FAD analogues as prosthetic groups of human glutathione reductase. Properties of the modified enzyme species and comparisons with the active site structure

Human glutathione reductase (NADPH + GSSG + H+ in equilibrium with NADP+ + 2 GSH) is a suitable enzyme for correlating spectroscopic properties and chemical reactivities of protein-bound FAD analogues with structural data. FAD, the prosthetic group of the enzyme, was replaced by FAD analogues, which were modified at the positions 8, 1, 2, 4, 5 and 6, respectively, of the isoalloxazine ring. When compared with a value of 100% for native glutathione reductase, the specific activities of most enzyme species ranged from 40% to 17%, in the order of the prosthetic groups 8-mercapto-FAD greater than 8-azido-FAD = 8-F-FAD = 8-C1-FAD greater than 4-thio-FAD = 1-deaza-FAD greater than 2-thio-FAD. The enzymic activities indicate a correct orientation of the bound analogues. The enzyme species containing 5-deaza-FAD and 6-OH-FAD, respectively, had no more glutathione reductase activity than the FAD-free apoenzyme. 5-Deaza-FAD X glutathione reductase was crystallized for X-ray diffraction analysis. Detailed studies were focussed on position 8 of the flavin. 8-Cl-FAD X glutathione reductase and 8-F-FAD X glutathione reductase reacted only poorly with HS- to give 8-mercapto-FAD X glutathione reductase, which suggests that the region around Val61 hinders the halogen anion from leaving the tetrahedral intermediate. Other experiments showed that position 8 is accessible to certain solvent-borne reagents. 8-Mercapto-FAD X glutathione reductase, for instance, reacted readily and stoichiometrically with the thiol reagent methylmethanethiosulfonate. 8-Mercapto-FAD X glutathione reductase does not exhibit a long wavelength charge transfer absorption band upon reduction, as it is the case for the 2-electron-reduced FAD-containing enzyme. This behaviour indicates that the charge transfer interaction between flavin and the thiolate of Cys63 in the native enzyme is not per se essential for catalysis. The absorption spectrum of the blue anionic 8-mercapto-FAD bound to glutathione reductase suggests that the protein concurs to the stabilization of a negative charge in the pyrimidine subnucleus. In light of the protein structure this effect is attributed to the dipole moment of alpha-helix 338-354 which starts out close to the N(1)/C(2)/O(2 alpha) region of the flavin. 1-Deaza-FAD binds as tightly as FAD to the apoenzyme. The resulting holoenzyme was found to be enzymically active but structurally unstable. In this respect 1-deaza-FAD . glutathione reductase mimics the properties of the enzyme species found in inborn glutathione reductase deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...     

Monooxygenation of aromatic compounds by flavin‐dependent monooxygenases

Many flavoenzymes catalyze hydroxylation of aromatic compounds especially phenolic compounds have been isolated and characterized. These enzymes can be classified as either single‐component or two‐component flavin‐dependent hydroxylases (monooxygenases). The hydroxylation reactions catalyzed by the enzymes in this group are useful for modifying the biological properties of phenolic compounds. This review aims to provide an in‐depth discussion of the current mechanistic understanding of representative flavin‐dependent monooxygenases including 3‐hydroxy‐benzoate 4‐hydroxylase (PHBH, a single‐component hydroxylase), 3‐hydroxyphenylacetate 4‐hydroxylase (HPAH, a two‐component hydroxylase), and other monooxygenases which catalyze reactions in addition to hydroxylation, including 2‐methyl‐3‐hydroxypyridine‐5‐carboxylate oxygenase (MHPCO, a single‐component enzyme that catalyzes aromatic‐ring cleavage), and HadA monooxygenase (a two‐component enzyme that catalyzes additional group elimination reaction). These enzymes have different unique structural features which dictate their reactivity toward various substrates and influence their ability to stabilize flavin intermediates such as C4a‐hydroperoxyflavin. Understanding the key catalytic residues and the active site environments important for governing enzyme reactivity will undoubtedly facilitate future work in enzyme engineering or enzyme redesign for the development of biocatalytic methods for the synthesis of valuable compounds.     

Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme

The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.     

Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis. Studies with nucleotide analogues

Previous studies have shown that the interaction of P450 reductase with bound NADP(H) is essential to ensure fast electron transfer through the two flavin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogues. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogue is inactive as electron donor, but its binding affinity and rate constant of release are very close to those for NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide does not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase binds NADPH with a K(d) of 0.3 microM, while the affinity of the reduced enzyme is considerably lower, K(d) = 1.9 microM. P450 reductase catalyzes a transhydrogenase reaction between NADPH and oxidized nucleotides, such as thionicotinamide-NADP(+), acetylpyridine-NADP(+), or [(3)H]NADP(+). The reverse reaction, reduction of [(3)H]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction as follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavoprotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semiquinone content, rate constant for NADPH release, and transhydrogenase turnover rates allowed us to estimate the steady-state distribution of P450 reductase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate that equilibrium redox potentials of the flavin cofactors are not the sole factor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.     

Crystal structure of NAD(P)H:flavin oxidoreductase from Escherichia coli.

Flavin reductases use flavins as substrates and are distinct from flavoenzymes which have tightly bound flavins. The reduced flavin can serve to reduce ferric complexes and iron proteins. In Escherichia coli, reactivation of ribonucleotide reductase is achieved by reduced flavins produced by flavin reductase. The crystal structure of E. coli flavin reductase reveals that the enzyme structure is similar to the structures of the ferredoxin reductase family of flavoproteins despite very low sequence similarities. The main difference between flavin reductase and structurally related flavoproteins is that there is no binding site for the AMP moiety of FAD. The direction of the helix in the flavin binding domain, corresponding to the phosphate binding helix in the flavoproteins, is also slightly different and less suitable for phosphate binding. Interactions for flavin substrates are instead provided by a hydrophobic isoalloxazine binding site that also contains a serine and a threonine, which form hydrogen bonds to the isoalloxazine of bound riboflavin in a substrate complex.     

The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases.

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."     

Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.     

Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis.

Genes encoding NAD(P)H-flavin oxidoreductases (flavin reductases) similar in both size and sequence to Fre, the most abundant flavin reductase in Escherichia coli, were identified in four species of luminous bacteria, Photorhabdus luminescens (ATCC 29999), Vibrio fischeri (ATCC 7744), Vibrio harveyi (ATCC 33843), and Vibrio orientalis (ATCC 33934). Nucleotide sequence analysis showed Fre-like flavin reductases in P. luminescens and V. fischeri to consist of 233 and 236 amino acids, respectively. As in E. coli Fre, Fre-like enzymes in luminous bacteria preferably used riboflavin as an electron acceptor when NADPH was used as an electron donor. These enzymes also were good suppliers of reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. In V. fischeri, the Fre-like enzyme is a minor flavin reductase representing < 10% of the total FMN reductase. That the V. fischeri Fre-like enzyme has no appreciable homology in amino acid sequence to the major flavin reductase in V. fischeri, FRase I, indicates that at least two different types of flavin reductases supply FMNH2 to the luminescence system in V. fischeri. Although Fre-like flavin reductases are highly similar in sequence to luxG gene products (LuxGs), Fre-like flavin reductases and LuxGs appear to constitute two separate groups of flavin-associated proteins.     

Dihydrodipicolinate reductases from Bacillus cereus and Bacillus megaterium.

Dyhydrodipicolinate reductases were purified 100-fold from crude extracts of B. cereus and B. megaterium and their properties were compared with those of the reductase from B. subtilis. The molecular weights of the reductases of B. cereus and B. megaterium were fount to be 155,000 and 150,000, respectively. These reductases were shown to be free of flavin, unlike the B. subtilis enzyme, which contains flavin. Both NADPH and NADH acted as coenzymes for these two reductases. NADPH being three or four times more effective than NADH. The Km values for NADPH and dihydrodipicolinate were 8 micrometer and 62 micrometer, respectively, with B. cereus reductase, and 13 micrometer and 59 micrometer with B. megaterium reductase. The pH optima of the enzymes from B. cereus and B. megaterium were pH 7.4 and 7.2, respectively. The reductases were inhibited by dipicolinate noncompetitively with respect to dihydrodipicolinate and the Ki values were 85 micrometer and 140 micrometer, respectively. Lysine and diaminopimelate were not inhibitory. The properties of the reductases from B. cereus and B. megaterium were similar, but they differed considerably from those of the B. subtilis enzyme. However, all three Bacillus reductases were markedly inhibited by dipicolinate, unlike the enzyme from E. coli.     

