Flavin Binding Research Articles

Apical meristem transcriptome analysis identifies a role for the blue light receptor gene GhFKF1 in cotton architecture development

Cotton architecture is determined by the differentiation fate transition of axillary meristem (AM), and influences cotton yield and the efficiency of mechanized harvesting. We observed that the initiation of flowering primordium was earlier in early-maturing than that in late-maturing cultivars during the differentiation and development of AM. The RNA-Seq and expression level analyses showed that genes FLAVIN BINDING, KELCH REPEAT, F-BOX1 (GhFKF1), and GIGANTEA (GhGI) were in response to circadian rhythms, and involved in the regulation of cotton flowering. The gene structure, predicted protein structure, and motif content analyses showed that in Arabidopsis, cotton, rapseed, and soybean, proteins GhFKF1 and GhGI were functionally conserved and share evolutionary origins. Compared to the wild type, in GhFKF1 mutants that were created by the CRISPR/Cas9 system, the initiation of branch primordium was inhibited. Conversely, the knocking out of GhGI increased the number of AM differentiating into flower primordium, and there were much more lateral branch differentiation and development. Besides, we investigated that proteins GhFKF1 and GhGI can interact with each other. These results suggest that GhFKF1 and GhGI are key regulators of cotton architecture development, and may collaborate to regulate the differentiation fate transition of AM, ultimately influencing plant architecture. We describe a strategy for using the CRISPR/Cas9 system to increase cotton adaptation and productivity by optimizing plant architecture.

Functional and structural characterization of a thermostable flavin reductase from Geobacillus mahadii Geo-05

Flavin reductases play a vital role in catalyzing the reduction of flavin through NADH or NADPH oxidation. The gene encoding flavin reductase from the thermophilic bacterium Geobacillus mahadii Geo-05 (GMHpaC) was cloned, overexpressed in Escherichia coli BL21 (DE3) pLysS, and purified to homogeneity. The purified recombinant GMHpaC (Class II) contains chromogenic cofactors, evidenced by maximal absorbance peaks at 370 nm and 460 nm. GMHpaC stands out as the most thermostable and pH-tolerant flavin reductase reported to date, retaining up to 95 % catalytic activity after incubation at 70 °C for 30 min and maintaining over 80 % activity within a pH range of 2–12 for 30 min. Furthermore, GMHpaC's catalytic activity increases by 52 % with FMN as a co-factor compared to FAD and riboflavin. GMHpaC, coupled with 4-hydroxyphenylacetate-3-monooxygenase (GMHpaB) from G. mahadii Geo-05, enhances the hydroxylation of 4-hydroxyphenylacetate (HPA) by 85 %. The modeled structure of GMHpaC reveals relatively conserved flavin and NADH binding sites. Modeling and docking studies shed light on structural features and amino acid substitutions that determine GMHpaC's co-factor specificity. The remarkable thermostability, high catalytic activity, and general stability exhibited by GMHpaC position it as a promising enzyme candidate for various industrial applications.

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.

'Seeing' the electromagnetic spectrum: spotlight on the cryptochrome photocycle.

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

Avian cryptochrome 4 binds superoxide

Flavin-binding cryptochromes are blue-light sensitive photoreceptors that have been implicated with magnetoreception in some species. The photocycle involves an intra-protein photo-reduction of the flavin cofactor, generating a magnetosensitive radical pair, and its subsequent re-oxidation. Superoxide (O2•−) is generated in the re-oxidation with molecular oxygen. The resulting O2•−-containing radical pairs have also been hypothesised to underpin various magnetosensitive traits, but due to fast spin relaxation when tumbling in solution would require immobilisation. We here describe our insights in the binding of superoxide to cryptochrome 4 from C. livia based on extensive all-atom molecular dynamics studies and density-functional theory calculations. The positively charged “crypt” region that leads to the flavin binding pocket transiently binds O2•− at 5 flexible binding sites centred on arginine residues. Typical binding times amounted to tens of nanoseconds, but exceptional binding events extended to several hundreds of nanoseconds and slowed the rotational diffusion, thereby realising rotational correlation times as large as 1 ns. The binding sites are particularly efficient in scavenging superoxide escaping from a putative generation site close to the flavin-cofactor, possibly implying a functional relevance. We discuss our findings in view of a potential magnetosensitivity of biological flavin semiquinone/superoxide radical pairs.

