FMN Cofactor Research Articles

The Flavin Reductase Activity of the Flavoprotein Component of Sulfite Reductase from Escherichia coli: A NEW MODEL FOR THE PROTEIN STRUCTURE

Sulfite reductase (SiR) from Escherichia coli has a alpha 8 beta 4 subunit structure, where alpha 8 is a flavoprotein (SiR-FP) containing both FAD and FMN as prosthetic groups. It also exhibits a NADPH:flavin oxidoreductase activity with exogenous riboflavin, FMN, and FAD serving as substrates. The flavin reductase activity may function during activation of ribonucleotide reductase or during ferrisiderophore reduction. A plasmid containing cysJ gene, coding for the alpha subunit, overexpresses flavin reductase activity by 100-fold, showing that alpha is the site of free flavin reduction. The overproducer allows a fast and simple preparation of large amounts of the flavoprotein. Kinetic studies of its flavin reductase activity demonstrates a ping-pong bisubstrate-biproduct reaction mechanism. NADP+ inhibition studies show that both substrates, NADPH and free flavins, bind to the same site. While the FAD cofactor mediates the electron transfer between NADPH and free flavins, the FMN cofactor is not essential since a FMN-depleted SiR-FP retains a large proportion of activity. In contradiction with previous reports, SiR-FP is found to contain 1.6-1.7 flavin per alpha subunit. This result, together with the sequence homology between SiR-FP and NADPH-cytochrome P-450 reductase, suggests a new model for the structure of the protein with one FMN and one FAD prosthetic group per alpha subunit.

Site-Directed Mutagenesis of Tyrosine-98 in the Flavodoxin from Desulfovibrio vulgaris (Hildenborough): Regulation of Oxidation-Reduction Properties of the Bound FMN Cofactor by Aromatic, Solvent, and Electrostatic Interactions

The contributions made by tyrosine-98 in establishing the redox properties of the flavodoxin from Desulfovibrio vulgaris were investigated by substituting a number of amino acids at this position using site-directed mutagenesis. Tyr98, which makes extensive van der Waals contacts with the isoalloxazine ring of the flavin mononucleotide cofactor, is often found in the cofactor binding site of flavodoxins and related flavoproteins. Solution studies suggest that tyrosine may assist in the stabilization of the neutral flavin semiquinone through preferential complex formation relative to the other oxidation states. In this study, the midpoint potentials of the oxidized/semiquinone couple of the Y98W and Y98F mutants were found to be very similar to the wild-type flavodoxin. However, significantly more negative midpoint potentials (by 25-60 mV) were observed in the Y98A, Y98H, and Y98R mutants. These results imply that it is the general apolar environment provided by the aromatic amino acids rather than preferential affinities suggested by solution studies that is at least partially responsible for the thermodynamic stabilization of the neutral flavin semiquinone in this flavodoxin. The midpoint potential of the semiquinone/hydroquinone couple is profoundly dependent on the properties of the amino acid at this position. Compared to phenylalanine, the more electron-rich aromatic side chains of tryptophan and tyrosine decrease the midpoint potential of this couple by 30-40 mV. Greater solvent exposure of the isoalloxazine ring in the Y98A mutant increases the midpoint potential by 140 mV relative to wild type. The positively charged amino acids increase the midpoint potential of this couple by > 180 mV, most probably through favorable electrostatic interactions with the flavin hydroquinone anion. These observations strongly support the proposition that the functional role of the electron-rich, apolar aromatic amino acid residues adjacent to the flavin isoalloxazine ring is to substantially destabilize the flavin hydroquinone anion, resulting in the very low oxidation-reduction potentials for the semiquinone/hydroquinone couple that typify the flavodoxin family.

Crystal structure of apo-glycolate oxidase

The crystal structure of the apoform of the α/β-barrel enzyme glycolate oxidase has been determined to 2.6 Å resolution. Removal of the tightly bound cofactor FMN has a very strong influence on the protein structure; it is converted into a very flexible state, verging on a molten globule type of structure. The asymmetric unit contains two subunits with different conformations to each other and to the holo-enzyme. The secondary structures are preserved, but their mutual arrangement has changed to some extent introducing cavities into the protein. The largest structural shifts are, however, found in the loops.

Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions.

The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin-NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (approximately 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.

Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding.

Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms.

The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major increase in P450 reductase protein or activity was detectable over a 3-day period. Together, these studies establish that thyroid hormone regulates P450 reductase expression by pretranslational mechanisms. They also suggest that other regulatory mechanisms, which may involve changes in P450 reductase protein stability and/or changes in the translational efficiency of its mRNA, are likely to occur.

Catalytic and regulatory properties of native and chymotrypsin-treated pyridoxine-5-phosphate oxidase.

