Identification, sequence determination, and expression of the flavodoxin gene from Desulfovibrio salexigens

Restriction fragments of genomic DNA from Desulfovibrio salexigens (ATCC 14822) containing the structural gene coding for the flavodoxin protein were identified using the entire coding region of the gene for the Desulfovibrio vulgaris (Hildenborough) flavodoxin as a probe (Krey, G.D., Vanin, E.F., and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). A 1.4-kb PstI-HindIII fragment was ultimately identified which contains an open reading frame coding for a polypeptide of 146 amino acid residues that was highly homologous to the D. vulgaris flavodoxin, sharing a sequence identity of 55%. When compared to the X-ray crystal structure of the D. vulgaris protein, the homologous regions were largely confined to those portions of the protein which are in the immediate vicinity of the flavin mononucleotide cofactor binding site. Tryptophan-60 and tyrosine-98, which reside on either side of the isoalloxazine ring of the cofactor, are conserved, as are the sequences of the polypeptide loop that interacts with the phosphate moiety of the flavin. Acidic residues forming the interface of model electron-transfer complexes with certain cytochrome c proteins are retained. The flavodoxin holoprotein is over-expressed in E. coli from the cloned gene using its endogenous promoter.     

Cloning and sequencing of the gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough

Abstract The gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough (148 amino acid residues), the first flavoprotein for which a three-dimensional structure has been determined, was cloned with the use of two synthetic oligonucleotides, designed to recognize the coding sequence for amino acid residues 11–19 and 98–103, respectively. The two oligonucleotides were used to screen a library of 900 λ-clones of the D. vulgaris chromosome. A single clone, λFL1, reacting with both probes was isolated. The entire structural gene for flavodoxin is contained in the 15 kb insert of λFL1 as found by nucleic acid sequencing. The codon usage in the flavodoxin gene is strongly biased towards G or C in the third codon position. A table in which codon usage information from all genes of D. vulgaris sequenced to date is combined is presented and should facilitate further gene cloning with oligonucleotide probes.     

Cloning, nucleotide sequence, and expression of the flavodoxin gene from Desulfovibrio vulgaris (Hildenborough)

The gene coding for the flavodoxin protein from Desulfovibrio vulgaris (Hildenborough) has been identified, cloned, and sequenced. DNA fragments containing the flavodoxin gene were identified by hybridization of a mixed synthetic heptadecanucleotide probe to Southern blots of SalI-digested genomic DNA. The nucleotide sequences of the probe were derived from the published protein primary structure (Dubourdieu, M., LeGall, J., and Fox, J. L. (1973) Biochem. Biophys. Res. Commun. 52, 1418-1425). The same oligonucleotide probe was used to screen libraries (in pUC19) containing size-selected SalI fragments. One recombinant, carrying a 1.6-kilobase (kb) insert which strongly hybridizes to the probe, was found to contain a nucleotide sequence which codes for the first 104 residues of the amino-terminal portion of the flavodoxin protein sequence but lacked the remainder of the gene. Therefore, a PstI restriction fragment from this clone was used as a probe to isolate the entire gene from a partial Sau3AI library in Charon 35. Of the plaques which continued to hybridize strongly to this probe through repeated screenings, one recombinant, containing a 16-kb insert, was further characterized. The entire flavodoxin gene was localized within a 1.4-kb XhoI-SacI fragment of this clone. The complete nucleotide sequence of the structural gene for the flavodoxin protein from Desulfovibrio vulgaris and flanking sequences which may include promoter and regulatory sequences are reported here. The cloned flavodoxin gene was placed behind the hybrid tac promoter for overexpression of the protein in Escherichia coli. Modification to the 5'-end of the gene, including substitutions at the second codon, were required to obtain high levels of expression. The expressed recombinant flavodoxin protein is isolated from E. coli cells as the holoprotein with physical and spectral properties similar to the protein isolated from D. vulgaris. To our knowledge, this is the first example of the expression of a foreign flavodoxin gene in E. coli using recombinant DNA methods.     

