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, overexpression, and characterization of peroxiredoxin and NADH peroxiredoxin reductase from Thermus aquaticus.

The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (PrxR) have been cloned from the thermophilic bacterium Thermus aquaticus. prx is located upstream from prxR, the two genes being separated by 13 bases. The amino acid sequences show that Prx is related to two-cysteine peroxiredoxins from a range of organisms and that PrxR resembles NADH-dependent flavoenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacteria. The sequence of PrxR also resembles those of thioredoxin reductases (TrxR) from thermophiles but with an N-terminal extension of about 200 residues. PrxR has motifs for two redox-active disulfides, one in the FAD-binding site, as occurs in TrxR, and the other in the N-terminal extension. The molecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respectively; both enzymes exist as multimers. The recombinant flavoenzyme requires 3 mol equivalents of dithionite for full reduction, as is consistent with 1 FAD and 2 disulfides per monomer. PrxR and Prx together catalyze the anaerobic reduction of hydrogen peroxide. The activity of Prx is much less than has been observed with homologous proteins. Prx appears to be inactivated by cumene hydroperoxide. PrxR itself has low peroxidase activity.

Novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of the gene.

A novel dehydratase that catalyzes the stoichiometric dehydration of Z-phenylacetaldoxime to phenylacetonitrile has been purified 483-fold to homogeneity from a cell-free extract of Bacillus sp. strain OxB-1 isolated from soil. It has a M(r) of about 40 000 and is composed of a single polypeptide chain with a loosely bound protoheme IX. The enzyme is inactive unless FMN is added to the assay, but low activity is also observed when sulfite replaces FMN. The activity in the presence of FMN is enhanced 5-fold under anaerobic conditions compared to the activity measured in air. The enzyme has maximum activity at pH 7.0 and 30 degrees C, and it is stable at up to 45 degrees C at around neutral pH. The aerobically measured activity in the presence of FMN is also enhanced by Fe(2+), Sn(2+), SO(3)(2)(-), and NaN(3). Metal-chelating reagents, carbonyl reagents, electron donors, and ferri- and ferrocyanides strongly inhibit the enzyme with K(i) values in the micromolar range. The enzyme is active with arylalkylaldoximes and to a lesser extent with alkylaldoximes. The enzyme prefers the Z-form of phenylacetaldoxime over its E-isomer. On the basis of its substrate specificity, the enzyme has been tentatively named phenylacetaldoxime dehydratase. The gene coding for the enzyme was cloned into plasmid pUC18, and a 1053 base-pair open reading frame that codes for 351 amino acid residues was identified as the oxd gene. A nitrilase, which participates in aldoxime metabolism in the organism, was found to be coded by the region just upstream from the oxd gene. In addition an open reading frame (orf2), whose gene product is similar to bacterial regulatory (DNA-binding) proteins, was found just upstream from the coding region of the nitrilase. These findings provide genetic evidence for a novel gene cluster that is responsible for aldoxime metabolism in this microorganism.

The effects of pH and semiquinone formation on the oxidation-reduction potentials of flavin mononucleotide. A reappraisal.

Calculation shows that there is poor agreement between frequently cited values for the midpoint redox potentials of the two one-electron steps in the reduction of flavin mononucleotide and equations for the lines that relate these potentials to pH and that use the published pKa values for the three redox states of the flavin [Draper, R. & Ingraham, L.L. (1969) Arch. Biochem. Biophys. 125, 802-808]. Equilibrium data for the first step in the reduction obtained by pulse radiolysis [Anderson, R.F. (1983) Biochim. Biophys. Acta 722, 158-162] show much closer agreement with theory and lead to values for the semiquinone formation constant of flavin mononucleotide that are close to those derived from measurements of the radical concentration using ESR spectroscopy. It is concluded that the data from the second method are more reliable. The redox potentials for flavin mononucleotide at pH 7.0 and 20 degrees C are calculated to be -0.207 V for the overall two-electron reduction (Em), -0.313 V for reduction of the oxidized flavin to the semiquinone (E2) and -0.101 V for the reduction of the semiquinone to the hydroquinone (E1). Information is provided to allow calculation of the three redox potentials at other pH values in the physiological range.

Crystallization and preliminary crystallographic analysis of an NADH oxidase that functions in peroxide reduction in Thermus aquaticus YT-1.

NADH oxidase from Thermus aquaticus is a thermostable flavoenzyme that is similar in amino-acid sequence and other properties to the flavoenzyme component of the NADH peroxidase systems from Salmonella typhimurium and Amphibacillus xylanus. The enzyme has been isolated from T. aquaticus and crystallized using the hanging-drop method of vapour diffusion with sodium citrate as a precipitant at pH 8.5. The crystals belong to the hexagonal space group P622 with unit-cell dimensions a = b = 89.9, c = 491.6 A, and diffract to 2.5 A resolution.

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.

Cloning and analysis of the genes for a novel electron-transferring flavoprotein from Megasphaera elsdenii. Expression and characterization of the recombinant protein.

