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PubMed1978

Chaotropic resolution of high molecular weight (type I) NADH dehydrogenase, and reassociation of flavin-rich (type II) and flavin-poor subunits.

1. Type-I NADH dehydrogenase (Complex I) was solubilized and dissociated into subunits by NaClO4. NADH slows the dissociation. On subsequent stepwise addition of (NH4)2SO4 the dissociation is partly reversed, as is to be expected from the opposing effects of ClO-4 and SO-24, which are on the salting-in and salting-out sides, respectively, of the lyotropic series. 2. In consequence, the aggregates of subunits that are separated by (NH4)2-SO4 fractionation consist of randomly associated subunits as well as fragments of Type I enzyme. The fraction precipitating at 27% satd. (NH4)2SO4 is flavin-poor, that remaining soluble at 55% satd. (NH4)2SO4 flavin-rich and those separating between 27 and 55% satd. (NH4)2SO4 intermediate in composition. 3. The fraction remaining soluble at 55% satd. (NH4)2SO4 contains the purified low-molecular-weight iron-sulphur flavoprotein (Type-II dehydrogenase). It is a dimer consisting of one molecule of FMN, one 28-kilodalton and one 56-kilodalton subunit per protomer. Work of others indicates that it contains 4 Fe and 4 acid-labile S atoms per molecule of FMN. Sometimes the fraction remaining soluble at 55% satd. (NH4)2SO4 contained an additional small subunit (12 kilodaltons) and four additional Fe and acid labile S atoms per protomer. The sedimentation coefficients (s020,w) of the two preparations were 5.3 and 6.6 S, respectively, with calculated frictional ratios of 1.5 and 1.24, respectively. 4. The intermediate fractions are mixtures of the various subunits present in Complex I. Specifically a fraction separating at 55% satd. (NH4)2SO4 was found to be a mixture of two fragments, the pure iron-sulphur flavoprotein and a 26-S fragment that contained per protomer four subunits of 12 kilodaltons, one each of 28, 32, 56 and 77 kilodaltons, one molecule of FMN and 20 Fe and acid-labile S atoms. It was probably tetrameric or even larger. 5. The oxidoreductase activity of the intermediate fractions is dependent on the protein concentration, the activity with ferricyanide increasing and that with ferricytochrome c decreasing with increasing protein concentration. This is interpreted as an increased association of subunits present in the intermediate fractions. Similar results are obtained when flavin-rich and flavin-poor fractions are mixed. The association is cooperative. NADH favours the association of the subunits. 6. Association of the subunits is accompanied by a 10-fold increase in k2 (rate constant for intramolecular electron flow), a 10-fold decrease of the accessibility of ferricyanide to the reduced enzyme and a 10(4)-fold decrease of the accessibility of ferricytochrome c. The Ks (NADH) is also decreased. Although the changes are in the direction to be expected from a conversion of Type II enzyme to Type I, the value of k2 is still much less than in the latter enzyme.

Flavin Mononucleotide↗
PubMed1996

Flavin reductase P: structure of a dimeric enzyme that reduces flavin.

We report the structure of an NADPH:FMN oxidoreductase (flavin reductase P) that is involved in bioluminescence by providing reduced FMN to luciferase. The 1.8 A crystal structure of flavin reductase P from Vibrio harveyi was solved by multiple isomorphous replacement and reveals that the enzyme is a unique dimer of interlocking subunits, with 9352 A2 of surface area buried in the dimer interface. Each subunit comprises two domains. The first domain consists of a four-stranded antiparallel beta-sheet flanked by helices on either side. The second domain reaches out from one subunit and embraces the other subunit and is responsible for interlocking the two subunits. Our structure explains why flavin reductase P is specific for FMN as cofactor. FMN is recognized and tightly bound by a network of 16 hydrogen bonds, while steric considerations prevent the binding of FAD. A flexible loop containing a Lys and an Arg could account for the NADPH specificity. The structure reveals information about several aspects of the catalytic mechanism. For example, we show that the first step in catalysis, which is hydride transfer from C4 of NADPH to cofactor FMN, involves addition to the re face of the FMN, probably at the N5 position. The limited accessibility of the FMN binding pocket and the extensive FMN-protein hydrogen bond network are consistent with the observed ping-pong bisubstrate--biproduct reaction kinetics. Finally, we propose a model for how flavin reductase P might shuttle electrons between NADPH and luciferase.

