Electron Transfer Proteins Research Articles

Association between an electron transfer flavoprotein alpha subunit polymorphism (rs321948383) and the meat quality of Berkshire pigs

ABSTRACTThe electron transfer flavoprotein α subunit gene (ETFA) encodes a protein that forms part of the ETF enzyme. This enzyme is normally active in mitochondria, the energy-producing centres of cells, to donate electrons derived from fatty acid oxidation to ETF- ubiquinone oxidoreductase. In the present study, we identified a non-synonymous single-nucleotide polymorphism (nsSNP) (rs321948383, c.569G > A) in the porcine ETFA gene and analyzed the association between this nsSNP and meat quality traits in 405 Berkshire pigs. The pigs with rs321948383 G/G had a higher meat quality from the aspects of meat colour (L*, p = 0.013), water-holding capacity (p = 0.001), drip loss (p = 0.000), carcass temperatures (T1, p = 0.000; T4, p = 0.000; T12, p = 0.000; T24, p = 0.001), and pH24 (p = 0.014). Therefore, this nsSNP (c.569G > A) could play an important role in improving the meat quality of pigs.

Membrane-spanning electron transfer proteins from electrogenic bacteria: Production and investigation.

Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation. Industrially relevant oxidation reactions can proceed exclusively when electrons are released to anodes. Reduced products such as fuels can be generated when electrons are provided from (photo)cathodes. Rational development of these opportunities and inspiration for novel technologies is underpinned by resolution at the molecular level of pathways supporting electron exchange across bacterial cell envelopes. This chapter describes methods for purification, engineering, and in vitro characterization of proteins providing the primary route for electron transport across the outer membrane lipid bilayer of Shewanella oneidensis MR-1, a well-described electrogenic bacterium and chassis organism for related biotechnologies.

Self-assembled nanocontainer mediated oxidation of Fe(ii) by Cu(ii)–neocuproine complex: a model system to emulate electron transfer proteins

Catalysis of coordination inspired Fe(ii) oxidation by Cu(ii)–neocuproine complex.

Molecular Basis for Converting (2S)-Methylsuccinyl-CoA Dehydrogenase into an Oxidase.

Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S)-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S)-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S)-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS) based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

Differential impact of amino acids on OXPHOS system activity following carbohydrate starvation in Arabidopsis cell suspensions.

Plant respiration mostly depends on the activity of glycolysis and the oxidation of organic acids in the tricarboxylic acid cycle to synthesize ATP. However, during stress situations plant cells also use amino acids as alternative substrates to donate electrons through the electron-transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (ETF/ETFQO) complex to the mitochondrial electron transport chain (mETC). Given this, we investigated changes of the oxidative phosphorylation (OXPHOS) system in Arabidopsis thaliana cell culture under carbohydrate starvation supplied with a range of amino acids. Induction of isovaleryl-CoA dehydrogenase (IVDH) activity was observed under carbohydrate starvation which was associated with increased amounts of IVDH protein detected by immunoblotting. Furthermore, activities of the protein complexes of the mETC were reduced under carbohydrate starvation. We also observed that OXPHOS system activity behavior is differently affected by different amino acids and that proteins associated with amino acids catabolism are upregulated in cells following carbohydrate starvation. Collectively, our results support the contention that ETF/ETFQO is an essential pathway to donate electrons to the mETC and that amino acids are alternative substrates to maintain respiration under carbohydrate starvation.

A novel mutation in ETFDH manifesting as severe neonatal-onset multiple acyl-CoA dehydrogenase deficiency

Neonatal-onset multiple acyl-CoA dehydrogenase deficiency (MADD type I) is an autosomal recessive disorder of the electron transfer flavoprotein function characterized by a severe clinical and biochemical phenotype, including congenital abnormalities with unresponsiveness to riboflavin treatment as distinguishing features. From a retrospective study, relying mainly on metabolic data, we have identified a novel mutation, c.1067G>A (p.Gly356Glu) in exon 8 of ETFDH, in three South African Caucasian MADD patients with the index patient presenting the hallmark features of type I MADD and two patients with compound heterozygous (c.1067G>A+c.1448C>T) mutations presenting with MADD type III. SDS-PAGE western blot confirmed the significant effect of this mutation on ETFDH structural instability. The identification of this novel mutation in three families originating from the South African Afrikaner population is significant to direct screening and strategies for this disease, which amongst the organic acidemias routinely screened for, is relatively frequently observed in this population group.

