Flavin-protein Interactions Research Articles

Modulation of the flavin-protein interactions in NADH peroxidase and mercuric ion reductase: a resonance Raman study.

NADH peroxidase (Npx) and mercuric ion reductase (MerA) are flavoproteins belonging to the pyridine nucleotide:disulfide oxidoreductases (PNDO) and catalyzing the reduction of toxic substrates, i.e., hydrogen peroxide and mercuric ion, respectively. To determine the role of the flavin adenine dinucleotide (FAD) in the detoxification mechanism, the resonance Raman (RR) spectra of these enzymes under various redox and ligation states have been investigated using blue and/or near-UV excitation(s). These data were compared to those previously obtained for glutathione reductase (GR), another enzyme of the PNDO family, but catalyzing the reduction of oxidized glutathione. Spectral differences have been detected for the marker bands of the isoalloxazine ring of Npx, MerA, and GR. They provide evidence for different catalytic mechanisms in these flavoproteins. The RR modes of the oxidized and two-electron reduced (EH2) forms of Npx are related to very tight flavin-protein interactions maintaining a nearly planar conformation of the isoalloxazine tricycle, a low level of H-bonding at the N1/N5 and O2/O4 sites, and a strong H-bond at N3H. They also indicate minimal changes in FAD structure and environment upon either NAD(H) binding or reduction of the sulfinic redox center. All these spectroscopic data support an enzyme functioning centered on the Cys-SO-/Cys-S- redox moiety and a neighbouring His residue. On the contrary, the RR data on various functional forms of MerA are indicative of a modulation of both ring II distortion and H-bonding states of the N5 site and ring III. The Cd(II) binding to the EH2-NADP(H) complexes, biomimetic intermediates in the reaction of Hg(II) reduction, provokes important spectral changes. They are interpreted in terms of flattening of the isoalloxazine ring and large decreases in H-bonding at the N5 site and ring III. The large flexibility of the FAD structure and environment in MerA is in agreement with proposed mechanisms involving C4a(flavin) adducts.

Ionic strength effects on the photodegradation reactions of riboflavin in aqueous solution

A study of the effect of ionic strength on the photodegradation reactions (photoreduction and photoaddition) of riboflavin (RF) in phosphate buffer (pH7.0) has been carried out using a specific multicomponent spectrometric method. It has been found that the rates of photodegradation reactions of RF are dependent upon the ionic strength of the solutions at different buffer concentrations. The apparent first-order rate constants (kobs) for the photodegradation of riboflavin at ionic strengths of 0.1–0.5 (0.5M phosphate) lie in the range of 7.35–30.32×10−3min−1. Under these conditions, the rate constants for the formation of the major products, lumichrome (LC) by photoreduction pathway, and cyclodehydroriboflavin (CDRF) by photoaddition pathway, are in the range of 3.80–16.03 and 1.70–6.07×10−3min−1, respectively. A linear relationship has been observed between log kobs and √μ/1+√μ. A similar plot of log k/ko against √μ yields a straight line with a value of ~+1 for ZAZB showing the involvement of a charged species in the rate determining step. NaCl appears to promote the photodegradation reactions of RF probably by an excited state interaction. The implications of ionic strength on RF photodegradation by different pathways and flavin–protein interactions have been discussed.

Nonresonance Raman Study of the Flavin Cofactor and Its Interactions in the Methylotrophic Bacterium W3A1 Electron-Transfer Flavoprotein

