A comparison of the urea-induced unfolding of apoflavodoxin and flavodoxin from Desulfovibrio vulgaris.

The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s(-1) in 3 m urea in 25 mm sodium phosphate at 25 degrees C), and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding, but they do not exclude the possibility of intermediates. Calculation suggests that 21% and 30% of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin, respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride, and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin, but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein.     

Redox and flavin-binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue. Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV. The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.     

Properties of the complexes of riboflavin 3',5'-bisphosphate and the apoflavodoxins from Megasphaera elsdenii and Desulfovibrio vulgaris

Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3',5'-bisphosphate. Several biochemical and biophysical properties of the complexes have been investigated and the results are compared with the properties of the native proteins. The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for the native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone-hydroquinone transition is about 20 mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein-bound artificial prosthetic group and the protein-bound natural prosthetic group are very similar. In both the oxidized and in the fully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15N NMR. 31P-NMR studies show that the 5'-phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3'-phosphate group in riboflavin 3',5'-bisphosphate is monoanionic or even neutral when bound to the protein. The 3'-phosphate group is also close to or on the surface of the protein. Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3',5'-bisphosphate which is 10 times higher as compared to Megasphaera elsdenii apoflavodoxin (Ka = 10(8) M-1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3'5'-bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3',5'-bisphosphate analog gives a value (Kd approximately equal to 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649-655]. The results are discussed in the light of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins and flavoproteins, pp. 493-496, Walter de Gruyter, Berlin].     

A comparative carbon-13, nitrogen-15, and phosphorus-31 nuclear magnetic resonance study on the flavodoxins from Clostridium MP, Megasphaera elsdenii, and Azotobacter vinelandii

The flavodoxins from Megasphaera elsdenii, Clostridium MP, and Azotobacter vinelandii were studied by 13C, 15N, and 31P NMR techniques by using various selectivity enriched oxidized riboflavin 5'-phosphate (FMN) derivatives. It is shown that the pi electron distribution in protein-bound flavin differs from that of free flavin and depends also on the apoflavoprotein used. In the oxidized state Clostridium MP and M. elsdenii flavodoxins are very similar with respect to specific hydrogen bond interaction between FMN and the apoprotein and the electronic structure of flavin. A. vinelandii flavodoxin differs from these flavodoxins in both respects, but it also differs from Desulfovibrio vulgaris flavodoxin. The similarities between A. vinelandii and D. vulgaris flavodoxins are greater than the similarities with the other two flavodoxins. The differences in the pi electron distribution in the FMN of reduced flavodoxins from A. vinelandii and D. vulgaris are even greater, but the hydrogen bond patterns between the reduced flavins and the apoflavodoxins are very similar. In the reduced state all flavodoxins studied contain an ionized prosthetic group and the isoalloxazine ring is in a planar conformation. The results are compared with existing three-dimensional data and discussed with respect to the various possible mesomeric structures in protein-bound FMN. The results are also discussed in light of the proposed hypothesis that specific hydrogen bonding to the protein-bound flavin determines the specific biological activity of a particular flavoprotein.     

Phosphates of riboflavin and riboflavin analogs: A reinvestigation by high-performance liquid chromatography

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5'-phosphate was 70-75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of greater than 99%-pure 5'-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4'-phosphate, riboflavin 3'-phosphate, riboflavin 4',5'-diphosphate; riboflavin 3',4'-diphosphate, and riboflavin 3',5'-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii (KD = 9.7 X 10(-9) M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.     

Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins

Flavodoxins from Clostridium beijerinckii and from Megasphaera elsdenii with 1-carba-1-deaza-FMN substituted for FMN have been used to study flavin-protein interactions in flavodoxins. The oxidized 1-deaza analogue of FMN binds to apoflavodoxins from M. elsdenii and C. beijerinckii (a.k.a. Clostridium MP) with association constants (Ka) of 1.0 x 10(7) M-1 and 3.1 x 10(6) M-1, values about 10(2) less than the corresponding Ka values for FMN. X-ray structure analysis of oxidized 1-deaza-FMN flavodoxin from C. beijerinckii at 2.5-A resolution shows that the analogue binds with the flavin atoms in the same locations as their equivalents in FMN but that the protein moves in the vicinity of Gly 89 to accommodate the 1-CH group, undergoing displacements which increase the distance between position 1 of the flavin ring and the main-chain atoms of Gly 89 and move the peptide hydrogen of Gly 89 by about 0.6 A. The X-ray analysis implies that protonation of normal flavin at N(1), as would occur in formation of the neutral fully reduced species, would result in a similar structural perturbation. The oxidation-reduction potentials of 1-deaza-FMN flavodoxin from M. elsdenii have been determined in the pH range 4.5-9.2. The oxidized/semiquinone equilibrium (E'0 = -160 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit; the semiquinone/reduced equilibrium (E'0 = -400 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit at low pH and is pH independent at high pH, with a redox-linked pK of 7.4. Spectral changes of fully reduced 1-deaza-FMN flavodoxin with pH suggest that this latter pK corresponds to protonation of the flavin ring system (the pK of free reduced 1-deaza-FMN is 5.6 [Spencer, R., Fisher, J., & Walsh, C. (1977) Biochemistry 16, 3586-3593]. The pK of reduced 1-deaza-FMN flavodoxin provides an estimate of the electrostatic interaction between the protein and the bound prosthetic group; the free energy of binding neutral reduced 1-deaza-FMN is more negative than that for binding the anionic reduced 1-deaza-FMN by 2.4 kcal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of flavin mononucleotide cofactor binding to the Desulfovibrio vulgaris flavodoxin. 1. Kinetic evidence for cooperative effects associated with the binding of inorganic phosphate and the 5'-phosphate moiety of the cofactor

The pathway(s) by which the flavin cofactor binds to the apoflavoprotein is the subject of some debate. The crystal and NMR structures of several different flavodoxins have provided some insight, although there is disagreement about the location of the initial interaction between the flavin mononucleotide (FMN) and the apoflavodoxin and the degree of protein conformational change associated with cofactor binding [Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332; Steensma, E., and van Mierlo, C. P. M. (1998) J. Mol. Biol. 282, 653-666]. Binding kinetics using stopped-flow spectrofluorimetry and phosphate competition studies were used to develop a model for flavin binding to the flavodoxin from Desulfovibrio vulgaris. In the presence of phosphate, the time course of fluorescence quenching associated with FMN binding to apoflavodoxin was biphasic, whereas riboflavin, which lacks the 5'-phosphate group of FMN, displayed monophasic binding kinetics. When the concentration of phosphate in solution was increased, the FMN binding rates of the two phases behaved differently; the rate of one phase decreased, while the rate of the other increased. A similar increase in the single phase associated with riboflavin binding was also observed. This has led to the following model. The binding of the flavin isoalloxazine ring to its subsite is dependent on the presence of a phosphate group in the phosphate-binding subsite. When phosphate is in the buffer solution, FMN can bind in either of two ways: by the initial insertion of the 5'-phosphate group followed by ring binding or, when inorganic phosphate from solution is bound, the insertion of the isoalloxazine ring first. Riboflavin, which lacks the phosphate moiety of FMN, binds only in the presence of inorganic phosphate, presumably due to the binding of this group in the phosphate-binding subsite. These results suggest that cooperative interactions exist between the phosphate subsite and the ring-binding region in the D. vulgaris flavodoxin that are necessary for isoalloxazine ring binding.     

Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp. PCC 7119.

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.     

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.     

pH-dependent spectroscopic changes associated with the hydroquinone of FMN in flavodoxins