Insights into the conversion potential of Theobroma cacao L. somatic embryos using quantitative proteomic analysis

Abstract Somatic embryogenesis (SE) has been routinely used as mass micropropagation technique, and as a model system for investigating the structural, physiological, and molecular events occurring during somatic embryo development. Successful in vitro SE is related to the quality and yield of somatic embryos obtained. In cacao (Theobroma cacao L.) SE, the efficiency of somatic embryo production has improved with secondary SE. However, the low number of somatic embryos able to conversion into viable plantlets is still low. Then, two morphological types of normal mature somatic embryos can be identified during cacao secondary SE. The first type shows white appearance and high conversion potential (75%), while the second type shows translucent appearance and exhibit low conversion potential (15%). In order to investigate the proteins that can be associated to conversion potential in cacao somatic embryos, the mass spectrometry HDMSE proteomic approach was used. At least 60 proteins showed differences in abundance levels in cacao white somatic embryos, when compared to translucent. An increased abundance of Beta-glucosidase, NAD(P)-linked oxidoreductase and Electron transfer flavoprotein proteins were observed in white somatic embryos. Moreover, in translucent somatic embryos were observed an increased abundance of Cytochrome P450 and Pathogenesis-related proteins. Using white somatic embryos as a model, we suggest that carbohydrate metabolism process and the redox regulation are involved in the control/regulation of somatic embryo quality. These new findings may improve cacao SE protocol, as well as the understanding of the role of pivotal metabolic pathways associated to this in vitro morphogenetic route.

Commonalities and differences in plants deficient in autophagy and alternative pathways of respiration on response to extended darkness

ABSTRACTAutophagy is a highly conserved cellular mechanism in eukaryotes allowing the degradation of cell constituents. It is of crucial significance in both cellular homeostasis and nutrient recycling. During energy limited conditions plant cells can metabolize alternative respiratory substrates, such as amino acids, providing electrons to the mitochondrial metabolism via the tricarboxylic acid (TCA) cycle or electron transfer flavoprotein/ electron transfer flavoprotein ubiquinone oxidoreductase (ETF/ETFQO) system. Our recent study reveals the importance of autophagy in the supply of amino acids to provide energy through alternative pathways of respiration during carbon starvation. This fact apart, autophagy seems to have more generalized effects related not only to amino acid catabolism but also to metabolism in general. By further comparing the metabolic data obtained with atg mutants with those of mutants involved in the alternative pathways of respiration, we observed clear differences between these mutants, pointing out additional effects of the autophagy deficiency on metabolism of Arabidopsis leaves. Collectively, our data point to an interdependence between mitochondrial metabolism and autophagy and suggest an exquisite regulation of primary metabolism under low energetic conditions.

A panel of protein candidates for comprehensive study of Caprine Arthritis Encephalitis (CAE) infection.

The caprine arthrite encephalite (CAE) is a disease that affects especially dairy goat. The virus shows compartmentalization features, that allows it to hide at certain times during the course of the disease, making it difficult to control. The present study was conducted to identify the major seminal plasma protein profile of goats infected by CAE and its associations with seroconversion using Western blotting. Two groups containing five males each, were used in this experiment. The first group was composed by seropositive animals and the control by seronegative confirmed by Western blotting and PCR. The semen was collected through artificial vagina and after that, two-dimensional electrophoresis and MALDI-TOF MS were used. Seventy-five spots were identified in the goat seminal plasma gels, equivalent to 13 different proteins with more expression. The similar proteins found in both groups and related to reproduction were spermadhesin Z13-like, bodhesin and bodhesin-2, Lipocalin, protein PDC-109-like, and albumin. In infected goats, proteases such as arisulfatase A have been identified, whose function probably is related to metabolism control of sulfatides, involved to virus control. The other ones were bifunctional ATP-dependent dihydroxyacetone kinase/FAD-AMP lyase, cathepsin F isoform X1, disintegrin and metalloproteinase domain-containing protein 2-like isoform X1, clusterin, carbonic anhydrase 2, electron transfer flavoprotein subunit beta, and epididymal secretory glutathione peroxidase. The results of this study show the reaction of the innate immune system against chronic infection of goats by CAE.

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family.