Nonresonance Raman spectroscopy has been used to investigate the protein-flavin interactions of the oxidized and anionic semiquinone states of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) in solution. Several unique features of oxidized wETF were revealed from the Raman data. The unusually high frequency of the Raman band for the C(4)=O of the flavin suggests that hydrogen-bonding interactions with the C(4)O are very weak or nonexistent in wETF. In contrast, hydrogen bonding with the C(2)=O is one of the strongest among the flavoproteins investigated thus far. According to the crystal structure, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized flavin cofactor in wETF. The replacement of alphaSer254 by cysteine by site-directed mutagenesis resulted in shifts in N(5)-relevant Raman bands in both the oxidized and anionic semiquinone states of the protein. These results confirm the presence of the hydrogen-bonding interaction at N(5) that is evident in the crystal structure of the oxidized protein and that it persists in the one-electron reduced state. The data suggest that these bands can serve as useful Raman markers for the N(5) interactions in both oxidation states of flavoproteins. The wETF displays unusually low frequencies of flavin ring I (o-xylene ring) relevant bands, which suggests a ring I microenvironment different from most of the other flavoproteins. As indicated by Raman data, the alphaS254C mutation changed the environment of ring I, perhaps as the consequence of changes in the mobility of the FAD domain of wETF. These unusual flavin-protein interactions may be associated with the unique redox properties of wETF.

Vanillyl-alcohol oxidase, a tasteful biocatalyst

The covalent flavoenzyme vanillyl-alcohol oxidase (VAO) is a versatile biocatalyst. It converts a wide range of phenolic compounds by catalysing oxidation, deamination, demethylation, dehydrogenation and hydroxylation reactions. The production of natural vanillin, 4-hydroxybenzaldehyde, coniferyl alcohol and enantiomeric pure phenol derivatives is of interest for biotechnological applications. The hydroxylation of 4-alkylphenols is highly stereospecific for the (R)-isomer, whereas dehydrogenation of these substrates is specific for the cis- or trans-isomer. On the basis of crystallographic data, we suggest that the stereospecificity is related to the active site residue Asp170. Another important feature of VAO is the covalent flavin attachment. Studies from site-directed mutants suggest that the covalent flavin–protein interaction improves the catalytic performance as well as the long-term stability of VAO.

Flavoprotein resolution and reconstitution.

AbstractFlavins (FAD and FMN) are generally tightly bound to proteins; they are prosthetic groups and not cofactors, which implies that their dissociation constants are usually fairly small, between 10−7 and 10−10 M. It is therefore impossible in most cases to remove the flavin by simple dialysis. It is however useful to be able to resolve the flavin from a protein and be able to reconstitute the latter, either with the normal prosthetic group, so as to study the flavin binding parameters, or with flavin analogues, for structural and mechanistic purposes (1,2). In addition, in recent years, the use of isotopically labeled flavins has enabled the examination of flavin-protein interactions by (NMR) spectroscopy (3) and led to the need for large-scale procedures.KeywordsAmmonium SulfateEquilibration BufferProsthetic GroupSalmon PinkStandard BufferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of the flavin–protein interaction in L‐Lactate oxidase and old yellow enzyme by resonance inverse Raman spectroscopy

AbstractResonance inverse Raman spectroscopy was used to examine the interactions between the flavin and surounding protein in L‐lactate oxidase (from Mycobacterium smegmatis) and Old Yellow Enzyme (from brewer's bottom yeast). Spectra were taken of the enzymes both free and bound to various ligands. For L‐lactate oxidase, the ligands consisted of substrate analogs (acetate, propionate) and inorganic anions (phosphate, sulfate and nitrate). For the inorganic anions, the expected attenuation with detuning was not observed for several bands which are associated with flavin rings II and III. This effect appears to be due to a disruption of ring stacking between the uracil–pyrazine end of the flavin moiety and an aromatic amino acid. A shift in the 1232 cm−1 band and changes in the bandwidths of bands in the 1450–1600 cm−1 region indicate minor reorganization of the hydrogen bonding structure around the isoalloxazine on ligand binding.For Old Yellow Enzyme, the ligand was chloride. Chloride caused a slight change to the Raman spectrum, indicative of decreased hydrogen bonding to the flavin.