Photoreduction with a 5-deazaflavin as the catalyst was used to convert flavodoxins from Desulfovibrio vulgaris, Megasphaera elsdenii, Anabaena PCC 7119, and Azotobacter vinelandii to their hydroquinone forms. The optical spectra of the fully reduced flavodoxins were found to vary with pH in the pH range of 5.0-8.5. The changes correspond to apparent pKa values of 6.5 and 5.8 for flavodoxins from D. vulgaris and M. elsdenii, respectively, values that are similar to the apparent pKa values reported earlier from the effects of pH on the redox potential for the semiquinone-hydroquinone couples of these two proteins (7 and 5.8, respectively). The changes in the spectra resemble those occurring with the free two-electron-reduced flavin for which the pKa is 6.7, but they are red-shifted compared with those of the free flavin. The optical changes occurring with flavodoxins from D. vulgaris and A. vinelandii flavodoxins are larger than those of free reduced FMN. The absorbance of the free and bound flavin increases in the region of 370-390 nm (Delta epsilon = 1-1.8 mM-1 cm-1) with increases of pH. Qualitatively similar pH-dependent changes occur when FMN in D. vulgaris flavodoxin is replaced by iso-FMN, and in the following mutants of D. vulgaris flavodoxin in which the residues mutated are close to the isoalloxazine of the bound flavin: D95A, D95E, D95A/D127A, W60A, Y98S, W60M/Y98W, S96R, and G61A. The 13C NMR spectrum of reduced D. vulgaris [2,4a-13C2]FMN flavodoxin shows two peaks. The peak due to C(4a) is unaffected by pH, but the peak due to C(2) broadens with decreasing pH; the apparent pKa for the change is 6.2. It is concluded that a decrease in pH induces a change in the electronic structure of the reduced flavin due to a change in the ionization state of the flavin, a change in the polarization of the flavin environment, a change in the hydrogen-bonding network around the flavin, and/or possibly a change in the bend along the N(5)-N(10) axis of the flavin. A change in the ionization state of the flavin is the simplest explanation, with the site of protonation differing from that of free FMNH-. The pH effect is unlikely to result from protonation of D95 or D127, the negatively charged amino acids closest to the flavin of D. vulgaris flavodoxin, because the optical changes observed with alanine mutants at these positions are similar to those occurring with the wild-type protein.     

A continuous fluorometric assay for flavokinase. Properties of flavokinase from Peptostreptococcus elsdenii.

A continuous fluorometric assay that utilizes apoflavodoxin as a trapping agent for riboflavin 5'-phosphate (FMN) has been developed for flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26). Use of this assay is illustrated in a procedure for the partial purification of flavokinase from the strict anaerobe Peptostreptococcus elsdenii. The purified enzyme catalyzed the formation of 8.3 nmol FMN - min-1 - mg-1 at 37 degrees C and had apparent Km values for riboflavin and ATP of 10 and 4.7 micronM, respectively. ATP could be replaced by ADP (22% of the rate observed with ATP) but not by GTP. The enzyme also phosphorylated 5-deaza- and 8-bromoriboflavin with activities of 15 and 70%, respectively, of that with riboflavin; it was inactive with iso riboflavin and deoxyriboflavin.     

Last in, first out: the role of cofactor binding in flavodoxin folding

Although many proteins require the binding of a ligand to be functional, the role of ligand binding during folding is scarcely investigated. Here, we have reported the influence of the flavin mononucleotide (FMN) cofactor on the global stability and folding kinetics of Azotobacter vinelandii holoflavodoxin. Earlier studies have revealed that A. vinelandii apoflavodoxin kinetically folds according to the four-state mechanism: I(1) <=> unfolded apoflavodoxin <=> I(2) <=> native apoflavodoxin. I(1)an off-pathway molten globule-like is intermediate that populates during denaturant-induced equilibrium unfolding; I(2) is a high energy on-pathway folding intermediate that never populates to a significant extent. Here, we have presented extensive denaturant-induced equilibrium unfolding data of holoflavodoxin, holoflavodoxin with excess FMN, and apoflavodoxin as well as kinetic folding and unfolding data of holoflavodoxin. All folding data are excellently described by a five-state mechanism: I(1) + FMN <=> unfolded apoflavodoxin + FMN <=> I(2) + FMN <=> native apoflavodoxin + FMN<=> holoflavodoxin. The last step in flavodoxin folding is thus the binding of FMN to native apoflavodoxin. I(1),I(2), and unfolded apoflavodoxin do not interact to a significantextent with FMN. The autonomous formation of native apoflavodoxin is essential during holoflavodoxin folding. Excess FMN does not accelerate holoflavodoxin folding, and FMN does not act as a nucleation site for folding. The stability of holoflavodoxin is so high that even under strongly denaturing conditions FMN needs to be released first before global unfolding of the protein can occur.     