The NAD(P)H:flavin oxidoreductase from Escherichia coli. Evidence for a new mode of binding for reduced pyridine nucleotides.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, named Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADPH or NADH as electron donor. The enzyme does not contain any prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin in a ternary complex prior to oxidoreduction. The specificity of the flavin reductase for the pyridine nucleotide was studied by steady-state kinetics using a variety of NADP analogs. Both the nicotinamide ring and the adenosine part of the substrate molecule have been found to be important for binding to the polypeptide chain. However, in the case of NADPH, the 2'-phosphate group destabilized almost completely the interaction with the adenosine moiety. Moreover, NADPH and NMNH are very good substrates for the flavin reductase, and we have shown that both these molecules bind to the enzyme almost exclusively by the nicotinamide ring. This provides evidence that the flavin reductase exhibits a unique mode for recognition of the reduced pyridine nucleotide. In addition, we have shown that the flavin reductase selectively transfers the pro-R hydrogen from the C-4 position of the nicotinamide ring and is therefore classified as an A-side-specific enzyme.     

The EH2 reduced intermediate of glutathione reductase contains oxidised flavin-while EH4 does not

Glutathione reductase is a flavoprotein whose x-ray structure has been established. Functional data and the x-ray structure are consistent with a mechanism of reaction in which NADPH reacts with the enzyme to produce a two electron, EH2, and four electron, EH4, intermediate. The former is competent for the transfer of electrons to the substrate glutathione. Several structures are possible for the two NADPH intermediates; in order to aid in the determination of the structure of these intermediates, we have determined their resonance Raman spectra at two excitation frequencies. These studies establish that the EH2 intermediate is an oxidized flavin species while the EH4 species is not. Furthermore, the most likely structure for EH2 involves a charge transfer donation of electrons from the anion of cys-63 to the N5 position of flavin.     

Interaction of nitrofurans with glutathione reductase

Nitrofurans inhibit the oxidation of NADPH by glutathione, catalyzed by yeast glutathione reductase (EC 1.6.4.2). acting as uncompetitive incomplete inhibitors for NADPH and glutathione. The quinoline-substituted nitrofurans were the most effective inhibitors. These compounds increased the turnover numbers of enzyme at fixed concentrations of reduced glutathione, in the reverse reaction of glutathione reductase, but in most cases diminished the affinity of the enzyme for NAD+. Nitrofurans are weak one-electron oxidants of glutathione reductase. Their reactivity is close to that of p-quinones possessing the analoguous one-electron reduction potential (Cénas, N.K., Rakauskiené, G.A. and Kulys, J.J. (1989) Biochim. Biophys. Acta 973, 399-404), and reaction is stimulated by NADP+. It is assumed, that nitrofurans bind to the 'regulative' site of glutathione reductase (Karplus, P.A., Pai, E.F. and Schulz, G.E. (1989) Eur. J. Biochem. 178, 693-703).     

