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 $.

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.

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.

Expression, purification and characterization of two Clostridium acetobutylicum flavodoxins: potential electron transfer partners for CYP152A2.

Two flavodoxin genes from Clostridium acetobutylicum, CacFld1 (CAC0587) and CacFld2 (CAC3417), were expressed in Escherichia coli and investigated for their ability to support activity of CYP152A2, a fatty acid hydroxylase from C. acetobutylicum. E. coli flavodoxin reductase (FdR) was used as a redox partner, since flavodoxin reductase CacFdR (CAC0196) from C. acetobutylicum could not be purified in a functional form. CacFld1 was shown to accept electrons from FdR and transfer them to CYP152A2. Since H2O2 was generated by uncoupling at different stages of the reconstituted electron transfer chain, catalase was used as H2O2 scavenger in order to exclude peroxygenation by CYP152A2. The reconstituted P450 system with CacFld1 and FdR oxidized myristic acid with a K(M) of 137 µM and a k(cat) of 36 min⁻¹. Furthermore, the hydroxylase activity of CYP152A2 towards myristic acid with CacFld1 was 17-fold higher than without CacFld1. Along with CYP152A2 and a physiological flavodoxin reductase, CacFld1 is therefore likely to be involved in oxygen detoxification in C. acetobutylicum. Flavodoxin CacFld2 did not accept electrons from NADPH-reduced FdR, though it cannot be excluded as a candidate redox partner for CYP152A2 in the presence of an appropriate physiological reductase.

Mapping solvation dynamics at the function site of flavodoxin in three redox states.

Flavoproteins are unique redox coenzymes, and the dynamic solvation at their function sites is critical to the understanding of their electron-transfer properties. Here, we report our complete characterization of the function-site solvation of holoflavodoxin in three redox states and of the binding-site solvation of apoflavodoxin. Using intrinsic flavin cofactor and tryptophan residue as the local optical probes with two site-specific mutations, we observed distinct ultrafast solvation dynamics at the function site in the three states and at the related recognition site of the cofactor, ranging from a few to hundreds of picoseconds. The initial ultrafast motion in 1-2.6 ps reflects the local water-network relaxation around the shallow, solvent-exposed function site. The second relaxation in 20-40 ps results from the coupled local water-protein fluctuation. The third dynamics in hundreds of picoseconds is from the intrinsic fluctuation of the loose loops flanking the cofactor at the function site. These solvation dynamics with different amplitudes well correlate with the redox states from the oxidized form, to the more rigid semiquinone and to the much looser hydroquinone. This observation of the redox control of local protein conformation plasticity and water network flexibility is significant, and such an intimate relationship is essential to the biological function of interprotein electron transfer.

Cloning, expression and purification of cindoxin, an unusual Fmn-containing cytochrome p450 redox partner.

Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450(cin) (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH-dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range 6

Noncooperative Formation of the off-pathway molten globule during folding of the alpha-beta parallel protein apoflavodoxin.

During folding of many proteins, molten globules are formed. These partially folded forms of proteins have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristic of native structures. Molten globules are ensembles of interconverting conformers and are prone to aggregation, which can have detrimental effects on organisms. Consequently, molten globules attract considerable attention. The molten globule that is observed during folding of flavodoxin from Azotobacter vinelandii is a kinetically off-pathway species, as it has to unfold before the native state of the protein can be formed. This intermediate contains helices and can be populated at equilibrium using guanidinium hydrochloride as denaturant, allowing the use of NMR spectroscopy to follow molten globule formation at the residue level. Here, we track changes in chemical shifts of backbone amides, as well as disappearance of resonances of unfolded apoflavodoxin, upon decreasing denaturant concentration. Analysis of the data shows that structure formation within virtually all parts of the unfolded protein precedes folding to the molten globule state. This folding transition is noncooperative and involves a series of distinct transitions. Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. Although hydrophobic, tryptophan side chains are not involved in the latter process. This ordered core is gradually extended upon decreasing denaturant concentration, but part of apoflavodoxin's molten globule remains random coil in the denaturant range investigated. The results presented here, together with those reported on the molten globule of alpha-lactalbumin, show that helical molten globules apparently fold in a noncooperative manner.

Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.

NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: alpha2 and beta2. The alpha subunit contains the site of nucleotide reduction, and the beta subunit contains the essential diferric-tyrosyl radical (Y*) cofactor. Escherichia coli contains genes encoding two class I RNRs (Ia and Ib) and a class III RNR, which is active only under anaerobic conditions. Its class Ia RNR, composed of NrdA (alpha) and NrdB (beta), is expressed under normal aerobic growth conditions. The class Ib RNR, composed of NrdE (alpha) and NrdF (beta), is expressed under oxidative stress and iron-limited growth conditions. Our laboratory is interested in pathways of cofactor biosynthesis and maintenance in class I RNRs and modulation of Y* levels as a means of regulating RNR activity. Our recent studies have implicated a [2Fe2S]-ferredoxin, YfaE, in the NrdB diferric-Y* maintenance pathway and possibly in the biosynthetic and regulatory pathways. Here, we report that NrdI is a flavodoxin counterpart to YfaE for the class Ib RNR. It possesses redox properties unprecedented for a flavodoxin (E(ox/sq) = -264 +/- 17 mV and E(sq/hq) = -255 +/- 17 mV) that allow it to mediate a two-electron reduction of the diferric cluster of NrdF via two successive one-electron transfers. Data presented support the presence of a distinct maintenance pathway for NrdEF, orthogonal to that for NrdAB involving YfaE.

Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus.

Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 66.49, c = 121.32 A. X-ray data sets have been collected to 2.17 A resolution.

Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redox partners.

In Clostridium acetobutylicum, [FeFe]-hydrogenase is involved in hydrogen production in vivo by transferring electrons from physiological electron donors, ferredoxin and flavodoxin, to protons. In this report, by modifications of the purification procedure, the specific activity of the enzyme has been improved and its complete catalytic profile in hydrogen evolution, hydrogen uptake, proton/deuterium exchange and para-H2/ortho-H2 conversion has been determined. The major ferredoxin expressed in the solvent-producing C. acetobutylicum cells was purified and identified as encoded by ORF CAC0303. Clostridium acetobutylicum recombinant holoflavodoxin CAC0587 was also purified. The kinetic parameters of C. acetobutylicum [FeFe]-hydrogenase for both physiological partners, ferredoxin CAC0303 and flavodoxin CAC0587, are reported for hydrogen uptake and hydrogen evolution activities.

Flavodoxin, a new fluorescent substrate for monitoring proteolytic activity of FtsH lacking a robust unfolding activity.

Escherichia coli FtsH, which belongs to the ATPases associated with diverse cellular activities (AAA) family, is an ATP-dependent and membrane-bound protease. FtsH degrades misassembled membrane proteins and a subset of cytoplasmic regulatory proteins. To elucidate the molecular mechanisms of the proteolysis, a system for precisely monitoring substrate degradation is required. We have exploited E. coli flavodoxin containing non-covalently bound flavin mononucleotide (FMN) as a model substrate for monitoring protein degradation. It was found that FtsH degrades FMN-free apo-flavodoxin but not holo-flavodoxin. However, degradation of a mutant flavodoxin carrying a substitution of Tyr94 to Asp with a lower affinity for FMN could be monitored by fluorimetry. This newly developed monitoring system will also be applicable for proteolysis by other ATP-dependent proteases.

Towards a new therapeutic target: Helicobacter pylori flavodoxin.

Helicobacter pylori flavodoxin is the electronic acceptor of the pyruvate-oxidoreductase complex (POR) that catalyzes pyruvate oxidative decarboxilation. Inactivation of this metabolic route precludes bacterial survival. Because flavodoxin is not present in the human host, substances interfering electronic transport from POR might be well suited for eradication therapies against the bacterium. H. pylori flavodoxin presents a peculiar cofactor (FMN) binding site, compared to other known flavodoxins, where a conserved aromatic residue is replaced by alanine. A cavity thus appears under the cofactor that can be filled with small organic molecules. We have cloned H. pylori fldA gene, expressed the protein in Escherichia coli and characterized the purified flavodoxin. Thermal up-shift assays of flavodoxin with different concentrations of benzylamine, as well as fluorescence titration experiments indicate benzylamine binds in the pocket near the FMN binding site. It seems thus that low affinity inhibitors of H. pylori flavodoxin can be easily found that, after improvement, may give rise to leads.

Role of neighboring FMN side chains in the modulation of flavin reduction potentials and in the energetics of the FMN:apoprotein interaction in Anabaena flavodoxin.

Flavodoxins (Flds) are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule (FMN) as a redox active center. A distinguishing feature of these flavoproteins is the dramatic change in the E(sq/rd) reduction potential of the FMN upon binding to the apoprotein (at pH 8.0, from -269 mV when free in solution to -438 mV in Anabaena Fld). In this study, the contribution of three neighboring FMN residues, Thr56, Asn58, and Asn97, and of three negatively charged surface residues, Glu20, Asp65, and Asp96, to modulate the redox properties of FMN upon its binding to the apoprotein has been investigated. Additionally, the role of these residues in the apoflavodoxin:FMN interaction has been analyzed. Concerning the redox potentials, the most noticeable result was obtained for the Thr56Gly mutant. In this Fld variant, the increased accessibility of FMN leads to an increase of +63 mV in the E(sq/rd) value. On the other hand, a correlation between the electrostatic environment of FMN and the E(sq/rd) has been observed. The more positive residues or the less negative residues present in the surroundings of the FMN N(1) atom, then the less negative the value for E(sq/rd). With regard to FMN binding to apoflavodoxin, breaking of hydrophobic interactions between FMN and residues 56, 58, and 97 seems to increase the K(d) values, especially in the Thr56Gly Fld. Such results suggest that the H-bond network in the FMN environment influences the FMN affinity.

A Rubredoxin based system for screening of protein expression conditions and on-line monitoring of the purification process.