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.     

Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello.

The genes encoding the periplasmic [Fe] hydrogenase from Desulfovibrio vulgaris subsp. oxamicus Monticello were cloned by exploiting their homology with the hydAB genes from D. vulgaris subsp. vulgaris Hildenborough, in which this enzyme is present as a heterologous dimer of alpha and beta subunits. Nucleotide sequencing showed that the enzyme is encoded by an operon in which the gene for the 46-kilodalton (kDa) alpha subunit precedes that of the 13.5-kDa beta subunit, exactly as in the Hildenborough strain. The pairs of hydA and hydB genes are highly homologous; both alpha subunits (420 amino acid residues) share 79% sequence identity, while the unprocessed beta subunits (124 and 123 amino acid residues, respectively) share 71% sequence identity. In contrast, there appears to be no sequence homology outside these coding regions, with the exception of a possible promoter element, which was found approximately 90 base pairs upstream from the translational start of the hydA gene. The recently discovered hydC gene, which may code for a 65.8-kDa fusion protein (gamma) of the alpha and beta subunits and is present immediately downstream from the hydAB genes in the Hildenborough strain, was found to be absent from the Monticello strain. The implication of this result for the possible function of the hydC gene product in Desulfovibrio species is discussed.     

Redox potential difference between Desulfovibrio vulgaris and Clostridium beijerinckii flavodoxins

The redox potential of the flavin mononucleotide (FMN) hydroquinones for one-electron reduction in the Desulfovibrio vulgaris ( D. vulgaris) flavodoxin ( E sq/hq for FMNH (*)/FMNH (-)) was calculated using the crystal structure of the relevant hydroquinone form and compared to the results of the Clostridium beijerinckii ( C. beijerinckii) flavodoxin. In D. vulgaris and C. beijerinckii flavodoxins, the protein side chain causes significant downshifts of 170 and 240 mV in E sq/hq, respectively. In the C. beijerinckii flavodoxin, the E sq/hq downshift because of the protein side chain is essentially compensated by the counter influence of the protein backbone ( E sq/hq upshift of 260 mV). However, in the D. vulgaris flavodoxin, the corresponding protein backbone influence on E sq/hq is significantly small, i.e., less than half of that in the C. beijerinckii flavodoxin. In particular, there is a significant difference in the influence of the protein backbone of the so-called 60s loop region between the two flavodoxins. The E sq/hq difference can be best explained by the lower compensation of the side chain influence by the backbone influence in the D. vulgaris flavodoxin than in the C. beijerinckii flavodoxin.     

Chemical synthesis and expression of a synthetic gene for the flavodoxin from Clostridium MP

A gene coding for the flavodoxin from Clostridium MP was designed, synthesized, and expressed in Escherichia coli. The sequence of the coding region was derived from the published amino acid sequence of the protein (Tanaka, M., Haniu, M., Yasunobu, K.T., and Mayhew, S. G. (1974) J. Biol. Chem. 249, 4393-4397) and was designed for optimal expression and for use of the cassette mutagenesis approach. The structural gene was subassembled in three sections, each of which was constructed by the enzymatic ligation of three complementary pairs of chemically synthesized oligodeoxyribonucleotides having short single-stranded ends complementary to that of the adjacent pair. Coligation of the three sections produced the final structural gene which consists of 420 nucleotides. The synthetic gene was cloned behind the hybrid tac promoter (Amman, E., Brosius, J., and Ptashne, M. (1983) Gene (Amst.) 25, 167-178) in the pKK223-3 vector or adjacent to the strong T7 RNA polymerase promoter in the pET-3a expression vector (Rosenberg, A.H., Lade, B. N., Chui, D-S., Lin, S-W., Dunn, J. J., and Studier, F. W. (1987) Gene (Amst.) 56, 125-135) for expression in E. coli. Upon induction with isopropyl-beta-D-thiogalactoside, the flavodoxin polypeptide was expressed from the artificial gene to levels approaching 20% of total extractable proteins using either expression system. The flavodoxin was purified from cellular extracts as the holoprotein containing bound flavin mononucleotide. The recombinant flavodoxin protein was found to have an ultraviolet/visible spectrum, amino-terminal sequence, and amino acid composition identical to the wild-type flavodoxin protein purified from Clostridium MP. This work represents the first chemical synthesis and expression in E. coli of an artificial gene coding for a bacterial flavodoxin.     