Mediating the performance of anaerobic granular sludge by exogenous flavin supplementation: Flavin binding capacity and behavior, interspecies electron transfer, and methane production

Although flavins are known as effective electron mediators, the binding capacity of exogenous flavins by anaerobic granular sludge (AGS) and their role in interspecies electron transfer (IET) remains unknown. In this study, AGS was mediated by using three exogenous flavins of riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Results showed that the total amounts of flavins associated with extracellular polymeric substance (EPS) of AGS increased by 2.03–2.42 and 3.83–4.94 folds, after exposure to 50 and 200 μM of exogenous flavins, respectively. A large portion of FMN and FAD was transformed into RF by AGS. Exogenous flavin mediation also stimulated the production of EPS and cytochrome c (c-Cyts) as well as cytochrome-bound flavins. The increased abundance of these electron mediators led to a reduced electrochemical impedance of EPS and improved extracellular electron transfer capacity. The methane production of AGS after mediation with exogenous RF, FMN, and FAD increased by 19.03–31.71%, 22.86–26.04%, and 28.51–33.44%, respectively. This study sheds new light on the role of exogenous flavins in promoting the IET process of a complex microbial aggregate of AGS.

Delineation of novel genomic loci and putative candidate genes associated with seed iron and zinc content in lentil (Lens culinaris Medik.)

The use of molecular breeding approaches for development of lentil genotypes biofortified with essential micro-nutrients such as iron and zinc, could serve as a promising solution to address the problem of global malnutrition. Thus, genome-wide association study (GWAS) strategy was adopted in this study to identify the genomic regions associated with seed iron and zinc content in lentil. A panel of 95 diverse lentil genotypes, grown across three different geographical locations and evaluated for seed iron and zinc content, exhibited a wide range of variation. Genotyping-by-sequencing (GBS) analysis of the panel identified 33,745 significant single nucleotide polymorphisms (SNPs) that were distributed across all the 7 lentil chromosomes. Association mapping revealed 23 SNPs associated with seed iron content that were distributed across all the chromosomes except chromosome 3. Similarly, 14 SNPs associated with seed zinc content were also identified that were distributed across chromosomes 1, 2, 4, 5 and 6. Further, 80 genes were identified in the proximity of iron associated markers and 36 genes were identified in the proximity of zinc associated markers. Functional annotation of these genes revealed their putative involvement in iron and zinc metabolism. For seed iron content, two highly significant SNPs were found to be located within two putative candidate genes namely iron-sulfur cluster assembly (ISCA) and flavin binding monooxygenase (FMO) respectively. For zinc content, a highly significant SNP was detected in a gene encoding UPF0678 fatty acid-binding protein. Expression analysis of these genes and their putative interacting partners suggests their involvement in iron and zinc metabolism in lentil. Overall, in this study we have identified markers, putative candidate genes and predicted putative interacting protein partners significantly associated with iron and zinc metabolism that could be utilized in future breeding studies of lentil for nutrient biofortification.

Integrated Computational Study of the Light-Activated Structure of the AppA BLUF Domain and Its Spectral Signatures.

We apply an integrated approach combining microsecond MD simulations and (polarizable) QM/MM calculations of NMR, FTIR, and UV-vis spectra to validate the structure of the light-activated form of the AppA photoreceptor, an example of blue light using flavin (BLUF) protein domain. The latter photoactivate through a proton-coupled electron transfer (PCET) that results in a tautomerization of a conserved glutamine residue in the active site, but this mechanism has never been spectroscopically proven for AppA, which has been always considered as an exception. Our simulations instead confirm that the spectral features observed upon AppA photoactivation are indeed directly connected to the tautomer form of glutamine as predicted by the PCET mechanism. In addition, we observe small but significant changes in the AppA structure, which are transmitted from the flavin binding pocket to the surface of the protein.

