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PubMed1995

Flavin-binding and protein structural integrity studies on NADPH-cytochrome P450 reductase are consistent with the presence of distinct domains.

NADPH-cytochrome P450 reductase (reductase) contains FMN and FAD in 1:1 stoichiometry as tightly bound cofactors. Electrons from NADPH are transferred to cytochrome P450 through the intermediacy of reductase. A knowledge of the interactions which must occur to allow the intermolecular and intramolecular transfer of electrons is not only of intrinsic interest but is necessary to understand the regulation of the overall oxidation-reduction processes in which cytochromes P450 participate in the endoplasmic reticulum of many organs. In the present study, urea has been employed as a chaotropic agent to study the dissociation of flavins from NADPH-cytochrome P450 reductase. The results show that dissociation of FMN occurs at concentrations of urea between 0 and 1 M and that, as the concentrations of urea approach 1 M, the intrinsic protein fluorescence increases, indicating a change in protein conformation. Above 2 M urea protein fluorescence increases, reaching a plateau at 3 M urea, and FAD begins to dissociate from the enzyme. In the range of 0-1 M urea, a completely reversible dissociation of FMN occurs and, at 3 M urea, the fluorescence values representing flavin dissociation and protein conformation changes have reached a maximum. Thus, the definition of various states of the flavoprotein with both, one, or no flavins bound and the ability to remove the flavins reversibly under specific conditions have permitted the construction of a simple model to explain the various unfolding intermediates of this enzyme. Our experiments suggest that reductase is composed of distinct domains which can be examined independently by the application of chaotropic agents.

Circular Dichroism↗
PubMed1992

Flavin levels in the rat retina.

Exposure of riboflavin and its coenzymes adenine dinucleotide (FAD) and riboflavin-5'-phosphate (FMN) to UV and visible light results in the generation of radicals and photodegradative products that can damage surrounding macromolecules. Vertebrates and invertebrates have lost the ability to synthesize riboflavin and must obtain it or its coenzymes from food. The present study evaluated the relationship between FAD, FMN, and riboflavin concentrations in retina and blood of male Sprague-Dawley rats. Rations were provided in the form of purified diets containing 0, 3, 6, 30, and 300 mg riboflavin kg-1 diet. Analysis of flavins by HPLC showed that saturation levels of FAD, FMN and riboflavin in the retina and blood were achieved with diets containing 3 mg riboflavin kg-1. Retinal flavins were not significantly elevated by further increases in dietary riboflavin concentration, but an unidentified flavin appeared in the blood of rats given rations containing concentrations above 3 mg kg-1. The concentration of this unknown flavin varied in proportion to the level of dietary riboflavin.

Animals↗
PubMed1985

Solvent accessibility to flavin in oxynitrilase.

Oxynitrilase containing 2-thioFAD [C(2) = S] in place of FAD exhibits catalytic activity similar to that of native enzyme. Reaction of methyl methanethiolsulfonate with 2-thioFAD bound to oxynitrilase results in the formation of the corresponding flavin disulfide [C(2)-SSCH3]. Normal flavin [C(2) = O] is formed by reacting 2-thioFAD oxynitrilase with m-chloroperoxybenzoate or H2O2. Both reactions proceed via a spectrally detectable flavin 2-S-oxide intermediate [C(2) = S+-O-], but sizable amounts of this intermediate accumulate only in the m-chloroperoxybenzoate reaction (about 40%). While similar reactions have been reported with free 2-thioflavin, kinetic and other data indicate that the oxynitrilase reactions occur with intact enzyme. This shows that the 2-position of the pyrimidine ring in the bound coenzyme is accessible to solvent. The data are consistent with previous studies on the reaction of peroxides with oxynitrilase-bound 5-deazaFAD which show that the pyrimidine ring is accessible at position 4. Analogous studies indicate that the pyrimidine ring is buried in the case of flavin bound to lactate oxidase, since the data indicate that both positions 2 and 4 are inaccessible to solvent.

Aldehyde-Lyases↗
PubMed1986

Modulation of rat polymorphonuclear leukocyte 5-lipoxygenase activity by 5-HPETE and NADH-dependent flavin inhibition.

The effect of nicotinamide and flavin coenzymes on the 5-lipoxygenase activity has been determined in cell-free extracts from rat polymorphonuclear leukocytes. 5-lipoxygenase was assayed in the presence of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which caused a 3 to 4-fold stimulation in the maximal conversion of radiolabeled arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) and 5,12-dihydroxyeicosatetraenoic acid (5,12-di-HETE). Addition of FMN or FAD to the assay mixture had little effect on the 5-lipoxygenase activity and caused inhibition only at high concentrations (IC50 greater than 100 microM). NADH markedly potentiated the inhibition of lipoxygenase by flavins with a 100-fold decrease in the FMN concentration required to inhibit the enzyme (IC50 approximately equal to 2 microM). Similar effects were observed for FAD although this flavin derivative was slightly less potent than FMN (IC50 congruent to 10 microM). NADH could be substituted by NADPH but not by NAD or NADP, indicating that the inhibition was not due to the production of the oxidized forms of these co-factors. These results show that the 5-lipoxygenase activity is stimulated by 5-HPETE and inhibited by flavin-dependent redox transformations.