Identification and quantitative determination of the flavin component of soluble hydrogenase from Alcaligeneseutrophus

The flavin component of soluble hydrogenase (hydrogen: NAD⁺ oxidoreductase, EC 1.12.1.2) from $\text{Alcaligenes}\text{eutrophus}$ was identified as FMN by thin layer chromatography in two solvent systems and by binding studies with apoflavodoxin from $\text{Megasphaera}\text{elsdenii}$. The flavin of hydrogenase reacted rapidly with apoflavodoxin with almost complete quenching of the fluorescence at 525 nm. Quantitative determination of FMN was performed by fluorimetric titration with a standardized solution of apoflavodoxin. From the determined FMN content of different enzyme preparations and from the percentage of stimulation of hydrogenase activity by exogenous FMN it is concluded that hydrogenase contains 2 FMN per molecule.     

Effect of pressure upon the fluorescence of various flavodoxins.

The effects of hydrostatic pressure in the range of 10(-3) to 11 kbar on the fluorescence of flavodoxins from Peptostreptococcus elsdenii, Desulfovibrio vulgaris, Azotobacter vinelandii, and Clostridium MP were investigated. The first three flavoproteins showed under high pressure enhancements of flavin fluorescence of over 50 times resulting from the release of flavin mononucleotide from the protein complex. The Clostridial flavodoxin showed a very much smaller fluorescence change. At pH 7.5 the high-pressure fluorescence changes of the flavodoxins of D. vulgaris and P. elsdenii were not reversed by decompression, but in A. Vinelandii the pressure changes were over 80% reversible. At pH 5 over 80% reversibility was restored to the flavodoxins of D. vulgaris and P. elsdenii, although the pressure dependence of the fluorescence changes was very similar in the reversible and irreversible cases. The midpoint pressures in the reversible reactions were 4.7 kbar (D. vulgaris), 8.7 kbar (P. elsdenii), and 10.6 kbar (A. vinelandii) indicating specific differences in the flavin binding regions. Apparent volume changes in these reactions were 65-75 mL/mol indicating participation of a large fraction of the protein in the pressure-induced changes. The irreversible changes are not related to protein aggregation and are believed to result from a pressure-dependent covalent modification, not yet characterized, of the flavin binding region of the protein.     

Azotobacter vinelandii flavodoxin: purification and properties of the recombinant, dephospho form expressed in Escherichia coli

The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 [Bennett, L. T., Jacobsen, M. R., & Dean, D. R. (1988) J. Biol. Chem. 263 1364-1369] and the resulting plasmid transformed and expressed in Escherichia coli strain DH5. Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E. coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions. Even higher levels were observed with flavodoxin expressed in E. coli under control of a tac promoter. Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form. The flavodoxin purified from E. coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein. The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate. Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions. The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV. This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin. Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

13C NMR of cyanylated flavodoxin from Megasphaera elsdenii and of thiocyanate model compounds.

Both of the thiol groups of Megasphaera elsdenii flavodoxin have been cyanylated using 13C-enriched cyanide. This chemical modification increases the dissociation constant of the apoflavodoxin-flavin mononucleotide (FMN) complex from 0.4 nM to 2 microM. The thiocyanate carbons of the cyanylated cysteine residues in apoflavodoxin had 13C chemical shifts of 109.4 ppm and 112.2 ppm, which were replaced by signals at 115.5 ppm and 109.6 ppm when FMN was bound. The signals at 109.4 ppm and 112.2 ppm due to the cyanylated apoflavodoxin were unstable at 28 degrees C, and they were slowly replaced signals at 114.5 ppm and 115.3 ppm which are attributed to an inactive form of the apoprotein, which does not bind FMN. At alkaline pH values or after prolonged incubation at neutral pH, the signals at 114.5 ppm and 115.3 ppm were replaced by signals at approximately 171 ppm. On the basis of results obtained with model compounds, the signals at 171 ppm are assigned to the 2-imino carbon of the 2-iminothiazolidine ring formed by the cyclization of the appropriate thiocyanate group. After determining the chemical shift of the thiocyanate carbon of model compounds in a range of solvents, we conclude that the thiocyanate carbons will have a minimal chemical shift of approximately 109 ppm in apolar solvents which do not contain hydrogen bond donors. In water, a more polar hydrogen-bonding solvent, the chemical shift increases to approximately 115 ppm. We also conclude that the chemical shift of a thiocyanate carbon can be used as a probe of its molecular environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen-1, carbon-13, and nitrogen-15 NMR spectroscopy of Anabaena 7120 flavodoxin: assignment of beta-sheet and flavin binding site resonances and analysis of protein-flavin interactions