OsCRY2 and OsFBO10 co-regulate photomorphogenesis and photoperiodic flowering in indica rice

Cryptochromes (CRYs) are a class of photoreceptors that perceive blue/ultraviolet-A light of the visible spectrum to mediate a vast number of physiological responses in bacteria, fungi, animals and plants. In the present study, we have characterized OsCRY2 in a photoperiod sensitive indica variety, Basmati 370, by generating and analyzing overexpression (OE) and knock-down (KD) transgenic lines. The OsCRY2OE lines displayed dwarfism as shown in their reduced plant height and leaf length, attributed largely by an overall reduction in their cell size. The OsCRY2OE lines flowered significantly earlier and showed shorter and broader seeds with an overall reduced seed weight. The OsCRY2KD lines showed contrasting phenotypes, such as increased plant height and delayed flowering, however, decreased seed size and weight were also observed in the KD lines, along with reduced spikelet fertility and high seed shattering rate in mature panicles. Novel interactions were confirmed between OsCRY2 and members of ZEITLUPE family of blue/ultraviolet-A light photoreceptors, encoded by OsFBO8, OsFBO9 and OsFBO10 which are orthologous to ZEITLUPE (ZTL), LOV KELCH PROTEIN2 (LKP2) and FLAVIN BINDING, KELCH REPEAT F-BOX1 (FKF1), respectively, of Arabidopsis thaliana. Since FKF1 is known to play a role in regulating photoperiodic flowering, OsFBO10 was chosen for further studies. OsCRY2 and OsFBO10 interacted in the nucleus and cytoplasm of the cell and cross-regulated the expression of each other. They were also found to regulate the expression of several genes involved in photoperiodic flowering in rice. Both OsCRY2 and OsFBO10 played a positive role in photomorphogenic responses in different light conditions. The physical interaction of OsCRY2 with OsFBO10, their involvement in common physiological and developmental pathways and their cross-regulation of each other suggest that the two photoreceptors may regulate common developmental pathways in plants, either jointly or redundantly.

Responses of anammox and sulfur/pyrite autotrophic denitrification in one-stage system to high nitrogen load: Performance, metabolic and bacterial community

To remove residual nitrate from anammox process and achieve efficient nitrogen removal, a two-stage system (TAS) with the two individual reactors and a one-stage system (OAS) with the spatial functional areas in one reactor were established via anammox coupling sulfur autotrophic denitrification. The total nitrogen removal efficiency (TNRE) of OAS system (97.85 ± 1.92%) was higher than that of TAS system (93.63 ± 1.87%) under the influent NH4+-N and NO2−-N of 227 and 300 mg/L. Meanwhile, the responses of microbial metabolism to high nitrogen load were investigated in term of microbial metabolites, electron transfer and metabolic activity. Microbial metabolites characteristics demonstrated that the OAS system secreted more EPS with lower protein (PN)/polysaccharide (PS) ratio than that in the TAS system, which was beneficial to protect bacteria from high nitrogen load. Electrochemical analysis suggested that the secretion of electron conductive substance (such as PN, PS) and redox active substances (such as flavin mononucleotide, the binding of flavins and cytochrome c on the outer membrane) were increased in the OAS system, which promoted the electron transfer efficiency. Moreover, the electron transport system activity (ETSA) values and ATP contents in OAS system were higher than that in the TAS system, which indicated that metabolic activity was improved in OAS system under the stimulation of high nitrogen load. Additionally, the bacterial community analysis indicated that the functional bacteria of Candidatus_Kuenenia and Armatimonadetes_gp5 had higher abundance in the OAS system than that in the TAS system, which was beneficial to realize the stable nitrogen removal performance. Overall, the responses mechanism of the OAS system was established to explain the resistant to high nitrogen load.

Understanding the effects of higher-order assembly on the structure of the sulfite reductase.