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

Effect of the anode potential on the physiology and proteome of Shewanella oneidensis MR-1

Shewanella species respire using iron and manganese oxides as well as electrodes as solid terminal electron acceptors. Shewanella oneidenis MR-1 exploits mediated as well as direct extracellular electron transfer (EET) modes to transfer electrons at different formal potentials. These different EET modes at different potentials may utilise alternate electron transfer pathways. Therefore, we investigated how different anode potentials, providing different maximum microbial energy gains impacted S. oneidensis microbial physiology. Using quantitative proteomics, comparative analysis of the cellular variations to different anode potentials was performed. A label-free proteomic mass spectrometric analysis method, SWATH-MS, was used to gather quantitative information to determine physiological changes of Shewanella oneidensis MR-1 grown at different anodic potentials. S. oneidensis was cultured and grown in electrochemical cells at the set anode potentials of +0.71V, +0.21V & −0.19V versus SHE reference electrode, while the current production was monitored. At maximum current, electrodes were removed and whole-cell proteins extracted. Subsequent SWATH-MS analysis revealed information on 740 identified proteins across the three electrode potentials. For the first time, we show the abundance of S. oneidensis electron transfer proteins differs with electrode potential.

Compound heterozygous mutations in electron transfer flavoprotein dehydrogenase identified in a young Chinese woman with late-onset glutaric aciduria type II

BackgroundGlutaric aciduria type II (GA II) is an autosomal recessive disorder affecting fatty acid and amino acid metabolism. The late-onset form of GA II disorder is almost exclusively associated with mutations in the electron transfer flavoprotein dehydrogenase (ETFDH) gene. Till now, the clinical features of late-onset GA II vary widely and pose a great challenge for diagnosis. The aim of the current study is to characterize the clinical phenotypes and genetic basis of a late-onset GAII patient.MethodsIn this study, we described the clinical and biochemical manifestations of a 23-year-old female Chinese patient with late-onset GA II, and performed genomic DNA-based PCR amplifications and sequence analysis of ETFDH gene of the whole pedigree. We also used in-silicon tools to analyze the mutation and evaluated the pathogenicity of the mutation according to the criteria proposed by American College of Medical Genetics and Genomics (ACMG).ResultsThe muscle biopsy of this patient revealed lipid storage myopathy. Blood biochemical test and urine organic acid analyses were consistent with GA II. Direct sequence analysis of the ETFDH gene (NM_004453) revealed compound heterozygous mutations: c.250G > A (p.A84T) on exon 3 and c.920C > G (p.S307C) on exon 8. Both mutations were classified as “pathogenic” according to ACMG criteria.ConclusionsIn conclusion, our study described the phenotype and genotype of a late-onset GA II patient, reiterating the importance of ETFDH gene screening in these patients.

Exome array analysis identifies ETFB as a novel susceptibility gene for anthracycline-induced cardiotoxicity in cancer patients.

Anthracyclines are widely used chemotherapeutic drugs that can cause progressive and irreversible cardiac damage and fatal heart failure. Several genetic variants associated with anthracycline-induced cardiotoxicity (AIC) have been identified, but they explain only a small proportion of the interindividual differences in AIC susceptibility. In this study, we evaluated the association of low-frequency variants with risk of chronic AIC using the Illumina HumanExome BeadChip array in a discovery cohort of 61 anthracycline-treated breast cancer patients with replication in a second independent cohort of 83 anthracycline-treated pediatric cancer patients, using gene-based tests (SKAT-O). The most significant associated gene in the discovery cohort was ETFB (electron transfer flavoprotein beta subunit) involved in mitochondrial β-oxidation and ATP production (P=4.16×10-4) and this association was replicated in an independent set of anthracycline-treated cancer patients (P=2.81×10-3). Within ETFB, we found that the missense variant rs79338777 (p.Pro52Leu; c.155C>T) made the greatest contribution to the observed gene association and it was associated with increased risk of chronic AIC in the two cohorts separately and when combined (OR 9.00, P=1.95×10-4, 95% CI 2.83-28.6). We identified and replicated a novel gene, ETFB, strongly associated with chronic AIC independently of age at tumor onset and related to anthracycline-mediated mitochondrial dysfunction. Although experimental verification and further studies in larger patient cohorts are required to confirm our finding, we demonstrated that exome array data analysis represents a valuable strategy to identify novel genes contributing to the susceptibility to chronic AIC.

Inner mechanism of protection of mitochondrial electron-transfer proteins against oxidative damage. Focus on hydrogen peroxide decomposition

It is generally recognized that the mitochondria are the major source of reactive oxygen species including hydrogen peroxide (H2O2). Although the local concentration of H2O2 near the electron-transfer chain is potentially quite high, the chain's components are rarely found to be significantly damaged by H2O2. Our experimental data, as well as the data published by others, suggest that mitochondrial electron-transfer proteins, which are in the first line to be harmed by ROS, are well prepared to defend themselves. One of such protection mechanism involves peroxidase/catalase-like activity of all major mitochondrial respiration chain players, which catalyze the decomposition of H2O2. Understanding the molecular mechanisms, by which mitochondrial electron-transfer proteins might defend themselves against an oxidative stress and therefore being a part of the mitochondrial antioxidant system, can help to clarify many controversial experimental data.