Cloning and sequencing of a [NiFe] hydrogenase operon from Desulfovibrio vulgaris Miyazaki F

A hydrogenase operon was cloned from chromosomal DNA isolated from Desulfovibrio vulgaris Miyazaki F with the use of probes derived from the genes encoding [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough. The nucleic acid sequence of the cloned DNA indicates this hydrogenase to be a two-subunit enzyme: the gene for the small subunit (267 residues; molecular mass = 28763 Da) precedes that for the large subunit (566 residues; molecular mass = 62495 Da), as in other [NiFe] and [NiFeSe] hydrogenase operons. The amino acid sequences of the small and large subunits of the Miyazaki hydrogenase share 80% homology with those of the [NiFe] hydrogenase from Desulfovibrio gigas. Fourteen cysteine residues, ten in the small and four in the large subunit, which are thought to co-ordinate the iron-sulphur clusters and the active-site nickel in [NiFe] hydrogenases, are found to be conserved in the Miyazaki hydrogenase. The subunit molecular masses and amino acid composition derived from the gene sequence are very similar to the data reported for the periplasmic, membrane-bound hydrogenase isolated by Yagi and coworkers, suggesting that this hydrogenase belongs to the general class of [NiFe] hydrogenases, despite its low nickel content and apparently anomalous spectral properties.     

Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus.

The genes coding for the large and small subunits of the periplasmic hydrogenase from Desulfovibrio baculatus have been cloned and sequenced. The genes are arranged in an operon with the small subunit gene preceding the large subunit gene. The small subunit gene codes for a 32 amino acid leader sequence supporting the periplasmic localization of the protein, however no ferredoxin-like or other characteristic iron-sulfur coordination sites were observed. The periplasmic hydrogenases from D. baculatus (an NiFeSe protein) and D. vulgaris (an Fe protein) exhibit no homology suggesting that they are structurally different, unrelated entities.     

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)     

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].     

Amino acid sequence of Desulfovibrio vulgaris flavodoxin.

The complete amino acid sequence for the 148-amino acid flavodoxin from Desulfovibrio vulgaris was determined to be: H3N+-Met-Pro-Lys-Ala-Leu-Ile-Val-Tyr-Gly-Ser-Thr-Thr-Gly-Asn-Thr-Glu-Tyr-Thr-Ala-Glu-Thr-Ile-Ala-Arg-Glu-Leu-Ala-Asn-Ala-Gly-Tyr-Glu-Val-Asp-Ser-Arg-Asp-Ala-Ala-Ser-Val-Glu-Ala-Gly-Gly-Leu-Phe-Glu-Gly-Phe-Asp-Leu-Val-Leu-Leu-Gly-Cys-Ser-Thr-Trp-Gly-Asp-Asp-Ser-Ile-Glu-Leu-Gln-Asp-Asp-Phe-Ile-Pro-Leu-Phe-Asp-Ser-Leu-Glu-Glu-Thr-Gly-Ala-Gln-Gly-Arg-Lys-Val-Ala-Cys-Phe-Gly-Cys-Gly-Asp-Ser-Ser-Tyr-Glu-Tyr-Phe-Cys-Gly-Ala-Val-Asp-Ala-IleGlu-Glu-Lys-Leu-Lys-Asn-Leu-Gly-Ala-Glu-Ile-Val-Gln-Asp-Gly-Leu-Arg-Ile-Asp-Gly-Asp-Pro-Arg-Ala-Ala-Arg-Asp-Asp-Ile-Val-Gly-Try-Ala-His-Asp-Val-Arg-Gly-Ala-Ile-COO. This protein is of interest as it was the first flavoenzyme for which high resolution x-ray diffraction studies were published (Watenpaugh, K.D., Sieker, L.C., and Jensen, L.H. (1973) Proc. NAtl. Acad. Sci. U.S.A. 70, 3857-3860). Ser(10), Thr(12), Asn(14), and Thr(15) were shown to bind the phosphate of the FMN while the isoalloxazine ring is positioned between Trp(60) and Tyr(98).     