The redox state of sulfur determines its bioavailbility and its reduction to sulfide is required for assimilation of sulfur into amino acids and cofactors.The NADPH-dependent assimilatory sulfite reductase (SiR) is a metabolic enzyme that performs the six-electron reduction of sulfite to sulfide. Escherichia coli SiR is composed of eight flavoprotein (SiRFP, ⍺) and four hemoprotein (SiRHP, β) subunits that associate in α8:β4 holoenzyme. This dodecameric assembly is unique because SiRs from other sulfur reducing organisms are dimers of reductase and oxidase subunits. Both SiR subunits are modular. SiRFP is a fusion between flavodoxin domain and a ferredoxin-NADP+ reductase domain that assembles through its 52 N-terminal amino acids into an octamer. SiRHP is a monomeric metalloenzyme that houses the active site. Despite almost 30 years of effort across many laboratories, structures of SiR complexes have remained recalcitrant to X-ray crystallography and cryo-EM because of its structural heterogeneity aising from intrinsically disordered regions throughout the complex, including the flexible linker joining SiRFP's flavin binding domain. Thus we do not know how the domains assemble, which leaves a gap in understanding about how these domains coordinate to perform electron transfer. Here, we use neutron contrast variation (NCV) and small angle neutron scattering (SANS) to observe the relative subunit positioning within the SiR complex. To better understand the electron transfer mechanism, we have generated conformationally restricted variant of SiRFP that either locks its flavin binding domains in an open conformation by shortening the linker between domains, or in closed conformation by engineering disulfide bonds. SANS and NCV study reveal SiR's asymmetric complex, and the resulting maps supports a redundant, cis/trans mechanism of electron transfer between the reductase subunits as well as between the tightly or transiently bound reductase and oxidase domains.

Characterization of two bacterial multi-flavinylated proteins harboring multiple covalent flavin cofactors

In recent years, studies have shown that a large number of bacteria secrete multi-flavinylated proteins. The exact roles and properties, of these extracellular flavoproteins that contain multiple covalently anchored FMN cofactors, are still largely unknown. Herein, we describe the biochemical and structural characterization of two multi-FMN-containing covalent flavoproteins, SaFMN3 from Streptomyces azureus and CbFMN4 from Clostridiaceae bacterium. Based on their primary structure, these proteins were predicted to contain three and four covalently tethered FMN cofactors, respectively. The genes encoding SaFMN3 and CbFMN4 were heterologously coexpressed with a flavin transferase (ApbE) in Escherichia coli, and could be purified by affinity chromatography in good yields. Both proteins were found to be soluble and to contain covalently bound FMN molecules. The SaFMN3 protein was studied in more detail and found to display a single redox potential (-184 mV) while harboring three covalently attached flavins. This is in line with the high sequence similarity when the domains of each flavoprotein are compared. The fully reduced form of SaFMN3 is able to use dioxygen as electron acceptor. Single domains from both proteins were expressed, purified and crystallized. The crystal structures were elucidated, which confirmed that the flavin cofactor is covalently attached to a threonine. Comparison of both crystal structures revealed a high similarity, even in the flavin binding pocket. Based on the crystal structure, mutants of the SaFMN3-D2 domain were designed to improve its fluorescence quantum yield by changing the microenvironment of the isoalloxazine moiety of the flavin cofactor. Residues that quench the flavin fluorescence were successfully identified. Our study reveals biochemical details of multi-FMN-containing proteins, contributing to a better understanding of their role in bacteria and providing leads to future utilization of these flavoprotein in biotechnology.

Heterologous Expression and Characterization of a Full-length Protozoan Nitroreductase from Leishmania orientalis isolate PCM2.

Leishmaniasis, a parasitic disease found in parts of the tropics and subtropics, is caused by Leishmania protozoa infection. Nitroreductases (NTRs), enzymes involved in nitroaromatic prodrug activation, are attractive targets for leishmaniasis treatment development. In this study, a full-length recombinant NTR from the Leishmania orientalis isolate PCM2 (LoNTR), which causes severe leishmaniasis in Thailand, was successfully expressed in soluble form using chaperone co-expression in Escherichia coli BL21(DE3). The purified histidine-tagged enzyme (His6-LoNTR) had a subunit molecular mass of 36kDa with no cofactor bound; however, the addition of exogenous flavin (either FMN or FAD) readily increased its enzyme activity. Bioinformatics analysis found that the unique N-terminal sequences of LoNTR is only present in Leishmania where the addition of this region might result in the loss of flavin binding. Either NADH or NADPH can serve as an electron donor to transfer electrons to nitrofurazone; however, NADPH was preferred. Molecular oxygen was identified as an additional electron acceptor resulting in wasteful electrons from NADPH for the main catalysis. Steady-state kinetic experiments revealed a ping-pong mechanism for His6-LoNTR with Km,NADPH, Km,NFZ, and kcat of 28µM, 68µM, and 0.84min-1, respectively. Besides nitroreductase activity, His6-LoNTR also has the ability to reduce quinone derivatives. The properties of full-length His6-LoNTR were different from previously reported protozoa and bacterial NTRs in many respects. This study provides information of NTR catalysis to be developed as a potential future therapeutic target to treat leishmaniasis.