Brain pyridoxine-5-P oxidase is activated by the tryptophan metabolites 3-hydroxyanthranilate and 3-hydroxykynurenine. 3-Hydroxyanthranilate at concentrations of 0.03 mM relieves the inhibition elicited by accumulation of the substrate pyridoxine-5-P (Ki = 60 microM). The results of fluorometric measurements indicate that four molecules of 3-hydroxyanthranilate bind to the dimeric enzyme (56 kDa) with an association constant of 5.5 x 10(4) M-1. Differential spectral measurements failed to detect any direct interaction between the cofactor FMN and the effector 3-hydroxyanthranilate. These results are consistent with the hypothesis that the effector molecules bind to sites of the dimeric protein distinct from the cofactor site. Limited chymotrypsin digestion of pyridoxine-5-P oxidase yields catalytically active species that are no longer susceptible to activation by 3-hydroxykynurenine. A polypeptide of 16 kDa containing FMN and endowed with full catalytic activity was isolated by ion-exchange chromatography. It is postulated that the structural domain associated with catalytic activity composes approximately one-half of the molecular mass of pyridoxine-5-P oxidase (28 kDa), whereas the remaining portion of the macromolecule contains regulatory binding sites.

Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding

The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rate constants (9 x 10(8) M-1 s-1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding identical second-order rate constants (1.1 x 10(7) M-1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvate binding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process.

Laser flash photolysis study of intermolecular and intramolecular electron transfer in trimethylamine dehydrogenase

Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119 are described. The first 36 residues of the amino-terminal amino acid sequence have been determined and show extensive homology with flavodoxins isolated from other sources. Anabaena flavodoxin exhibits a net negative change (—3) in the helix-1 segment as found with other cyanobacterial flavodoxins Synechococcus 6301 ( Anacystis nidulans) and Nostoc MAC, but in contrast to the net positive charge found in this region in the case of flavodoxins isolated from nitrogen-fixing bacteria ( Azotobacter and Klebsiella). The FMN cofactor can be reversibly resolved from the apoprotein by trichloroacetic acid treatment. Apoflavodoxin, thus prepared, binds FMN with a K d value of 0.1 nM and binds riboflavin with a decreased affinity ( K d = 5 μM) at pH 7.2. The apoprotein is stable dilute solutions at pH values around 7 but readily denatures at pH 8 as judged from loss in flavin-binding ability and by ultraviolet circular dichroism spectroscopy. Oxidation-reduction potential studies at pH values of 7 and 8 show OX/SQ couples of −195 mV and −255 mV, respectively, and show SQ/HQ couples of −390 mV and −418 mV, respectively. From these data, the binding constant for the FMN semiquinone is calculated to be approx. 5-fold tighter and the binding of the FMN hydroquinone is approx. 10 5-fold weaker than that of the oxidized FMN to the apoprotein. Anabaena flavodoxin functions as an effective mediator of electron transfer from ferredoxin-NADP +-reductase to cytochrome c with a turnover number (((4.5–5)·10 3 min -1); a values similar to that determined for Anabaena ferredoxin. The flavodoxin binds tightly to the reductase with K d values of 6.4 and 8.5 μM at pH values of 7.0 and 8.0, respectively.

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000 X g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000 X g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of Mr 26,000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.

Influence of 8 alpha-imidazole substitution of the FMN cofactor on the rate of electron transfer from the neutral semiquinones of two flavodoxins to cytochrome c.

The effects of substituting an imidazole ring onto the 8 alpha-position of the FMN cofactor on the kinetics of electron transfer from the neutral semiquinone forms of Azotobacter and Clostridium flavodoxins to oxidized horse heart cytochrome c have been investigated by stopped-flow methods. Although 8 alpha-substitution does not alter the mechanistic pathway of the reaction, the rate constants are decreased by factors of 10-30, without significant changes in the equilibrium association constants of the intermediate electron-transfer complexes. Protonation of the imidazole ring further decreases the observed second-order rate constants for the electron-transfer reaction by factors of 20-50. The pKa values for the 8 alpha-imidazole ring in both flavodoxin semiquinones were determined to be approximately 7. In contrast, the reactions of the native flavodoxins with cytochrome c are pH independent. The results are consistent with a structural model of the intermediate complex [Simondsen, R. P., Weber, P. C., Salemme, F. R., & Tollin, G. (1982) Biochemistry 21, 6366-6375], which postulates a close fit between the exposed dimethylbenzene ring of the FMN and the heme edge within a nonpolar interface region. The results further indicate that electron transfer is uncoupled from proton transfer, that it is the rate-limiting step, and that it occurs prior to proton transfer at all pH values. Finally, the results do not provide support for a direct role of the imidazole ring in the facilitation of one-electron transfer in those enzymes containing 8 alpha-N-histidylflavin coenzymes.

Three-dimensional structure of the iron-sulfur flavoprotein trimethylamine dehydrogenase at 2.4-A resolution.

The three-dimensional structure of trimethylamine dehydrogenase from the methylotrophic bacterium W3A1 has been determined to 2.4-A resolution. The enzyme is composed of two identical 83,000-dalton subunits, each of which is folded into three structural domains. The largest domain, at the NH2 terminus of the molecule, is folded as an eight-stranded parallel alpha/beta barrel. It contains the [4Fe-4S] and covalently bound FMN cofactors separated by about 4 A. The folding topology of the large domain and orientation of the FMN cofactor are very similar to those found in glycolate oxidase. The other two domains contain alpha/beta parallel beta sheet topologies with similar folding patterns. The topologies and spatial arrangements of these two domains are remarkably similar to the FAD- and NADPH-binding domains of glutathione reductase.