A rate-limiting conformational change of the flavin in p-hydroxybenzoate hydroxylase is necessary for ligand exchange and catalysis: studies with 8-mercapto- and 8-hydroxy-flavins

The FAD of p-hydroxybenzoate hydroxylase (PHBH) is known to exist in two conformations. The FAD must be in the in-position for hydroxylation of p-hydroxybenzoate (pOHB), whereas the out-position is essential for reduction of the flavin by NADPH. In these investigations, we have used 8-mercapto-FAD and 8-hydroxy-FAD to probe the movement of the flavin in catalysis. Under the conditions employed, 8-mercapto-FAD (pK(a) = 3.8) and 8-hydroxy-FAD (pK(a) = 4.8) are mainly anionic. The spectral characteristics of the anionic forms of these flavins are very sensitive to their environment, making them sensitive probes for detecting movement of the flavin during catalysis. With these flavin analogues, the enzyme hydroxylates pOHB efficiently, but at a rate much slower than that of enzyme with FAD. Reaction of oxygen with reduced forms of these modified enzymes in the absence of substrate appears to proceed through the formation of the flavin-C4a-hydroperoxide intermediate, as with normal enzyme, but the decay of this intermediate is so fast compared to its formation that very little accumulates during the reaction. However, after elimination of H2O2 from the flavin-C4a-hydroperoxide, a perturbed oxidized enzyme spectrum is observed (Eox*), and this converts slowly to the spectrum of the resting oxidized form of the enzyme (Eox). In the presence of pOHB, PHBH reconstituted with 8-mercapto-FAD also shows the additional oxidized intermediate (Eox*) after the usual oxygenated C4a-intermediates have formed and decayed in the course of the hydroxylation reaction. This Eox* to Eox step is postulated to be due to flavin movement. Furthermore, binding of pOHB to resting (Eox) follows a three-step equilibrium mechanism that is also consistent with flavin movement being the rate-limiting step. The rate for the slowest step during pOHB binding is similar to that observed for the conversion of Eox* to Eox during the oxygen reaction in the absence or presence of substrate. Steady-state kinetic analysis of PHBH substituted with 8-mercapto-FAD demonstrated that the apparent k(cat) is also similar to the rate of Eox* conversion to Eox. Presumably, the protein environment surrounding the flavin in Eox* differs slightly from that of the final resting form of the enzyme (Eox).     

Inhibition of pea ferredoxin-NADP(H) reductase by Zn-ferrocyanide.

Ferredoxin-NADP(H) reductases (FNRs) represent a prototype of enzymes involved in numerous metabolic pathways. We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn2+ (Ki 1.57 microM). Dichlorophenolindophenol diaphorase activity was also inhibited by Zn2+ (Ki 1.80 microM), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions. Escherichia coli FNR was also inhibited by Zn-ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes. The inhibitor behaves in a noncompetitive manner for NADPH and for artificial electron acceptors. Analysis of the oxidation state of the flavin during catalysis in the presence of the inhibitor suggests that the electron-transfer process between NADPH and the flavin is not significantly altered, and that the transfer between the flavin and the second substrate is mainly affected. Zn-ferrocyanide interacts with the reductase, probably increasing the accessibility of the prosthetic group to the solvent. Ferredoxin reduction was also inhibited by Zn-ferrocyanide in a noncompetitive manner, but the observed Ki was about nine times higher than those for the diaphorase reactions. The electron transfer to Anabaena flavodoxin was not affected by Zn-ferrocyanide. Binding of the apoflavodoxin to the reductase was sufficient to overcome the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect.     

Determination of the redox properties of human NADPH-cytochrome P450 reductase

Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) [oxidized/semiquinone] = -286 +/- 6 mV, E(2) [semiquinone/reduced] = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) [FMN] = -66 +/- 8 mV, E(2) [FMN] = -269 +/- 10 mV; E(1) [FAD] = -283 +/- 5 mV, E(2) [FAD] = -382 +/- 8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples. Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase. However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes.     

8-Mercaptoflavins as active site probes of flavoenzymes.

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.     