Animals↗
PubMed1976

A study of solvent effects on the phosphorescence properties of flavins.

A combination of zero field triplet state techniques are used to study the excited electronic states of a series of flavin and flavin related molecules both in single crystals and glass matrices. Particular attention is given to the effects of solvent interaction on the triplet state properties of the flavin molecules. The total phosphorescence decay rate constants at 1.4 degrees K are reported for the flavin molecules in polar and nonpolar solvents. The rate constants are then correlated to the degree of solvent interaction. Results indicate possible complex formation between the isoalloxazine and adenine groups in FAD. Finally, the results and possible interpretation on the study of a flavoenzyme, L-amino acid oxidase are presented.

Amino Acid Oxidoreductases↗
PubMed1982

Reduction, oxidation, and addition reactions between free radicals and flavins.

Flavins and reduced flavins were reacted with a variety of free radicals produced in dilute aqueous solution at pH 7 +/- 0.1 by radiation chemical methods. The radical .CH2C(CH3)2OH and the aliphatic beta, gamma, and delta radicals of ethanol, 2-propanol, and 1-butanol added to the radical form of flavin adenine dinucleotide (FAD) (FH.) to yield products that could not be reoxidized to flavin by oxygen. The first radical also added to FAD but with a much lower efficiency. In contrast, the alpha-carbon radicals .CH(OH)CH2OH, CH3(-3) CHOH, and (CH3)2COH appeared to undergo two reactions: FH. + RR'COH leads to FH- + RR'C==O FH. + RR'COH leads to FH- + RR'C==O + H+ The formate radical anion .CO2(-) reacted similarly, producing stoichiometric two-electron reduction of riboflavin and lumiflavin as well as FAD. While eaq- also seemed capable of this, it was found to react irreversibly with FADH2, which makes it a poor reagent for producing the dihydroflavin. The dihydro form of FAD was reoxidized to FAD by the species RS. and .BR2(-). In contrast to FAD, the alloxazine lumichrome underwent only one-electron reduction and oxidation by .CO2(-) and .Br2(-), respectively.

Alcohols↗
PubMed1986

Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme.

Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.

Bacillus subtilis↗
PubMed1980

Magnetic circular dichroism studies on the active-site flavin of lipoamide dehydrogenase.

The magnetic circular dichroic (MCD) spectra of oxidized and reduced flavins are recorded in various solvents. They are shown to be sensitive to flavin environment. The MCD spectra of oxidized and reduced lipoamide dehydrogenase are reported. In the oxidized enzyme the sign of the B term associated with the 27 000-cm-1 band is reversed from free flavins. This is attributed to interaction of the disulfide with the short-axis dipole of FAD. The sign reversal is also present in a closely related disulfide enzyme, glutathione reductase, but absent in glucose oxidase. In the half-reduced enzyme, the appearance of an A term at 18 180 cm-1 is attributable to a charge-transfer complex with a thiolate anion as donor. Insensitivity of the term's energy and intensity to the redox state of flavin suggests that a protein residue may accept or stabilize the thiolate charge transfer.

Animals↗
PubMed1976

15N nuclear magnetic resonance of flavins.

Ninety-nine percent 15N-enriched flavins were synthesized and their proton decoupled 15N resonances were observed. The enriched compounds were [1,3-15N]riboflavin, [1,3,5-15N]riboflavin, [1,3-15N]riboflavin 5'-phosphate, [1,3,5-15N]riboflavin 5'-phosphate, and [1,3,5-15N] flavin adenine dinucleotide, [1,3,5-15N] lumiflavin, and [1,3,5-15N] lumichrome. By comparison of their spectra and from th- nuclear Overhauser effect data each 15N resonance peak could be assigned to each 15N nucleus. The order of the chemical shifts well corresponds to that of the calculated pi-electron densities. The N-3 nucleus gives the most intense inverted peak and the N-5 nucleus a small noninverted peak. By changing pH from neutral to alkaline, the chemical shift and the intensity of signal were mostly affected in the N-3 resonance of riboflavin 5'-phosphate. The N-5 signal of flavin adenine dinucleotide showed a fairly large downfield shift with the increase of temperature. These observations can be well interpreted by the chemical structure and the proposed conformation of riboflavin 5'-phosphate and flavin adenine dinucleotide.

Flavin-Adenine Dinucleotide↗
PubMed1997

Flavin mononucleotide-binding domain of the flavoprotein component of the sulfite reductase from Escherichia coli.

The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FMN-binding domain. It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.