Sequence-specific 1H and 13C NMR assignments have been made for residues that form the five-stranded parallel beta-sheet and the flavin mononucleotide (FMN) binding site of oxidized Anabaena 7120 flavodoxin. Interstrand nuclear Overhauser enhancements (NOEs) indicate that the beta-sheet arrangement is similar to that observed in the crystal structure of the 70% homologous long-chain flavodoxin from Anacystis nidulans [Smith et al. (1983) J. Mol. Biol. 165, 737-755]. A total of 62 NOEs were identified: 8 between protons of bound FMN, 29 between protons of the protein in the flavin binding site, and 25 between protons of bound FMN and protons of the protein. These constraints were used to determine the localized solution structure of the FMN binding site. The electronic environment and conformation of the protein-bound flavin isoalloxazine ring were investigated by determining 13C chemical shifts, one-bond 13C-13C and 15N-1H coupling constants, and three-bond 13C-1H coupling constants. The carbonyl edge of the flavin ring was found to be slightly polarized. The xylene ring was found to be nonplanar. Tyrosine 94, located adjacent to the flavin isoalloxazine ring, was shown to have a hindered aromatic ring flip rate.     

Structure of the oxidized long-chain flavodoxin from Anabaena 7120 at 2 A resolution.

The structure of the long-chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 A resolution by the molecular replacement method using the atomic coordinates of the long-chain flavodoxin from Anacystis nidulans. The structure of a third long-chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A. 7120 flavodoxin belong to the monoclinic space group P2(1) with a = 48.0, b = 32.0, c = 51.6 A, and beta = 92 degrees, and one molecule in the asymmetric unit. The 2 A intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2 sigma and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R-value of 0.185 for a model containing atoms for protein residues 2-169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5'-phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long-chain structures are similar except for the O4' of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9-15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A. 7120, whereas in A. nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 A and 3.5 A, respectively, whereas in the third flavodoxin the distance is 3.1 A, close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin.     

Protein folding and stability investigated by fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story

In this review, the experimental results obtained on the folding and stability of Azotobacter vinelandii flavodoxin are summarised. By doing so, three main spectroscopic techniques used to investigate protein folding and stability are briefly introduced. These techniques are: circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy in combination with the hydrogen exchange methodology. Results on the denaturant-induced and thermal equilibrium unfolding of apoflavodoxin from A. vinelandii, i.e. flavodoxin in the absence of the riboflavin-5'-monophosphate (FMN) cofactor, are discussed. A scheme for the equilibrium unfolding of apoflavodoxin is presented which involves a relatively stable molten globule-like intermediate. Denaturant-induced apoflavodoxin (un)folding as followed at the residue-level by NMR shows that the transition of native A. vinelandii apoflavodoxin to its molten globule state is highly co-operative. However, the unfolding of the molten globule to the unfolded state of the protein is non-co-operative. A comparison of the folding of A. vinelandii flavodoxin with the folding of flavodoxin from Anaboena PCC 7119 is made. The local stabilities of apo- and holoflavodoxin from A. vinelandii as measured by NMR spectroscopy are compared. Both Che Y and cutinase, which have no sequence homology with apoflavodoxin but which share the flavodoxin-like topology, have stabilisation centres different from that of apoflavodoxin from A. vinelandii. The stable centres of structurally similar proteins can thus reside in different parts of the same protein topology. Insight in the variations in (local) unfolding processes of structurally similar proteins can be used to stabilise proteins with a flavodoxin-like fold. Finally, the importance of some recent experimental and theoretical developments for the study of flavodoxin folding is briefly discussed.