid-binding site in Dokdonia sp. PRO95 Na+-translocating rhodopsin by a single amino acid substitution.

Light-driven H+, Cl- and Na+ rhodopsin pumps all use a covalently bound retinal molecule to capture light energy. Some H+-pumping rhodopsins (xanthorhodopsins; XRs) additionally contain a carotenoid antenna for light absorption. Comparison of the available primary and tertiary structures of rhodopsins pinpointed a single Thr residue (Thr216) that presumably prevents carotenoid binding to Na+-pumping rhodopsins (NaRs). We replaced this residue in Dokdonia sp. PRO95 NaR with Gly, which is found in the corresponding position in XRs, and produced a variant rhodopsin in a ketocarotenoid-synthesising Escherichia coli strain. Unlike wild-type NaR, the isolated variant protein contained the tightly bound carotenoids canthaxanthin and echinenone. These carotenoids were visible in the absorption, circular dichroism and fluorescence excitation spectra of the Thr216Gly-substituted NaR, which indicates their function as a light-harvesting antenna. The amino acid substitution and the bound carotenoids did not affect the NaR photocycle. Our findings suggest that the antenna function was recently lost during NaR evolution but can be easily restored by site-directed mutagenesis.