Inter- and Intramolecular Electron Transfer in Copper Complexes: Electronic Entatic State with Redox-Active Guanidine Ligands.

Fast and efficient electron transfer in blue copper proteins is realized by a structural harmonization between the CuI and CuII complex pair ("entatic state" model). Herein, we present now a CuI /CuII complex pair with redox-active guanidine ligands showing almost perfect match between both redox states. By modifying the ligand electron donor strength, the redox chemistry of the copper complex can be controlled to be either metal-centered or to cross the borderline to ligand-centered. This work is the first systematic study of complexes with redox-active ligands within the concept of the entatic state.

Electron Bifurcation: Thermodynamics and Kinetics of Two-Electron Brokering in Biological Redox Chemistry.

How can proteins drive two electrons from a redox active donor onto two acceptors at very different potentials and distances? And how can this transaction be conducted without dissipating very much energy or violating the laws of thermodynamics? Nature appears to have addressed these challenges by coupling thermodynamically uphill and downhill electron transfer reactions, using two-electron donor cofactors that have very different potentials for the removal of the first and second electron. Although electron bifurcation is carried out with near perfection from the standpoint of energy conservation and electron delivery yields, it is a biological energy transduction paradigm that has only come into focus recently. This Account provides an exegesis of the biophysical principles that underpin electron bifurcation. Remarkably, bifurcating electron transfer (ET) proteins typically send one electron uphill and one electron downhill by similar energies, such that the overall reaction is spontaneous, but not profligate. Electron bifurcation in the NADH-dependent reduced ferredoxin: NADP+ oxidoreductase I (Nfn) is explored in detail here. Recent experimental progress in understanding the structure and function of Nfn allows us to dissect its workings in the framework of modern ET theory. The first electron that leaves the two-electron donor flavin (L-FAD) executes a positive free energy "uphill" reaction, and the departure of this electron switches on a second thermodynamically spontaneous ET reaction from the flavin along a second pathway that moves electrons in the opposite direction and at a very different potential. The singly reduced ET products formed from the bifurcating flavin are more than two nanometers distant from each other. In Nfn, the second electron to leave the flavin is much more reducing than the first: the potentials are said to be "crossed." The eventually reduced cofactors, NADH and ferredoxin in the case of Nfn, perform crucial downstream redox processes of their own. We dissect the thermodynamics and kinetics of electron bifurcation in Nfn and find that the key features of electron bifurcation are (1) spatially separated transfer pathways that diverge from a two-electron donor, (2) one thermodynamically uphill and one downhill redox pathway, with a large negative shift in the donor's reduction potential after departure of the first electron, and (3) electron tunneling and activation factors that enable bifurcation, producing a 1:1 partitioning of electrons onto the two pathways. Electron bifurcation is found in the CO2 reducing pathways of methanogenic archaea, in the hydrogen pathways of hydrogenases, in the nitrogen fixing pathway of Fix, and in the mitochondrial charge transfer chain of complex III, cytochrome bc1. While crossed potentials may offer the biological advantage of producing tightly regulated high energy reactive species, neither kinetic nor thermodynamic considerations mandate crossed potentials to generate successful electron bifurcation. Taken together, the theoretical framework established here, focusing on the underpinning electron tunneling barriers and activation free energies, explains the logic of electron bifurcation that enables energy conversion and conservation in Nfn, points toward bioinspired schemes to execute multielectron redox chemistry, and establishes a roadmap for examining novel electron bifurcation networks in nature.

A Highly Sensitive Scanning Electrochemical Microscope Based Method for Studying Enzymatic Proton-Coupled Electron Transfer