Rubredoxin (Rub) from Thermotoga maritima, a 6.1-kDa red protein containing an Fe(III)-cysteine(4) center, was evaluated for its usefulness as a colored fusion tag for expression of recombinant proteins in E. coli. Here, we describe the Rub features relevant to accelerating screening for optimal high yield soluble expression conditions and automating the ensuing purification process. Spectroscopic properties and the yield of Rub fused to a typical target protein were compared to analogous GFP and Flavodoxin constructs, showing Rub absorption to be sufficient for structural genomics purposes while being produced at much higher soluble levels than GFP constructs. Based entirely on Rub absorption at 380 nm, both generic and affinity purification of crude cell lysate were performed: thus guided anion exchange purification of a Rub fusion construct as well as automated Ni-NTA purification resulted in pure protein. Rub is stable over a wide range of pH, temperature, and buffer environments, enabling robust purification protocols. Across a variety of fusion constructs, including N- and C-terminal Rub, quantitation via the Rub signal was shown to reliably correlate with analytical HPLC data obtained at 220 nm. We propose the "RubyTag" as an alternative to conventional protein fusion tags, as it combines a specific absorption signal with convenient biochemical and biological properties. Further, it allows direct on-line readout on conventional chromatography systems, holding promise for automated multi-step chromatography.

Cloning, sequencing and expression of the gene for flavodoxin from Megasphaera elsdenii and the effects of removing the protein negative charge that is closest to N(1) of the bound FMN.

The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin. A site-directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to < or = 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and < or = 5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone.

Cloning and expression of the gene encoding flavodoxin from Desulfovibrio vulgaris (Miyazaki F).

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli. A 1.6-kbp DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SalI and EcoRI, contained the flavodoxin gene and its regulatory region. An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E. coli. The purified protein was soluble and exhibited a characteristic visible absorption spectrum. HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D. vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM. In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1 = -434 and E2 = -151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule.

Modulation of the redox potentials of FMN in Desulfovibrio vulgaris flavodoxin: thermodynamic properties and crystal structures of glycine-61 mutants.

Mutants of the electron-transfer protein flavodoxin from Desulfovibrio vulgaris were made by site-directed mutagenesis to investigate the role of glycine-61 in stabilizing the semiquinone of FMN by the protein and in controlling the flavin redox potentials. The spectroscopic properties, oxidation-reduction potentials, and flavin-binding properties of the mutant proteins, G61A/N/V and L, were compared with those of wild-type flavodoxin. The affinities of all of the mutant apoproteins for FMN and riboflavin were less than that of the wild-type apoprotein, and the redox potentials of the two 1-electron steps in the reduction of the complex with FMN were also affected by the mutations. Values for the dissociation constants of the complexes of the apoprotein with the semiquinone and hydroquinone forms of FMN were calculated from the redox potentials and the dissociation constant of the oxidized complex and used to derive the free energies of binding of the FMN in its three oxidation states. These showed that the semiquinone is destabilized in all of the mutants, and that the extent of destabilization tends to increase with increasing bulkiness of the side chain at residue 61. It is concluded that the hydrogen bond between the carbonyl of glycine-61 and N(5)H of FMN semiquinone in wild-type flavodoxin is either absent or severely impaired in the mutants. X-ray crystal structure analysis of the oxidized forms of the four mutant proteins shows that the protein loop that contains residue 61 is moved away from the flavin by 5-6 A. The hydrogen bond formed between the backbone nitrogen of aspartate-62 and O(4) of the dimethylisoalloxazine of the flavin in wild-type flavodoxin is absent in the mutants. Reliable structural information was not obtained for the reduced forms of the mutant proteins, but if the mutants change conformation when the flavin is reduced to the semiquinone, to facilitate hydrogen bonding between N(5)H and the carbonyl of residue 61, then the change must be different from that known to occur in wild-type flavodoxin.

Flavodoxin-dependent pyruvate oxidation, acetate production and metronidazole reduction by Helicobacter pylori.

Helicobacter pylori flavodoxin was purified to homogeneity from cell extracts of strain NCTC 11637. The molecular weight of the protein was estimated by gel electrophoresis to be 18 kDa. Oxidized flavodoxin showed an absorption spectrum with maxima at 378 nm and 453 nm, and it was reduced to a neutral form of flavin semiquinone by the electrons generated in the oxidation of pyruvate. This coenzyme A dependent pyruvate:flavodoxin oxidoreductase activity of H. pylori was also detected as a reduction of methyl viologen or cytochrome c by bacterial extracts. The apparent Km of pyruvate was 310 microM. Anaerobically incubated bacteria (10[9]) of strain NCTC 11637 produced acetate (96 +/- 16 nmol/h) from pyruvate concomitantly reducing metronidazole (17 +/- 5 nmol/h). In anaerobic conditions both sensitive and resistant H. pylori strains reduced metronidazole, and there was a significant positive correlation between acetate production and metronidazole activation (r = 0.77, P < 0.01, n = 11). In the presence of atmospheric oxygen, H. pylori excreted twice as much acetate but metronidazole was not activated. These results suggest that the pyruvate:flavodoxin oxidoreductase complex catalyses pyruvate oxidation in H. pylori. Electrons generated in this reaction are transferred to flavodoxin and under anaerobic conditions further to metronidazole (imidazoles) thus reducing the drug to its bactericidal form.