The genes that encode the two different subunits of the novel electron-transferring flavoprotein (ETF) from Megasphaera elsdenii were identified by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase chain reaction. The cloned genes are arranged in tandem with the coding sequence for the beta-subunit in the position 5' to the alpha-subunit coding sequence. Amino acid sequence analysis of the two subunits revealed that there are two possible dinucleotide-binding sites on the alpha-subunit and one on the beta-subunit. Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and with fixAB gene products from nitrogen-fixing bacteria. The recombinant ETF was isolated from extracts of Escherichia coli. It is a heterodimer with subunits identical in size to the native protein. The isolated enzyme contains approximately 1 mol of FAD, but like the native protein it binds additional flavin to give a total of about 2 mol of FAD/dimer. It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.

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.

Purification and characterisation of NADH oxidase from Thermus aquaticus YT-1 and evidence that it functions in a peroxide-reduction system.

A thermostable enzyme previously identified as an NADH oxidase has been purified from Thermus aquaticus YT-1 by chromatography on DEAE-cellulose and AMP-Sepharose. The enzyme is dimeric with subunits of 54 kDa and one molecule FAD/subunit. The FAD is tightly bound, but it can be removed reversibly by hydrophobic chromatography at low pH. The blue flavin semiquinone is stabilised during photo-chemical reduction of the enzyme. Chemical reduction by static titration with dithionite ion showed that the enzyme requires about 5 mol dithionite/mol FAD for full reduction, and that reduction occurs in four phases. Reduction by the substrate NADH is incomplete, with the formation of a new long-wavelength absorption underlying the semiquinone absorption. Amino acid sequencing showed that the T aquaticus enzyme resembles other microbial flavoenzymes that function in two-enzyme systems for the reduction of peroxides, and which contain two redox-active disulphide groups in addition to the flavin. The enzyme catalyses the reduction of O2, ferricyanide ion, 2,6-dichloroindophenol, and 5,5'dithiobis(2,2'-dinitrobenzoate), and of cumene hydroperoxide in the presence of the small protein component (AhpC) of the peroxide-reducing system of Salmonella typhimurium. The reduction of O2 is slow in the absence of exogenous flavin while dye reduction is fast, suggesting that the free flavin that is added to the usual assay for T. aquaticus NADH oxidase functions by mediating electron transfer from enzyme-bound reduced flavin to O2. The physiological function of the enzyme is probably in peroxide reduction with a small protein analogous to AhpC as the natural electron acceptor.

X-ray crystal structure of the Desulfovibrio vulgaris (Hildenborough) apoflavodoxin-riboflavin complex.

The apoprotein of flavodoxin from Desulfovibrio vulgaris forms a complex with riboflavin. The ability to bind riboflavin distinguishes this flavodoxin from other short-chain flavodoxins which require the phosphate of FMN for flavin binding. The redox potential of the semiquinone/hydroquinone couple of the bound riboflavin is 180 mV less negative than the corresponding complex with FMN. To elucidate the binding of riboflavin, the complex has been crystallized and the crystal structure solved by molecular replacement using native flavodoxin as a search model to a resolution of 0.183 nm. Compared to the FMN complex, the hydrogen-bonding network at the isoalloxazine sub-site of the riboflavin complex is severely disrupted by movement of the loop residues Ser58-Ile64 (60-loop) which contact the isoalloxazine by over 0.35 nm, and by a small displacement of the isoalloxazine moiety. The 60-loop movement away from the flavin increases the solvent exposure of the flavin-binding site. The conformation of the site at which 5'-phosphate of FMN normally binds is similar in the two complexes, but in the riboflavin complex a sulphate or phosphate ion from the crystallization buffer occupies the space. This causes small structural perturbations in the phosphate-binding site. The flexibility of the 60-loop in D. vulgaris flavodoxin appears to be a contributing factor to the binding of riboflavin by the apoprotein, and a feature that distinguishes the protein from other 'short chain' flavodoxins. In the absence of the terminal phosphate group, free movement at the 5'-OH group of the ribityl chain can occur. Thus, the 5'-phosphate of FMN secures the cofactor at the binding site and positions it optimally. The structural changes which occur in the 60-loop in the riboflavin complex probably account for most of the positive shift that is observed in the midpoint potential of the semiquinone/hydroquinone couple of the riboflavin complex compared to that of the FMN complex.

Differential stabilization of the three FMN redox forms by tyrosine 94 and tryptophan 57 in flavodoxin from Anabaena and its influence on the redox potentials.