Cloning of genes encoding redox proteins of known amino acid sequence from a library of the Desulfovibrio vulgaris (Hildenborough) genome

A library of 900 recombinant phages has been constructed for the genome of Desulfovibrio vulgaris Hildenborough (1.7 x 10(6) bp) by cloning size-fractionated Sau3A fragments (15-20 kb) into the replacement vector lambda-2001. When a hydrogenase gene probe, a 4.7-kb SalI-EcoRI fragment of known nucleotide sequence, was used to screen the plaque lifted library, 23 positive clones were found, which together span 31 kb of D. vulgaris DNA. To facilitate the cloning of genes with oligodeoxynucleotides as probes, DNA was purified for all clones in the library and spotted on a 16 x 16-cm grid of nitrocellulose. This grid was incubated sequentially to identify lambda clones containing the gene for redox proteins of known amino acid sequence: cytochrome c3 (one 18-mer----four clones), flavodoxin (one 17-mer and one 26-mer----one clone) and rubredoxin (one 44-mer----21 clones). The four cyc-positive clones are also recognized by the rubredoxin oligodeoxynucleotide probe. Restriction mapping defines a 35-kb region of the D. vulgaris chromosome in which the rub and cyc loci are separated by 17.5 kb. The nucleotide sequence of the rubredoxin gene was determined and the deduced amino acid sequence found to agree with that determined in Bruschi [Biochim. Biophys. Acta 434 (1976) 4-17] with the exception of Thr-21 which is found to be encoded by GAC, an Asp codon. A plausible ribosome-binding site precedes the N-terminal initiator methionine residue. Rubredoxin does not have an N-terminal signal sequence which is in agreement with the cytoplasmic location of this redox protein.     

The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio vulgaris (Hildenborough).

The gene encoding a protein containing a putative [6Fe-6S] prismane cluster has been cloned from Desulfovibrio vulgaris (Hildenborough) and sequenced. The gene encodes a polypeptide composed of 553 amino acids (60,161 Da). The DNA-derived amino acid sequence was partly confirmed by N-terminal sequencing of the purified protein and of fragments of the protein generated by CNBr cleavage. Furthermore, the C-terminal sequence was verified by digestion with carboxypeptidases A and B. The polypeptide contains nine Cys residues. Four of these residues are gathered in a Cys-Xaa2-Cys-Xaa7-Cys-Xaa5-Cys motif located towards the N-terminus of the protein. No relevant sequence similarity was found with other proteins, including those with high-spin Fe-S clusters (nitrogenase, hydrogenase), with one significant exception: the stretch containing the first four Cys residues spans two submotifs, Cys-Xaa2-Cys and Lys-Gly-Xaa-Cys-Gly, separated by 11 residues, that are also present in high-spin Fe-S cluster containing CO dehydrogenase. Western-blot analysis demonstrates cross-reactivity of antibodies raised against the purified protein both in Desulfovibrio strains and other sulfate-reducing bacteria. Hybridization of the cloned gene with genomic DNA of several other Desulfovibrio species indicates that homologous sequences are generally present in the genus Desulfovibrio.     

The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio desulfuricans (ATCC 27774).

The gene encoding a protein containing a novel iron sulfur cluster ([6Fe-6S]) has been cloned from Desulfovibrio desulfuricans ATCC 27774 and sequenced. An open reading frame was found encoding a 545 amino acid protein (M(r) 58,496). The amino acid sequence is highly homologous with that of the corresponding protein from D. vulgaris (Hildenborough) and contains a Cys-motif that may be involved in coordination of the Fe-S cluster.     

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.     

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.     

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.