Phylogenomics-Based Reconstruction and Molecular Evolutionary Histories of Brassica Photoreceptor Gene Families.

Photosensory proteins known as photoreceptors (PHRs) are crucial for delineating light environments in synchronization with other environmental cues and regulating their physiological variables in plants. However, this has not been well studied in the Brassica genus, which includes several important agricultural and horticultural crops. Herein, we identified five major PHR gene families—phytochrome (PHY), cryptochrome (CRY), phototropin (PHOT), F-box containing flavin binding proteins (ZTL/FKF1/LKP2), and UV RESISTANCE LOCUS 8 (UVR8)—genomic scales and classified them into subfamilies based on their phylogenetic clustering with Arabidopsis homologues. The molecular evolution characteristics of Brassica PHR members indicated indirect expansion and lost one to six gene copies at subfamily levels. The segmental duplication was possibly the driving force of the evolution and amplification of Brassica PHRs. Gene replication retention and gene loss events of CRY, PHY, and PHOT members found in diploid progenitors were highly conserved in their tetraploid hybrids. However, hybridization events were attributed to quantitative changes in UVR8 and ZTL/FKF1/LKP2 members. All PHR members underwent purifying selection. In addition, the transcript expression profiles of PHR genes in different tissue and in response to exogenous ABA, and abiotic stress conditions suggested their multiple biological significance. This study is helpful in understanding the molecular evolution characteristics of Brassica PHRs and lays the foundation for their functional characterization.

Flavin binding affinity and initial kinetic characterization of DnmZ, a flavin‐dependent N‐oxygenase

Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius. It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a derivatized form of doxorubicin with an additional seven‐carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N‐oxygenase that oxidizes a sugar‐linked exocyclic amine, triggering a nonenzymatic retro oxime‐aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ‐catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases.Our transient‐state experiments confirm that DnmZ catalyzes the formation of the C4a‐(hydro)peroxyflavin intermediate (C4a‐OOH). We have identified the appropriate wavelengths to monitor to observe C4a‐OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 mM, which compares well with other Class D monooxygenases. Our preliminary results suggest that increasing pH speeds up C4a‐OOH elimination but has no effect on the intermediate’s formation. We are also investigating the effect of mutating active site residues on C4a‐OOH formation and elimination.

The bare bones of bifurcation: small flavoproteins as artificial electron bifurcases

Flavin‐based electron bifurcation (FBEB) is an essential mode of energy conservation in bacteria and archaea wherein specific flavoenzymes couple exergonic and endergonic metabolic electron flux from a single electron donor. While our understanding of the role of FBEB in metabolism continues to grow, the mechanism by which the flavin cofactor and the protein scaffold coordinate electron bifurcation remains elusive, largely due to the complexity of native bifurcases. We have leveraged the light‐oxygen‐voltage (LOV) domain flavoprotein as a robust and straight‐forward scaffold to interrogate the operant principles of electron bifurcation, including the role of the flavin binding pocket on tuning the flavin redox potential, proton‐coupled electron transfer, and potential “inversion,” as well as the role of conformational gating. Using bioinformatics and structural homology, we have remodeled the LOV flavin binding site to mimic that of native bifurcases and demonstrated key residues stabilize the anionic hydroquinone and increases potential inversion by 60 mV relative to wild‐type LOV. Furthermore, reactions with single electron donors/acceptors implicates a reactive neutral semiquinone, and lay the groundwork for kinetic electron bifurcation studies. Using cytochrome c as a model one‐electron acceptor, we can model the conformational gating mechanism by controlling donor‐acceptor concentrations, and have demonstrated the facile inter‐protein electron transfer, and photochemical crosslinking indicates a unique electron transfer interface. Kinetic modeling, including short‐circuit pathways, suggests that a bifurcation prone state should be long enough lived for direct observation, and studies are ongoing to spectroscopically identify this species. These results illustrate the utility of this reductionist approach in gaining insight into the physical principles that dictate FBEB and inform novel bifurcase design.