Use of a site-directed triple mutant to trap intermediates: demonstration that the flavin C(4a)-thiol adduct and reduced flavin are kinetically competent intermediates in mercuric ion reductase

A mutant form of mercuric reductase, which has three of its four catalytically essential cysteine residues replaced by alanines (ACAA: Ala135Cys140Ala558Ala559), has been constructed and used for mechanistic investigations. With disruption of the Hg(II) binding site, the mutant enzyme is devoid of Hg(II) reductase activity. However, it appears to fold properly since it binds FAD normally and exhibits very tight binding of pyridine nucleotides as is seen with the wild-type enzyme. This mutant enzyme allows quantitative accumulation of two species thought to function as intermediates in the catalytic sequence of the flavoprotein disulfide reductase family of enzymes. NADPH reduces the flavin in this mutant, and a stabilized E-FADH- form accumulates. The second intermediate is a flavin C(4a)-Cys140 thiol adduct, which is quantitatively accumulated by reaction of oxidized ACAA enzyme with NADP+. The conversion of the Cys135-Cys140 disulfide in wild-type enzyme to the monothiol Cys140 in ACAA and the elevated pKa of Cys140 (6.7 vs 5.0 in wild type) have permitted detection of these intermediates at low pH (5.0). The rates of formation of E-FADH- and the breakdown of the flavin C(4a)-thiol adduct have been measured and indicate that both intermediates are kinetically competent for both the reductive half-reaction and turnover by wild-type enzyme. These results validate the general proposal that electrons flow from NADPH to FADH- to C(4a)-thiol adduct to the FAD/dithiol form that accumulates as the EH2 form in the reductive half-reaction for this class of enzymes.     

Structure-function correlations of the reaction of reduced nicotinamide analogues with p-hydroxybenzoate hydroxylase substituted with a series of 8-substituted flavins

Structural and kinetic studies have revealed two flavin conformations in p-hydroxybenzoate hydroxylase (PHBH), the in-position and the out-position. Conversion between these two conformations is believed to be essential during catalysis. Although substrate hydroxylation occurs while the flavin in PHBH is in the in-conformation, the position of the flavin during reduction by NADPH is uncertain. To investigate the catalytic importance of the out-conformation of the flavin and to clarify the mechanism of flavin reduction in PHBH, we report quantitative structure-reactivity relationships (QSAR) using PHBH substituted separately with nine derivatives of FAD modified in the 8-position and four dihydronicotinamide analogues as reducing agents. The 8-position of the FAD isoalloxazine ring was chosen for modification because in PHBH it has minimal interactions with the protein and is accessible to solvent. The chemical sequence of events during catalysis by PHBH was not altered when using any of the modified flavins, and normal products were obtained. Although the rate of reduction of PHBH reconstituted with flavin derivatives is expected to be dependent on the redox potential of the flavin, no strict correlation was observed. Instead, the rate of reduction correlated with the kappa-substituent constant, which is based on size and hydrophobicity of the 8-substituent on the FAD. Substituents that sterically hinder attainment of the out-conformation decreased the rate of flavin reduction much more than expected on the basis of the redox potential of the flavin. The results of this QSAR analysis are consistent with the hypothesis that the flavin in PHBH must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction.     

Purification and characterization of NADPH-dependent flavin reductase. An enzyme required for the activation of chorismate synthase in Bacillus subtilis.

NADPH-dependent flavin reductase (required for the activation of chorismate synthase) was purified to homogeneity from cell-free extracts of Bacillus subtilis. The enzyme has a molecular weight of 13,000 as determined by sodium dodecyl sulfate-gel electrophoresis, is specific for NADPH, and requires a divalent metal ion and either FMN or FAD for maximal rates of NADPH oxidation. The enzyme is able to reduce 2,6-dichlorophenolindophenol (DCIP) in the presence of NADPH and a divalent metal ion. Both catalytic activities were completely inhibited by EDTA. The Km for FMN is 1.25 X 10(-5) M and for NADPH 7.8 X 10(-5) M with oxygen as the final electron acceptor, and 3.85 X 10(-4) M with DCIP as the final electron acceptor. The enzyme was also isolated in association with chorismate synthase and dehydroquinate synthase. The enzyme associated with the complex has the same catalytic properties as the dissociated enzyme except that it requires both a divalent metal ion and FMN for DCIP reduction. Maximal enzyme activity was observed when the enzyme was preincubated with FMN and the divalent metal ion. The enzyme complex is easily dissociable and the dissociation of the enzyme complex resulted in the failure of NADPH-dependent flavin reductase to adsorb to phosphocellulose.