Structure, dynamics, and redox reactivity of an all-purpose flavodoxin.

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial enzymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld's ability to work with diverse partners, we solved its crystal structure. We observed the canonical flavodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize partner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies of protein-protein associations and conformational consequences, we assigned the 19F NMR signals of all five tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3-19F-Tyr in place of Tyr has only a modest effect on Rp9Fld's redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the 19F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However, the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We propose that the loops surrounding the flavin adopt altered conformations upon binding to partners and may even participate actively in electron transfer.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

A dimer-monomer transition captured by the crystal structures of cyanobacterial apo flavodoxin.

In cyanobacteria and algae (but not plants), flavodoxin (Fld) replaces ferredoxin (Fd) under stress conditions to transfer electrons from photosystem I (PSI) to ferredoxin-NADP+ reductase (FNR) during photosynthesis. Fld constitutes a small electron carrier noncovalently bound to flavin mononucleotide (FMN), and also an ideal model for revealing the protein/flavin-binding mechanism because of its relative simplicity compared to other flavoproteins. Here, we report two crystal structures of apo-Fld from Synechococcus sp. PCC 7942, one dimeric structure of 2.09 Å and one monomeric structure of 1.84 Å resolution. Analytical ultracentrifugation showed that in solution, apo-Fld exists both as monomers and dimers. Our dimer structure contains two ligand-binding pockets separated by a distance of 45 Å, much longer than the previous structures of FMN-bound dimers. These results suggested a potential dimer-monomer transition mechanism of cyanobacterial apo-Fld. We further propose that the dimer represents the "standby" state to stabilize itself, while the monomer constitutes the "ready" state to bind FMN. Furthermore, we generated a new docking model of cyanobacterial Fld-FNR complex based on the recently reported cryo-EM structures, and mapped the special interactions between Fld and FNR in detail.

A look at the face of the molten globule: Structural model of the Helicobacter pylori apoflavodoxin ensemble at acidic pH.

Molten globule (MG) is the name given to a compact, non-native conformation of proteins that has stimulated the imagination and work in the protein folding field for more than 40 years. The MG has been proposed to play a central role in the folding reaction and in important cell functions, and to be related to the onset of misfolding diseases. Due to its inherent intractability to high-resolution studies, atomistic structural models have not yet been obtained. We present here an integrative atomistic model of the MG formed at acidic pH by the apoflavodoxin from the human pathogen Helicobacter pylori. This MG has been previously shown to exhibit the archetypical expansion, spectroscopic and thermodynamic features of a molten conformation. To obtain the model, we have analyzed the stability of wild-type and 55 apoflavodoxin mutants to derive experimental equilibrium Φ values that have been used in biased molecular dynamics simulations to convert the native conformation into an MG ensemble. The ensemble has been refined to reproduce the experimental hydrodynamic radius and circular dichroism (CD) spectrum. The refined ensemble, deposited in PDB-Dev, successfully explains the characteristic 1 H-nuclear magnetic resonance (NMR) and near-UV CD spectral features of the MG as well as its solvent-accessible surface area (SASA) change upon unfolding. This integrative model of an MG will help to understand the energetics and roles of these elusive conformations in protein folding and misfolding. Interestingly, the apoflavodoxin MG is structurally unrelated to previously described partly unfolded conformations of this protein, exemplifying that equilibrium MGs need not to reflect the properties of kinetic intermediates.

Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the ß subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.

Ultrafast Dynamics of Nonequilibrium Short-Range Electron Transfer in Semiquinone Flavodoxin.

Short-range protein electron transfer (ET) is crucially important in light-induced biological processes such as in photoenzymes and photoreceptors and often occurs on time scales similar to those of environment fluctuations, leading to a coupled dynamic process. Herein, we use semiquinone Anabaena flavodoxin to characterize the ultrafast photoinduced redox cycle of the wild type and seven mutants by ultrafast spectroscopy. We have found that the forward and backward ET dynamics show stretched behaviors in a few picoseconds (1-5 ps), indicating a coupling with the local protein fluctuations. By comparison with the results from semiquinone D. vulgaris flavodoxin, we find that the electronic coupling is crucial to the ET rates. With our new nonergodic model, we obtain smaller values of the outer reorganization energy (λoγ) of environment fluctuations and the reaction free energy force (ΔGγ), a signature of nonequilibrium ET dynamics.

Ultrafast nonequilibrium dynamics of short-range protein electron transfer in flavodoxin.