The effects of reversible freezing inactivation and inhibitor binding on redox properties of l-amino-acid oxidase

We measured the redox potentials of frozen inactivated l-amino-acid oxidase ( l-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) and inhibitor-bound (anthranilic acid) enzyme, and compared these redox properties to those of active l-amino-acid oxidase and benzoate-bound d-amino-acid oxidase (EC 1.4.3.3), respectively. The redox properties of the inactive enzyme are similar to the properties of free flavin; the potential is within 0.015 V of free flavin and no radical stabilization is seen. This corresponds to the loss of most interactions between apoprotein and flavin. In contrast, the anthranilic acid lowers the amount of radical stabilized from 85% to 35%. The potentials are still 0.150 V positive of free flavin, indicating that in the presence of inhibitor, many flavin-protein interactions remain intact. The difference between this behavior and that of d-amino-acid oxidase bound to benzoate, where the amount of radical declined from 95% to 5%, is explained on the basis of the relative tightness of binding of apoprotein to FAD. d-Amino-acid oxidase apoprotein has a relatively low K a (10 6) for FAD, and benzoate has a relatively high K a (10 5) for the enzyme. Therefore, the binding of benzoate increases the tightness of FAD binding to apo- d-amino-acid oxidase (10 11), indicating significant changes in flavin-protein interactions. In contrast, apo- l-amino-acid oxidase binds flavin tightly (the K a is greater than 10 7) and the enzyme binds to anthranilate much less tightly, with a K a of 10 3. The l-amino-acid oxidase apoprotein binding to FAD is tight initially, and the binding of anthranilate changes it only slightly. Therefore, redox studies indicate that the ability of a flavoprotein to be regulated may be influenced by the strength of the interaction of flavin with the apoprotein, as well as the strength of interaction of the substrate or activator.

Flavins of NADPH-cytochrome P-450 reductase: evidence for structural alteration of flavins in their one-electron-reduced semiquinone states from resonance Raman spectroscopy.

The mechanism of electron transfer from NADPH to cytochrome P-450 through FAD and FMN of the reductase is largely unknown. In this paper, we report the resonance Raman spectral properties of the oxidized and the semiquinonoid states of the flavins in the holoenzyme and the FMN-depleted forms, respectively, of detergent-solubilized rabbit liver microsomal NADPH-cytochrome P-450 reductase. The resonance Raman spectra of the oxidized forms [FAD; FMN] and [FAD;-] were essentially identical, indicating similar binding interactions of these flavins with the protein. To the contrary, the spectra of the semiquinonoid FADH. and FMNH. forms revealed significant spectral differences. Both O2-unstable species, characterized as [FADH.; FMNH2] and [FADH.;-] excited at 568.2 nm, have dominant spectral peaks at approximately 1611, 1539-1543, 1377, 1305, 1263, and 1226 cm-1. However, in the O2-stable [FAD; FMNH.] species, resonance Raman bands were located at 1611, 1532, 1388, 1304, 1268, and 1227 cm-1 when excited at the same wavelength. The approximately 10-cm-1 shifts of the 1532- and 1388-cm-1 bands suggest that the environments surrounding rings II and III of the isoalloxazines change upon reduction to semiquinonoid forms. It is proposed that N1 of FADH. (as a hydrogen-bond acceptor) and N5 of FMNH. (as donor) provide the distinguishing flavin-protein interactions in the semiquinonoid states. Furthermore, the resonance Raman spectra of the semiquinonoid species appear to be missing a number of bands assigned to ring I vibrations in the spectra of the oxidized flavins.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Fluoro-8-demethylriboflavin as a 19F-probe for flavin-protein interaction. A 19F NMR study with egg white riboflavin binding protein

Binding of 8-fluoro-8-demethylriboflavin to riboflavin binding protein was studied 19F-NMR spectroscopically. The pH dependence of 19F chemical shift of this fluoro derivative both free and bound to the protein revealed a shift of p K a associated with N(3)-H to alkali when bound to the protein. 19F chemical shift of the fluoro derivative bound to the protein is in a higher magnetic field than that of the free derivative in the neutral to acidic pH region. These results are interpreted in terms of the environmental changes at the 8- and 3-positions of isoalloxazine, indicating the applicability of this derivative for probing flavin-protein interaction at these positions.

Reconstitution of liver NADH: cytochrome b5 oxidoreductase and of Desulfovibvio vulgaris flavodoxin with 1-carba-1-deazaflavin.