Binding Sites↗
PubMed1990

Evidence for direct interaction between cysteine 138 and the flavin in thioredoxin reductase. A study using flavin analogs.

The flavoenzyme thioredoxin reductase from Escherichia coli contains an oxidation-reduction active disulfide made up of Cys135 and Cys138. Mutations changing each Cys residue to a Ser residue have been effected (Prongay, A. J., engelke, D. R., and Williams, C. H., Jr. (1989) J. Biol. Chem. 264, 2656-2664). The FAD prosthetic group of each altered thioredoxin reductase has been replaced with 1-deaza-FAD (a flavin analog with carbon substituted for nitrogen at position 1), 4-thio-FAD (a flavin analog with sulfur substituted for oxygen at position 4), and 6-thiocyanato-FAD. 1-Deaza-FAD-TRR(Cys135,Ser138) has absorbance and fluorescence spectral properties similar to the oxidized form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD. The absorbance spectrum of 1-deaza-FAD-TRR(Ser135,Cys138) is similar to the spectrum of the two-electron reduced form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD, indicating that it is a mixture of two species (O'Donnell, M. E., and Williams, C. H., Jr. (1984) J. Biol. Chem. 259, 2243-2251). The spectrum of one of these species of 1-deaza-FAD-TRR(Ser135,Cys138) resembles the spectrum of oxidized 1-deaza-FAD bound to wild type apothioredoxin reductase. The other species has an absorbance spectrum with a single peak at 400 nm (epsilon 400 = 11,100 M-1 cm-1) and resembles the spectrum of a thiolate adduct at the C4a position of the 1-deaza-FAD. The equilibrium between these species is pH-dependent, with a maximum of 50% C4a-adduct formation at low pH, and is linked to pK alpha values at 8.2 and 9.3. The absorbance spectrum of 4-thio-FAD-TRR(Cys135,Ser138) resembles the spectrum of the unbound 4-thio-FAD, whereas 4-thio-FAD-TRR(Ser135,Cys138) has a spectrum indicative of a mixture of 4-thio-FAD and FAD, suggesting a reaction between the 4-position of the flavin and Cys138. The binding of 6-thiocyanato-FAD to the apoprotein of the mutated enzymes showed no evidence for a reaction between the thiols and the group at the 6-position of the flavin.

Binding Sites↗
PubMed1998

The flavoprotein component of the Escherichia coli sulfite reductase: expression, purification, and spectral and catalytic properties of a monomeric form containing both the flavin adenine dinucleotide and the flavin mononucleotide cofactors.

The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain. SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E. coli and purified as a monomer. The N-terminal part of the native protein contains thus all the determinants required for the polymerization. SiR-FP60 retains both FAD and FMN with comparable contributions of the two flavins and the catalytic properties of SiR-FP. Thus, SiR-FP60 can be considered as a reliable simplified model of the sulfite reductase flavoprotein component. The formation and the stabilization of the neutral FMN semiquinone is thermodynamically favorable in SiR-FP60 upon reduction with photoreduced deazaflavin, dithionite, or NADPH. Generation of FMNH* is explained from a disproportionation of electrons between the reduced and oxidized FMN moieties during an intermolecular reaction, as shown with SiR-FP23, the FMN-binding domain of SiR-FP. The neutral FAD semiquinone can be observed only within SiR-FP43, the isolated FAD-binding domain. NADPH was used as a titrant or in excess to demonstrate that electron transfer is possible only because the FMN cofactor is coupled to FAD as an electron acceptor in the protein. The electron distribution within the various reduced forms of SiR-FP60 has been compared with that of the reduced forms of cytochrome P450 reductase, bacterial cytochrome P450, and nitric-oxide synthase. Despite the conservation of the bi-flavin-domain structure between these proteins over evolutionary time, each of them provides significantly different flavin reactivities.

Catalysis↗
PubMed2001

Flavin specificity and subunit interaction of Vibrio fischeri general NAD(P)H-flavin oxidoreductase FRG/FRase I.