Short-range protein electron transfer (ET) is ubiquitous in biology and is often observed in photosynthesis, photoreceptors and photoenzymes. These ET processes occur on an ultrafast timescale from femtoseconds to picoseconds at a short donor-acceptor distance within 10 Å, and thus couple with local environmental fluctuations. Here, we use oxidized Anabaena flavodoxin as a model system and have systematically studied the photoinduced redox cycle of the wild type and seven mutant proteins by femtosecond spectroscopy. We observed a series of ultrafast dynamics from the initial charge separation in 100-200 fs, subsequent charge recombination in 1-2 ps and final vibrational cooling process of the products in 3-6 ps. We further characterized the active-site solvation and observed the relaxations in 1-200 ps, indicating a nonergodic ET dynamics. With our new ET model, we uncovered a minor outer (solvent) reorganization energy and a large inner (donor and acceptor) reorganization energy, suggesting a frozen active site in the initial ultrafast ET while the back ET couples with the environment relaxations. The vibronically coupled back ET dynamics was first reported in D. vulgaris flavodoxin and here is observed in Anabaena flavodoxin again, completely due to the faster ET dynamics than the cooling relaxations. We also compared the two flavodoxin structures, revealing a stronger coupling with the donor tyrosine in Anabaena. All ultrafast ET dynamics are from the large donor-acceptor couplings and the minor activation barriers due to the reaction free energies being close to the inner reorganization energies. These observations should be general to many redox reactions in flavoproteins.

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Assignments of 19F NMR resonances and exploration of dynamics in a long-chain flavodoxin.

Flavodoxin is a small protein that employs a non-covalently bound flavin to mediate single-electron transfer at low potentials. The long-chain flavodoxins possess a long surface loop that is proposed to interact with partner proteins. We have incorporated 19F-labeled tyrosine in long-chain flavodoxin from Rhodopseudomonas palustris to gain a probe of possible loop dynamics, exploiting the presence of a Tyr in the long loop in addition to Tyr residues near the flavin. We report 19F resonance assignments for all four Tyrs, and demonstration of a pair of resonances in slow exchange, both corresponding to a Tyr adjacent to the flavin. We also provide evidence for dynamics affecting the Tyr in the long loop. Thus, we show that 19F NMR of 19F-Tyr labeled flavodoxin holds promise for monitoring possible changes in conformation upon binding to partner proteins.

An aspartyl protease-mediated cleavage regulates structure and function of a flavodoxin-like protein and aids oxidative stress survival.

A family of eleven glycosylphosphatidylinositol-anchored aspartyl proteases, commonly referred to as CgYapsins, regulate a myriad of cellular processes in the pathogenic yeast Candida glabrata, but their protein targets are largely unknown. Here, using the immunoprecipitation-mass spectrometry approach, we identify the flavodoxin-like protein (Fld-LP), CgPst2, to be an interactor of one of the aspartyl protease CgYps1. We also report the presence of four Fld-LPs in C. glabrata, which are required for survival in kidneys in the murine model of systemic candidiasis. We further demonstrated that of four Fld-LPs, CgPst2 was solely required for menadione detoxification. CgPst2 was found to form homo-oligomers, and contribute to cellular NADH:quinone oxidoreductase activity. CgYps1 cleaved CgPst2 at the C-terminus, and this cleavage was pivotal to oligomerization, activity and function of CgPst2. The arginine-174 residue in CgPst2 was essential for CgYps1-mediated cleavage, with alanine substitution of the arginine-174 residue also leading to elevated activity and oligomerization of CgPst2. Finally, we demonstrate that menadione treatment led to increased CgPst2 and CgYps1 protein levels, diminished CgYps1-CgPst2 interaction, and enhanced CgPst2 cleavage and activity, thereby implicating CgYps1 in activating CgPst2. Altogether, our findings of proteolytic cleavage as a key regulatory determinant of CgPst2, which belongs to the family of highly conserved, electron-carrier flavodoxin-fold-containing proteins, constituting cellular oxidative stress defense system in diverse organisms, unveil a hidden regulatory layer of environmental stress response mechanisms.

Computational approaches to amino acid side-chain conformation using combined NMR theoretical and experimental results: leucine-67 in Desulfovibrio vulgaris flavodoxin.