Flavodoxins are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule as the redox-active center. The redox potentials of the flavin nucleotide are profoundly altered upon interaction with the protein. In Anabaena flavodoxin, as in many flavodoxins, the flavin is sandwiched between two aromatic residues (Trp57 and Tyr94) thought to be implicated in the alteration of the redox potentials. We have individually replaced these two residues by each of the other aromatic residues, by alanine and by leucine. For each mutant, we have determined the redox potentials and the binding energies of the oxidized FMN--apoflavodoxin complexes. From these data, the binding energies of the semireduced and reduced complexes have been calculated. Comparison of the binding energies of wild-type and mutant flavodoxins at the three redox states suggests that the interaction between Tyr94 and FMN stabilizes the apoflavodoxin--FMN complex in all redox states. The oxidized and semireduced complexes are, however, more strongly stabilized than the reduced complex, making the semiquinone/hydroquinone midpoint potential more negative in flavodoxin than in unbound FMN. Trp57 also stabilizes all redox forms of FMN, thus cooperating with Tyr94 in strong FMN binding. On the other hand, Trp57 seems to slightly destabilize the semireduced complex relative to the oxidized one. Finally, we have observed that reduction of mutants lacking Trp57 is slow relative to that of wild-type or mutants lacking Tyr94, which suggests that Trp57 could play a role in the kinetics of flavodoxin redox reactions.

Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution.

Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escherichia coli. A complete backbone NMR assignment for the two-electron reduced protein revealed significant changes of chemical shift values compared to the oxidized protein, in particular for the flavine mononucleotide (FMN)-binding site. A comparison of homo- and heteronuclear NOESY spectra for the two redox states led to the assumption that reduction is not accompanied by significant changes of the global fold of the protein. The backbone dynamics of both the oxidized and reduced forms of D. vulgaris flavodoxin were investigated using two-dimensional 15N-1H correlation NMR spectroscopy. T1, T2 and NOE data are obtained for 95% of the backbone amide groups in both redox states. These values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4559, 4559-4570]. A comparison of the two redox states indicates that in the reduced species significantly more flexibility occurs in the two loop regions enclosing FMN. Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the oxidized state.

Crystallization and preliminary X-ray crystallographic analysis of the electron-transferring flavoprotein from Megasphaera elsdenii.

Electron-transferring flavoprotein from the rumen bacterium Megasphaera elsdenii is a heterodimer (M(r) = 75 kDa) containing FAD as cofactor and functioning solely to mediate electron transfer between the prosthetic groups of other proteins. The enzyme was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as precipitant. The crystals obtained belong to the space group P2(1)2(1)2(1) with unit-cell dimensions of a = 58.75, b = 61.77 and c = 122.27 A. Interestingly the crystals exhibit a low solvent content. Crystals diffracted to beyond 2.5 A using synchrotron radiation.

Analysis of the oxidation-reduction potentials of recombinant ferredoxin-NADP+ reductase from spinach chloroplasts.

Midpoint oxidation-reduction potentials for the two-electron reduction of the bound FAD in spinach ferredoxin-NADP+ reductase were measured by potentiometry (Em = -342 +/- 1 mV at pH 7 and 10 degrees C). They were used with the semiquinone formation constant, obtained by spectroscopic measurement of the semiquinone concentration, to calculate values for the redox potentials of the two one-electron steps in the reduction. The redox potential for the oxidized enzyme/enzyme semiquinone couple (EOX/SQ) at pH 7 is -350 +/- 2 mV (10 degrees C) while the value for the enzyme semiquinone/enzyme hydroquinone couple (ESQ/HQ) under the same conditions is -335 +/- 1 mV. These values correspond to a semiquinone formation constant of 0.55. Measurement of the effects of pH on the potentials showed that EOX/SQ varies linearly with pH (slope -46 +/- 4 mV), while ESQ/HQ is independent of pH at high pH values, but below about pH 7.5 the potential becomes less negative with decreasing pH. indicating that there is a redox-linked protonation of the fully reduced enzyme (pKa = 7.2, 10 degrees C). The absorption spectrum of the fully reduced enzyme was found to depend on pH with the changes giving a calculated pKa of 7.5 (at 15 degrees C). The spectrum at high pH is similar to that of the anionic form of free flavin hydroquinone. The observations suggest that at physiological pH, the enzyme FAD cycles between the three redox states: oxidized, neutral semiquinone and hydroquinone anion.

NMR investigation of the solution conformation of oxidized flavodoxin from Desulfovibrio vulgaris. Determination of the tertiary structure and detection of protein-bound water molecules.

Desulfovibrio vulgaris flavodoxin has been investigated with a combination of homo- and hetero-nuclear two-dimensional and three-dimensional NMR spectroscopy. The analysis of NOE, hydrogen exchange and J-coupling data led to a set of 1349 NOE, 63 hydrogen bond and 109 backbone phi-angle restraints which were used to determine the solution structure of the oxidized flavodoxin applying the distance geometry program DIANA combined with restrained energy minimization methods. Flavodoxin in solution consists of a five-stranded parallel beta-sheet which is pairwise flanked by four alpha-helices. The solution structure has been compared with the known crystal structure. While the global fold is identical, differences have been detected concerning local conformations. In addition, protein-bound water molecules have been localized by NOE effects which were detected in NMR experiments avoiding solvent suppression. The locations of these water molecules have been compared with those found in the X-ray structure.