Apoenzyme of the major NAD(P)H-utilizing flavin reductase FRG/FRase I from Vibrio fischeri was prepared. The apoenzyme bound one FMN cofactor per enzyme monomer to yield fully active holoenzyme. The FMN cofactor binding resulted in substantial quenching of both the flavin and the protein fluorescence intensities without any significant shifts in the emission peaks. In addition to FMN binding (K(d) 0.5 microM at 23 degrees C), the apoenzyme also bound 2-thioFMN, FAD and riboflavin as a cofactor with K(d) values of 1, 12, and 37 microM, respectively, at 23 degrees C. The 2-thioFMN containing holoenzyme was about 40% active in specific activity as compared to the FMN-containing holoenzyme. The FAD- and riboflavin-reconstituted holoenzymes were also catalytically active but their specific activities were not determined. FRG/FRase I followed a ping-pong kinetic mechanism. It is proposed that the enzyme-bound FMN cofactor shuttles between the oxidized and the reduced form during catalysis. For both the FMN- and 2-thioFMN-containing holoenzymes, 2-thioFMN was about 30% active as compared to FMN as a substrate. FAD and riboflavin were also active substrates. FRG/FRase I was shown by ultracentrifugation at 4 degrees C to undergo a monomer-dimer equilibrium, with K(d) values of 18.0 and 13.4 microM for the apo- and holoenzymes, respectively. All the spectral, ligand equilibrium binding, and kinetic properties described above are most likely associated with the monomeric species of FRG/FRase I. Many aspects of these properties are compared with a structurally and functionally related Vibrio harveyi NADPH-specific flavin reductase FRP.

Apoenzymes↗
PubMed1994

Detection of a reduced-flavin triplet state in free flavins and DNA photolyase.

A transient species was observed upon laser excitation (lambda exc = 355 nm) of reduced flavins (cFIH2) under anaerobic conditions. The transient decayed with a lifetime of about 1 microseconds, was quenched by iodide ions and was assigned as the reduced flavin triplet state 3cFIH2. The triplet-state absorption spectrum assumed three forms, depending on the pH of the solution, corresponding to 3cFIH2 and its monoprotonated and monodeprotonated forms. The triplet-state lifetime was not sensitive to the presence of pyrimidine cyclobutane dimers, apparently ruling out its involvement in the dimer splitting reaction observed with model systems of reduced flavins. An analogous species is apparently formed in the reduced form of Escherichia coli DNA photolyase.

Anaerobiosis↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1999

Role of glutamate-59 hydrogen bonded to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin. Glutamate-59 is not responsible for the pH dependency but contributes to the stabilization of the flavin semiquinone.

The midpoint potentials for both redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor in the flavodoxin are known to be pH dependent. While the pH dependency for the oxidized-semiquinone (ox/sq) couple is consistent with the formation of the blue neutral form of the flavin semiquinone, that of the semiquinone-hydroquinone (sq/hq) couple is more enigmatic. The apparent pK(a) of 6.7 for this couple in the flavodoxin from Clostridium beijerinckii has been attributed to the ionization of the FMN(HQ); however, nuclear magnetic resonance data strongly suggest the FMN(HQ) remains anionic over the entire pH range testable. As an alternative explanation, a specific glutamate residue (Glu59 in this flavodoxin), which is hydrogen-bonded to N(3)H of the FMN, has been postulated to be the primary redox-linked proton acceptor responsible for the pH effect in some flavodoxins. This model was directly tested in this study by permanently neutralizing Glu59 by its replacement with glutamine. This conservative substitution resulted in an increase of 86 mV (at pH 7) in midpoint potential of the sq/hq couple; however, the pH dependency of this couple was not altered. Thus, the redox-linked protonation of Glu59 clearly cannot be responsible for this effect as proposed. The pH dependency of the ox/sq couple was also similar to wild type, but the midpoint potential has decreased by 65 mV (pH 7). The K(d) values for the oxidized, semiquinone, and hydroquinone complexes increased by 43-, 590-, and 20-fold, respectively, relative to the wild type. Thus, the Glu59 to glutamine substitution substantially effects the stability of the semiquinone but, on a relative basis, slightly favors the formation of the hydroquinone. On the basis of (1)H-(15)N HSQC nuclear magnetic resonance spectroscopic studies, the increased temperature coefficients for the protons on N(3) and N(5) of the reduced FMN in E59Q suggest that the hydrogen-bonding interactions at these positions are significantly weakened in this mutant. The increase for N(5)H correlates with the reduced stability of the FMN(SQ) and the more negative midpoint potential for the ox/sq couple. On the basis of the X-ray structure, an "anchoring" role is proposed for the side chain carboxylate of Glu59 that stabilizes the structure of the 50's loop in such a way so as to promote the crucial hydrogen-bonding interaction that stabilizes the flavin semiquinone, contributing to the low potential of this flavodoxin.