Flavin-free cytochrome b5 reductase was reconstituted with 1-deazaflavin and 5-deazaflavin mononucleotides and dinucleotides. The 5-deazaenzyme functioned in transhydrogenation reactions but lacked electron transferase activity. The 1-deazaenzyme was fully competent for both input and output reactions. The flavin reduction rate was lowered about sevenfold upon N-1 substitution of FAD, but hydrogen abstraction from NADH remained the limiting step. Autoxidation of the reduced enzyme was more rapid than with the normal cofactor. Oxidation was accompanied by appearance of a transient blue-type semiquinone and superoxide ion production. Flavin-free apoflavodoxin was reconstituted with 1-deaza-1-carbaflavin mononucleotide (1-deaza-FMN). Its behaviour toward dithionite and oxygen was qualitatively highly similar to that of native flavodoxin. These observations contrast with the fact that apoflavocytochrome b2 could not be reconstituted with 1-deaza-FMN [Pompon. D. and Lederer, F. (1979) Eur. J. Biochem. 96. 571-579]. These results, as well as other data from the literature, are discussed in the light of existing hypotheses, which try to correlate flavin protein interactions and flavoprotein function.

Thermodynamics of association of 8-substituted riboflavins with egg white riboflavin binding protein.

Association constants of 14 species of riboflavin derivatives with hen egg-white riboflavin binding protein were measured by a fluorometric titration method using flavin and protein fluorescence in a temperature range of 10--42 degrees C. The derivatives were riboflavins whose 8-CH3 group was substituted with alkylamino, alkyloxy, halogen or hydrogen. The flavin derivatives were classified into four groups, i.e., RR'N, HRN, RO, and halogen, on the basis of the linear relation between enthalpy and entropy changes (linear free energy relationship), and from the chemical structures of the 8-substituents. The relation between the entropy change and the bulkiness of 8-substituents suggested burying of the substituents in a cavity of the protein, and provided an indication of the size of the substituent-accepting cavity. The relation also suggested an electric repulsion between halogen substituents and the binding site. A very good correlation was found between the constant term of the linear free energy relation and the wavelength of the visible absorption peak of flavins, and this correlation suggested the electronic nature of the flavin-protein interaction.

MO Study of flavin–protein interactions in flavodoxin catalysis

AbstractMINDO/3 calculations have been performed on the Clostridium MP flavodoxin active site (a complex of the redox active coenzyme flavin mononucleotide sandwiched between the side chains of methionine and tryptophan) at various redox levels using coordinates derived from x‐ray diffraction studies of the holoenzyme. Frontier orbital indices were calculated and indicate that reduction of the flavin is accompanied by induced polar states in the amino acid side chains. This stabilization of charge by the amino acid side chains could account for the reaction rate enhancement of flavin reduction catalyzed by flavodoxin. Frontier orbitals for free flavin, for the flavodoxin bound flavin without the amino acid side chains, and for the oxidized Desulfovibrio vulgaris flavodoxin active site were computed for comparison.

Flavin-protein interaction in bound glucose oxidase

Glucose oxidase was bound to Sepharose, Sephadex, gelatin, and dextran, yielding immobilized soluble and insoluble derivatives of the enzyme. The soluble preparations possessed higher enzymic activity than the analogous insoluble ones. The reversible dissociation process of the bound enzyme into apoenzyme and flavin adenine dinucleotide (FAD) was studied with the soluble and insoluble glucose oxidase in relation to enzymic activity and conformational changes as measured by circular dichroism and fluorescence methods. Bound apoenzyme was found to be more stable than the apoenzyme obtained from the unmodified glucose oxidase. The binding constant of FAD in bound glucose oxidase (Kdiss≈10-8M) calculated from fluorescent studies was lower than that of FAD in the native enzyme (Kdiss10-10M). The circular dichroism measurements indicated that dextran-bound glucose oxidase has a conformation similar to that of the native enzyme.