Proton-coupled electron transfer (PCET) reactions, such as those used in the electron transport chain in mitochondria for adenosine triphosphate production, photosynthesis or nucleic acid repair, are often important to proper cellular functioning and survival. Despite this, many of the mechanisms and energetics that govern PCET in proteins are not fully understood and call for further examination. One of the limitations to understanding enzymatic PCET reactions includes the limited availability of sensitive bioelectroanalytical techniques for studying this class of biological redox chemistry. In this presentation, we report a new, highly sensitive approach to probe PCET in biochemical systems through the use of a scanning electrochemical microscope (SECM) based chronoamperometric method. The proof-of-concept and feasibility measurements were carried out on surface-immobilized cytochrome c oxidase (CcO) and coenzyme Q10 / cytochrome c reductase (bc 1). The proteins were embedded into unilamellar liposomes and then immobilized onto the surface of a gold-coated silicon wafer electrode through integration of the membrane with various gold-bonded tethers or supported lipid membranes, such as long-chain alkanethiol or thiolated-PEG-cholesterol, while in HEPES-Na buffer (at various pH). Upon contact with the surface of the lipid membrane, the platinum ultramicroelectrode SECM tip (~600 nm) was held at a potential (–0.8 V vs. Ag/AgCl) so that diffusion-controlled hydrogen evolution can take place. This reaction current is proportional to the near-tip proton concentration. Upon application of a potential via the gold-coated silicon wafer, or injection of the protein’s native electron donor, such as reduced cytochrome c, a change in the localized proton concentration was measured (shown in Figure 1). The density of immobilized protein on the surface of the electrode was analyzed by AFM in order to normalize current responses. The technique proved to be successful in detecting sub-picoampere current changes in response to proton-pumping of the transmembrane proteins towards the platinum ultramicroelectrode, which could be used for further investigations in the kinetics of other proton-coupled electron transfer proteins. Figure 1

The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface.

Achieving fast electron transfer between a material and protein is a long-standing challenge confronting applications in bioelectronics, bioelectrocatalysis, and optobioelectronics. Interestingly, naturally occurring extracellular electron transfer proteins bind to and reduce metal oxides fast enough to enable cell growth, and thus could offer insight into solving this coupling problem. While structures of several extracellular electron transfer proteins are known, an understanding of how these proteins bind to their metal oxide substrates has remained elusive because this abiotic-biotic interface is inaccessible to traditional structural methods. Here, we use advanced footprinting techniques to investigate binding between the Shewanella oneidensis MR-1 extracellular electron transfer protein MtrF and one of its substrates, α-Fe2O3 nanoparticles, at the molecular level. We find that MtrF binds α-Fe2O3 specifically, but not tightly. Nanoparticle binding does not induce significant conformational changes in MtrF, but instead protects specific residues on the face of MtrF likely to be involved in electron transfer. Surprisingly, these residues are separated in primary sequence, but cluster into a small 3D putative binding site. This binding site is located near a local pocket of positive charge that is complementary to the negatively charged α-Fe2O3 surface, and mutational analysis indicates that electrostatic interactions in this 3D pocket modulate MtrF-nanoparticle binding. Strikingly, these results show that binding of MtrF to α-Fe2O3 follows a strategy to connect proteins to materials that resembles the binding between donor-acceptor electron transfer proteins. Thus, by developing a new methodology to probe protein-nanoparticle binding at the molecular level, this work reveals one of nature's strategies for achieving fast, efficient electron transfer between proteins and materials.

Protein complex formation during denitrification by Pseudomonas aeruginosa.

SummaryThe most efficient means of generating cellular energy is through aerobic respiration. Under anaerobic conditions, several prokaryotes can replace oxygen by nitrate as final electron acceptor. During denitrification, nitrate is reduced via nitrite, NO and N2O to molecular nitrogen (N2) by four membrane‐localized reductases with the simultaneous formation of an ion gradient for ATP synthesis. These four multisubunit enzyme complexes are coupled in four electron transport chains to electron donating primary dehydrogenases and intermediate electron transfer proteins. Many components require membrane transport and insertion, complex assembly and cofactor incorporation. All these processes are mediated by fine‐tuned stable and transient protein–protein interactions. Recently, an interactomic approach was used to determine the exact protein–protein interactions involved in the assembly of the denitrification apparatus of Pseudomonas aeruginosa. Both subunits of the NO reductase NorBC, combined with the flavoprotein NosR, serve as a membrane‐localized assembly platform for the attachment of the nitrate reductase NarGHI, the periplasmic nitrite reductase NirS via its maturation factor NirF and the N2O reductase NosZ through NosR. A nitrate transporter (NarK2), the corresponding regulatory system NarXL, various nitrite (NirEJMNQ) and N2O reductase (NosFL) maturation proteins are also part of the complex. Primary dehydrogenases, ATP synthase, most enzymes of the TCA cycle, and the SEC protein export system, as well as a number of other proteins, were found to interact with the denitrification complex. Finally, a protein complex composed of the flagella protein FliC, nitrite reductase NirS and the chaperone DnaK required for flagella formation was found in the periplasm of P. aeruginosa. This work demonstrated that the interactomic approach allows for the identification and characterization of stable and transient protein–protein complexes and interactions involved in the assembly and function of multi‐enzyme complexes.

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II.

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of −483 ± 1 mV and −187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.