Circular Dichroism↗
PubMed2000

Flavin-protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C- and 15N-labelled flavin.

A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C- and 15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the pi-electron distribution. The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins. With respect to N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane. In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N-chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins. 31P-NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme.

Binding Sites↗
PubMed1977

X-ray structures of two oxidation states of a flavin-nicotinamide biscoenzyme and models for flavin--nicotinamide interactions.

The flavin nicotinamide biscoenzymes Flox(-)--C3--Nic+ and H2Flred--C3--Nic+ assume extended conformations in the solid state. In both derivatives the nicotinamide and flavin groups associate through hydrogen bonding. The bending angle of the reduced flavin moiety is less than half that in any previously reported 1,5-dihydroflavin structure. This effect is apparently due to ring stacking interactions.

Chemical Phenomena↗
PubMed2006

A new flavin radical signal in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae. An EPR/electron nuclear double resonance investigation of the role of the covalently bound flavins in subunits B and C.

The Na(+)-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8alpha-methyl proton splittings, compared with the first anionic radical.

Base Sequence↗
PubMed1998

Synthesis, DNA-binding properties and cytotoxic activity of flavin-oligopyrrolecarboxamide and flavin-oligoimidazolecarboxamide conjugates.

The aim of this study was to develop novel series of photosensitizer-DNA minor groove binder hybrids composed of a flavin (isoalloxazine) chromophore linked to a moiety related to netropsin or distamycin. Three series (Fla-Pyr, Fla-Gly-Pyr and Fla-Gly-Im) were synthesized which differ by the number and the nature of the heterocyclic nuclei in the oligopeptide units, the nature of the linker and its anchoring position on the flavin. In terms of DNA binding and DNA specificity, satisfactory data are obtained in the Fla-Pyr and Fla-Gly-Pyr series; in terms of photo-induced cytotoxicity, the results are disappointing. The present study allows us to draw the following structure-activity relationships: (i) substitution of the flavin nucleus in either the N3 or the N10 position does not affect the activity; (ii) tris-pyrrolic hybrids are more efficient than bis- and tetra-pyrrolic analogs; (iii) the presence of a glycin in the linking chain does not suppress the DNA binding properties or the cytotoxic activities of the hybrids; and (iv) the replacement of the pyrrole nuclei by imidazoles has a drastic effect since it results in the loss of DNA affinity and cytotoxicity.

Aminoimidazole Carboxamide↗
PubMed2001

Determination of riboflavin, flavin mononucletide and flavin adenine dinucleotide in biological tissues by capillary zone electrophoresis and laser-induced fluorescence detection.

The separation of riboflavin, flavin mononucleotide and flavin adenine dinucleotide was investigated by capillary zone electrophoresis using laser-induced fluorescence detection. In the systematic approach developed, the differential electrophoretic mobilities were first maximized by adjusting the pH. Increasing the buffer concentration improved the separation at the expense of migration times. A buffer consisting of 50 mM phosphate adjusted to pH 8.5 was found to provide a very efficient and stable electrophoretic system. Responses were linear within the range 0.1-100 micromol L(-1), and the detection limits of B2 vitamers were 0.23 nmol L(-1) or less. The method was successfully applied to a variety of biological tissues from different animals.

Calibration↗
PubMed1998

Determination of riboflavin, flavin mononucleotide and flavin-adenine dinucleotide in wine and other beverages by high-performance liquid chromatography with fluorescence detection.