Flavine-protein interactions in flavoenzymes. Effects of aggregation of the apoprotein of Azotobacter flavodoxin on coenzyme binding.

Flavine-protein interactions in flavoenzymes. Effects of aggregation of the apoprotein of Azotobacter flavodoxin on coenzyme binding.

Effect of complexation of flavin radical with tryptophan on electron transfer rates: A model for flavin-protein interactions

The rates of oxidation of lumiflavin radical by ferricyanide, indole radical and oxygen are decreased by factors of four to ten as a result of complexation with tryptophan. Tyrosine, methionine and glycine were found not to measurably alter the flavin radical reactivity. Similar results were obtained using flavinyl peptides in which tryptophan or methionine were covalently linked to the flavin. These observations suggest that one of the consequences of the interaction between the flavin and a tryptophan side chain in the coenzyme binding site of the flavodoxins is to deactivate the semiquinone form of the enzyme towards oxidizing agents, thereby increasing its stability.

Flavin-protein interactions in flavoenzymes. Studies of FMN binding in Azotobacter flavodoxin using protein fluorescence lifetime measurements

Summary Frequency doubling of a nitrogen laser-pumped Rhodamine G dye laser has been used to provide an excitation pulse for the measurement of the tryptophan fluorescence decay of Azotobacter flavodoxin, apoflavodoxin and N-bromosuccinimide (NBS) oxidized derivatives. The fluorescence decay of the apoprotein can be resolved into two components. The major contribution (∼95%) has a lifetime of 4 nsec; the minor emission has a decay time of 1 nsec. The holoprotein fluorescence decay is non-exponential with lifetime components close to 1 nsec. These data, along with results obtained with a series of NBS oxidized derivatives, are interpreted in terms of the presence of a highly fluorescent tryptophan in the apoprotein which is strongly quenched upon FMN binding and which probably is part of the coenzyme binding site.

Flavine-protein interactions in flavoenzymes. pH dependence of the binding of flavine mononucleotide and riboflavine to Azotobacter flavodoxin.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFlavine-protein interactions in flavoenzymes. The pH dependence of the binding of flavine mononucleotide and riboflavine to Azotobacter flavodoxinMartha L. MacKnight, James M. Gillard, and Gordon TollinCite this: Biochemistry 1973, 12, 21, 4200–4206Publication Date (Print):October 1, 1973Publication History Published online1 May 2002Published inissue 1 October 1973https://doi.org/10.1021/bi00745a025RIGHTS & PERMISSIONSArticle Views52Altmetric-Citations19LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (808 KB) Get e-Alerts Get e-Alerts

Flavine-protein interactions in flavoenzymes. Thermodynamics and kinetics of reduction of Azotobacter flavodoxin.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFlavine-protein interactions in flavoenzymes. Thermodynamics and kinetics of reduction of Azotobacter flavodoxinBruce G. Barman and Gordon TollinCite this: Biochemistry 1972, 11, 25, 4755–4759Publication Date (Print):December 1, 1972Publication History Published online1 May 2002Published inissue 1 December 1972https://doi.org/10.1021/bi00775a019RIGHTS & PERMISSIONSArticle Views96Altmetric-Citations43LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Flavine-protein interactions in flavoenzymes. Temperature-jump and stopped-flow studies of flavine analog binding to the apoprotein of Azotobacter flavodoxin.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFlavine-protein interactions in flavoenzymes. Temperature-jump and stopped-flow studies of flavine analog binding to the apoprotein of Azotobacter flavodoxinBruce G. Barman and Gordon TollinCite this: Biochemistry 1972, 11, 25, 4746–4754Publication Date (Print):December 1, 1972Publication History Published online1 May 2002Published inissue 1 December 1972https://doi.org/10.1021/bi00775a018RIGHTS & PERMISSIONSArticle Views73Altmetric-Citations20LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-Alerts Get e-Alerts

Flavoprotein chemistry. III. Flavine protein interactions and the redox properties of the Shethna flavoprotein

Flavoprotein chemistry. III. Flavine protein interactions and the redox properties of the Shethna flavoprotein