The ins and outs of the flavin mononucleotide cofactor of respiratory complex I.

The flavin mononucleotide (FMN) cofactor of respiratory complex I occupies a key position in the electron transport chain. Here, the electrons coming from NADH start the sequence of oxidoreduction reactions, which drives the generation of the proton-motive force necessary for ATP synthesis. The overall architecture and the general catalytic proprieties of the FMN site are mostly well established. However, several aspects regarding the complex I flavin cofactor are still unknown. For example, the flavin binding to the N-module, the NADH-oxidizing portion of complex I, lacks a molecular description. The dissociation of FMN from the enzyme is beginning to emerge as an important regulatory mechanism of complex I activity and ROS production. Finally, how mitochondria import and metabolize FMN is still uncertain. This review summarizes the current knowledge on complex I flavin cofactor and discusses the open questions for future research.

Role of Plasma Membrane Redox Activities in Immune Response Mechanisms

Abstract: The plasma membrane redox system (PMRS) is an important component of the cell's ability to defend itself against oxidative stress. Many immune signaling pathways are regulated through redox reactions. Biological systems utilize oxidationreduction reactions to modulate their responses to environmental cues. The role of redox molecules such as NO and ROS as key mediators of immunity has recently gathered a lot of interest and attention. Beyond the chemical interactions of NO and ROS that combine to eradicate pathogens, these redox small molecules are effective immune-modulators that regulate cellular metabolism as well as multiple pro-inflammatory and repair/tissue-restoration pathways. Redox molecules such as peroxide, superoxide, NO, and RNS, once thought to be only toxic, are essential in tissue repair. These species are generated, converted and metabolized during host microbe interaction involving the innate immune system. Cytochrome b558 is the flavin binding component of the NADPH oxidase. NADPH oxidases are key producers of ROS. A variety of RNS and ROS is produced in the acidic mileu of phagosomes, which provide an environment conducive to the redox chemistry, which is the first line in fighting infection. Bacterial cell immune response also involves NO. Thus understanding the plasma membrane redox activities can help unravel the mechanisms of immune response. Keywords: Plasma membrane, Redox activities, oxidative stress, NO, ROS, RNS. Nitrous Oxide, Reactive Oxygen Species, Reactive Nitrogen species.

Tailoring Surface Properties of Electrodes for Synchronous Enhanced Extracellular Electron Transfer and Enriched Exoelectrogens in Microbial Fuel Cells.

An extracellular electron transfer (EET) process between an electroactive biofilm and an electrode is a crucial step for the performance of microbial fuel cells (MFCs), which is highly related to the enrichment of exoelectrogens and the electrocatalytic activity of the electrode. Herein, an efficient N- and Fe-abundant carbon cloth (CC) electrode with the comodification of iron porphyrin (FePor) and polyquaternium-7 (PQ) was synthesized using a facile solvent evaporation and immersion method and developed as an anode (named FePor-PQ) in MFCs. The surface structural characterizations confirmed the successful introduction of N and Fe atoms, whereas FePor-PQ achieved the N content of 9.59 at %, which may offer various active sites for EET. The introduction of PQ contributed to improving the surface hydrophilicity, providing the composite electrode good biocompatibility for bacterial attachment and colonization as well as substrate diffusion. Based on the advantages, the MFC with the FePor-PQ anode produced a maximum power density of 2165.7 mW m-2, strikingly higher than those of CC (1124.0 mW m-2), PQ (1668.8 mW m-2), and FePor (1978.9 mW m-2). Furthermore, with the EET mediated by the binding of flavins and c-type cytochromes on the outer membrane was enhanced prominently, the typical exoelectrogen Geobacter was enriched up to 55.84% in the FePor-PQ anode biofilm. This work reveals a synergistic effect from heteroatom coating and surface properties tailoring to boost both the EET efficiency and exoelectrogen enrichment for enhancing MFC performance, which also provides valuable insights for designing electrodes in other bio-electrochemical systems.