A new direct HPLC method with fluorescence detection has been developed for the routine analysis of riboflavin, flavin mononucleotide and flavin-adenine dinucleotide, in wines and other beverages. These compounds are the main agents responsible for the "taste of light" that some white wines and other beverages develop when they are exposed to the light, due to the formation of sulfur compounds that produce an anion/garlic odor. A Hewlett-Packard 1100 gradient liquid chromatograph with 1046A fluorescence detector was used. To improve the selectivity, each compound was monitored to fit the best lambda excitation/lambda emission (265/525 nm). A 500 nm cut-off filter was used. The column was a Hypersil C18 ODS, 200 x 2.1 mm, 5 microns particle size. The volume injected was 20 microliters. A constant flow-rate of 0.6 ml/min was used with two solvents: solvent A, 0.05 M buffer NaH2PO4 at pH = 3.0 with H3PO4 and solvent B, acetonitrile. The precision, linearity and sensitivity of this method have been established.

Beverages↗
PubMed2004

Redox properties of the isolated flavin mononucleotide- and flavin adenine dinucleotide-binding domains of neuronal nitric oxide synthase.

Electron transfer through neuronal nitric oxide synthase (nNOS) is regulated by the reversible binding of calmodulin (CaM) to the reductase domain of the enzyme, the conformation of which has been shown to be dependent on the presence of substrate, NADPH. Here we report the preparation of the isolated flavin mononucleotide (FMN)-binding domain of nNOS with bound CaM and the electrochemical analysis of this and the isolated flavin adenine dinucleotide (FAD)-binding domain in the presence and absence of NADP(+) and ADP (an inhibitor). The FMN-binding domain was found to be stable only in the presence of bound CaM/Ca(2+), removal of which resulted in precipitation of the protein. The FMN formed a kinetically stabilized blue semiquinone with an oxidized/semiquinone reduction potential of -179 mV. This is 80 mV more negative than the potential of the FMN in the isolated reductase domain, that is, in the presence of the FAD-binding domain. The FMN semiquinone/hydroquinone redox couple was found to be similar in both constructs. The isolated FAD-binding domain, generated by controlled proteolysis of the reductase domain, was found to have similar FAD reduction potentials to the isolated reductase domain. Both formed a FAD-hydroquinone/NADP(+) charge-transfer complex with a long-wavelength absorption band centered at 780 nm. Formation of this complex resulted in thermodynamic destabilization of the FAD semiquinone relative to the hydroquinone and a 30 mV increase in the FAD semiquinone/hydroquinone reduction potential. Binding of ADP, however, had little effect. The possible role of the nicotinamide/FADH(2) stacking interaction in controlling electron transfer and its likely dependence on protein conformation are discussed.

Animals↗
PubMed1980

Photodehalogenation of 7- and 8-halogen-substituted flavins. Photochemistry of the reduced flavin chromophore.

Flavodoxin was reconstituted with 8-chloro- and 7-bromo-FMN and p-hydroxybenzoate hydroxylase with the analogous FAD derivatives. In all cases, the spectral properties of the artificial enzymes changed as a result of photoreduction in the presence of ethylenediaminetetraacetate or oxalate as sources of reducing equivalents. The same changes were found to occur on irradiation of the enzymes which had been reduced previously in the dark under anaerobic conditions with dithionite. Using analogous 7- and 8-chlorolumiflavins, the observed changes were shown to be due to a novel photoreaction of the reduced flavin chromophore, in which either the 7- or 8-halogen substituent is eliminated and replaced by a proton derived from the solvent. The same reaction was shown to occur with 7,8-bis-norlumiflavin where 1 deuterium atom was incorporated into the molecule as a result of photoirradiation of the reduced flavin in deuterated medium. In the case of p-hydroxybenzoate hydroxylase, both the 8-chloro-FAD and 7-bromo-FAD enzymes, as well as their 8-nor-FAD and 7-nor-FAD photoproducts, possessed catalytic activity comparable to that of the native enzyme.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1987

Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time-resolved flavin fluorescence.

Subnanosecond-resolved fluorescence measurements of the FAD bound in glutathione reductase and lipoamide dehydrogenase revealed characteristic differences in dynamic properties of both enzymes, which are considered to have common structural features. The flavin fluorescence in glutathione reductase is quenched mainly via a dynamic mechanism, in agreement with enhanced flexibility of the flavin as inferred from rapid depolarization of the fluorescence.

Allosteric Site↗