In this paper, we show that the combination of NMR theoretical and experimental results can help to solve the molecular structure of peptides, here it is used as an example the residue Leucine-67 in Desulfovibrio vulgaris flavodoxin. We apply a computational protocol based on the leucine amino acid dipeptide, which, using calculated and experimental spin-spin coupling constants, allows us to obtain the conformation of the amino acid side chain. Calculated results show that the best agreement is obtained when three conformers around the lateral chain angle $\chi _1$ are considered or when the dynamic effect in the torsional angles is included. The population of each structure is estimated and analyzed according to the correlation between those two approaches. Independently of the approach, the estimated $\chi _1$ angle in solution is close to the staggered value of -60$^\circ $ and deviates significantly from the average x-ray angle of -90$^\circ $.

Mechanism of the small ATP-independent chaperone Spy is substrate specific.

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy's action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.

Flavodoxin hydroquinone provides electrons for the ATP-dependent reactivation of protein-bound corrinoid cofactors.

Corrinoid-dependent enzyme systems rely on the super-reduced state of the protein-bound corrinoid cofactor to be functional, for example, in methyl transfer reactions. Due to the low redox potential of the [CoII ]/[CoI ] couple, autoxidation of the corrinoid cofactor occurs and leads to the formation of the inactive [CoII ]-state. For the reactivation, which is an energy-demanding process, electrons have to be transferred from a physiological donor to the corrinoid cofactor by the help of a reductive activator protein. In this study, we identified reduced flavodoxin as electron donor for the ATP-dependent reduction of protein-bound corrinoid cofactors of bacterial O-demethylase enzyme systems. Reduced flavodoxin was generated enzymatically using pyruvate:ferredoxin/flavodoxin oxidoreductase rather than hydrogenase. Two of the four flavodoxins identified in Acetobacterium dehalogenans and Desulfitobacterium hafniense DCB-2 were functional in supplying electrons for corrinoid reduction. They exhibited a midpoint potential of about -400 mV (ESHE , pH 7.5) for the semiquinone/hydroquinone transition. Reduced flavodoxin could be replaced by reduced clostridial ferredoxin. It was shown that the low-potential electrons of reduced flavodoxin are first transferred to the iron-sulfur cluster of the reductive activator and finally to the protein-bound corrinoid cofactor. This study further highlights the importance of reduced flavodoxin, which allows maintaining a variety of enzymatic reaction cycles by delivering low-potential electrons.

Structure and function of an unusual flavodoxin from the domain Archaea.

Flavodoxins, electron transfer proteins essential for diverse metabolisms in microbes from the domain Bacteria, are extensively characterized. Remarkably, although genomic annotations of flavodoxins are widespread in microbes from the domain Archaea, none have been isolated and characterized. Herein is described the structural, biochemical, and physiological characterization of an unusual flavodoxin (FldA) from Methanosarcina acetivorans, an acetate-utilizing methane-producing microbe of the domain Archaea In contrast to all flavodoxins, FldA is homodimeric, markedly less acidic, and stabilizes an anionic semiquinone. The crystal structure reveals an flavin mononucleotide (FMN) binding site unique from all other flavodoxins that provides a rationale for stabilization of the anionic semiquinone and a remarkably low reduction potentials for both the oxidized/semiquinone (-301 mV) and semiquinone/hydroquinone couples (-464 mV). FldA is up-regulated in acetate-grown versus methanol-grown cells and shown here to substitute for ferredoxin in mediating the transfer of low potential electrons from the carbonyl of acetate to the membrane-bound electron transport chain that generates ion gradients driving ATP synthesis. FldA offers potential advantages over ferredoxin by (i) sparing iron for abundant iron-sulfur proteins essential for acetotrophic growth and (ii) resilience to oxidative damage.

Reconstructing the Remote Origins of a Fold Singleton from a Flavodoxin-Like Ancestor.

Evolutionary processes that led to the emergence of structured protein domains left footprints in the sequences of modern proteins. We searched for such hints employing state-of-the-art sequence analysis and found evidence that the HemD-like fold emerged from the flavodoxin-like fold through segment swap and gene duplication. To verify this hypothesis, we reverted these evolutionary steps experimentally, constructing a HemD-half that resulted in a protein with the canonical flavodoxin-like architecture. These results of fold reconstruction from the sequence of a different fold strongly support our hypothesis of common ancestry. It further illustrates the plasticity of modern proteins to form new folded proteins.

Long-chain flavodoxin FldX1 improves Paraburkholderia xenovorans LB400 tolerance to oxidative stress caused by paraquat and H2O2.

Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for flavodoxin from P. xenovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth ß-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2.

Structural insight into the high reduction potentials observed for Fusobacterium nucleatum flavodoxin.

Flavodoxins are small flavin mononucleotide (FMN)-containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate-binding loop (T/S-X-T-G-X-T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The Eox/sq (oxidized/semiquinone; -43 mV) and Esq/hq (semiquinone/hydroquinone; -256 mV) are the highest recorded reduction potentials of known long-chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with ~70-fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive Eox/sq . The more electropositive Esq/hq is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.

A Research-inspired biochemistry laboratory module-combining expression, purification, crystallization, structure-solving, and characterization of a flavodoxin-like protein.

Many laboratory courses consist of short and seemingly unconnected individual laboratory exercises. To increase the course consistency, relevance, and student engagement, we have developed a research-inspired and project-based module, "From Gene to Structure and Function". This 2.5-week full-day biochemistry and structural biology module covers protein expression, purification, structure solving, and characterization. The module is centered around the flavodoxin-like protein NrdI, involved in the activation of the bacterial ribonucleotide reductase enzyme system. Through an in-depth focus on one specific protein, the students will learn the basic laboratory skills needed in order to generate a broader knowledge and breadth within the field. With respect to generic skills, the students report their findings as a scientific article, with the aim to learn to present concise research results and write scientific papers. The current research-inspired project has the potential of being further developed into a more discovery-driven project and extended to include other molecular biological techniques or biochemical/biophysical characterizations. In student evaluations, this research-inspired laboratory course has received very high ratings and been highly appreciated, where the students have gained research experience for more independent future work in the laboratory. © 2019 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 47(3):318-332, 2019.

Reverse Micelle Encapsulation of Proteins for NMR Spectroscopy.

Reverse micelle (RM) encapsulation of proteins for NMR spectroscopy has many advantages over standard NMR methods such as enhanced tumbling and improved sensitivity. It has opened many otherwise difficult lines of investigation including the study of membrane-associated proteins, large soluble proteins, unstable protein states, and the study of protein surface hydration dynamics. Recent technological developments have extended the ability of RM encapsulation with high structural fidelity for nearly all proteins and thereby allow high-quality state-of-the-art NMR spectroscopy. Optimal conditions are achieved using a streamlined screening protocol, which is described here. Commonly studied proteins spanning a range of molecular weights are used as examples. Very low-viscosity alkane solvents, such as propane or ethane, are useful for studying very large proteins but require the use of specialized equipment to permit preparation and maintenance of well-behaved solutions under elevated pressure. The procedures for the preparation and use of solutions of RMs in liquefied ethane and propane are described. The focus of this chapter is to provide procedures to optimally encapsulate proteins in reverse micelles for modern NMR applications.

The Characterization of Different Flavodoxin Reductase-Flavodoxin (FNR-Fld) Interactions Reveals an Efficient FNR-Fld Redox Pair and Identifies a Novel FNR Subclass.

Flavodoxins (Flds) are small, bacterial proteins that transfer electrons to various redox enzymes. Flavodoxins are reduced by ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs), but little is known of the FNR-Fld interaction. Here, we compare the interactions of two flavodoxins (Fld1-2), one flavodoxin-like protein (NrdI), and three different thioredoxin reductase (TrxR)-like FNRs (FNR1-3), all from Bacillus cereus. Steady-state kinetics shows that the FNR2-Fld2 electron transfer pair is particularly efficient, and redox potential measurements also indicate that this is the most favorable electron donor/acceptor pair. Furthermore, crystal structures of FNR1 and FNR2 show that the proteins have crystallized in different conformations, a closed and an open conformation, respectively. We suggest that a large-scale conformational rearrangement takes place during the FNR catalytic cycle to allow for the binding and reduction of the Fld and, subsequently, the re-reduction of the FNR by NADPH. Finally, inspection of the residues surrounding the FAD cofactor in the FNR active site shows that a key isoalloxazine ring-stacking residue is different in FNR1 and FNR2, which could explain the large difference in catalytic efficiency between the two FNRs. To date, all of the characterized TrxR-like FNRs have a residue with aromatic character stacking against the FAD isoalloxazine ring, and this has been thought to be a conserved feature of this class of FNRs. FNR1, however, has a valine in this position. Bioinformatic analysis shows that the TrxR-like FNRs can actually be divided into two groups, one group where the FAD-stacking residue has aromatic character and another group where it is valine.