Binding Sites↗
PubMed1997

Involvement of a flavin iminoquinone methide in the formation of 6-hydroxyflavin mononucleotide in trimethylamine dehydrogenase: a rationale for the existence of 8alpha-methyl and C6-linked covalent flavoproteins.

In trimethylamine dehydrogenase, substrate is bound in the active site via cation-pi bonding to three aromatic residues (Tyr-60, Trp-264, and Trp-355). Mutation of one of these residues (Trp-355 --> Leu, mutant W355L) influences the chemistry of the flavin mononucleotide in the active site, enabling derivatization to 6-hydroxy-FMN. The W355L mutant is purified as a mixture of deflavo, natural 6-S-cysteinyl-FMN, and inactive 6-hydroxy-FMN forms, and the enzyme is severely compromised in its ability to oxidatively demethylate trimethylamine. Analysis of samples of the native and recombinant wild-type trimethylamine dehydrogenases also revealed the presence of 6-hydroxy-FMN, but at much reduced levels compared with that of the W355L enzyme. Unlike that for a C30A mutant of trimethylamine dehydrogenase, addition of substrate to the W355L trimethylamine dehydrogenase is not required for the production of 6-hydroxy-FMN. A mechanism is proposed for the 6-hydroxylation of FMN in trimethylamine dehydrogenase that involves an electrophilic flavin iminoquinone methide. The proposed mechanism involving the flavin iminoquinone methide could apply to the flavinylation of trimethylamine dehydrogenase at the C6 position but also to the flavinylation of enzymes via the 8alpha position, thus providing a rationale for the evolution of covalent flavoproteins in general. Covalent linkage at C6 or the 8alpha-methyl prevents 6-hydroxylation by direct modification at the C6 atom or by preventing formation of the flavin iminoquinone methide, respectively.

Binding Sites↗
PubMed1998

Identification of the covalent flavin attachment site in sarcosine oxidase.

Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric enzyme (alphabetagammadelta) that contains two noncovalently bound coenzymes (FAD, NAD+) and covalently bound FMN [8alpha-(N3-histidyl)FMN] which is attached to the beta subunit. Chlumsky et al. [(1995) J. Biol. Chem. 270, 18252-18259] tentatively identified His175 as the covalent FMN attachment site in the beta subunit, based on an alignment of the sequence of C. sp. P-1 beta subunit with a highly homologous flavin-containing peptide from another corynebacterial sarcosine oxidase (C. sp. U-96). To test this hypothesis, His175 in the C. sp. P-1 beta subunit was mutated to an alanine. Unexpectedly, the mutant enzyme was found to contain 1 mol of covalently bound flavin and to exhibit catalytic activity similar to wild-type enzyme. Covalent flavin-containing peptides were isolated from wild-type and mutant enzymes and analyzed by electrospray mass spectrometry. The mass observed for the mutant peptide (1152.4 Da) matched that predicted for an FMN-containing hexapeptide, corresponding to residues 173-178 (1152.1 Da). In the mutant, this region (HDAVAW) contains a single histidine (His173) which must be the covalent flavin attachment site. The mass observed for the wild-type peptide (1218.6 Da) matched that predicted for an FMN-containing hexapeptide, also corresponding to residues 173-178 in the beta subunit (1218.2 Da). This region in the wild-type enzyme includes two histidine residues (HDHVAW). Attempts to sequence the wild-type or mutant peptides by automated Edman degradation were unsuccessful. Instead, the peptide sequences were investigated by collisional-activated dissociation (CAD) and tandem mass spectrometry. The CAD mass spectral data with the mutant peptide confirmed the sequence deduced based on the mass of the intact peptide. The CAD mass spectral results with the wild-type peptide showed that FMN was covalently attached to the N-terminal histidine in the hexapeptide, which corresponds to His173 in the beta subunit.

Amino Acid Sequence↗
PubMed1998

Role of methionine 56 in the control of the oxidation-reduction potentials of the Clostridium beijerinckii flavodoxin: effects of substitutions by aliphatic amino acids and evidence for a role of sulfur-flavin interactions.

Flavodoxins are small electron transferases that participate in low-potential electron transfer pathways. The flavodoxin protein is able to separate the two redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor through the differential thermodynamic stabilization or destabilization of each of its redox states. In the flavodoxin from Clostridium beijerinckii, the sulfur atom of methionine 56 is in direct contact with the re or inner face of the isoalloxazine ring of the FMN cofactor. In this study, evidence was sought for a possible role for sulfur-aromatic (flavin) interactions in the regulation of one-electron reduction potentials in flavoproteins. Met56 was systematically replaced with all the naturally occurring aliphatic amino acids by site-directed mutagenesis. Replacement of Met56 with alanine or glycine increased the midpoint potentials at pH 7 for the oxidized-semiquinone couple by up to 20 mV compared to that of the wild type, while replacement by the longer chain aliphatic residues decreased the midpoint potential by >30 mV. The midpoint potential for the semiquinone-hydroquinone couple was less negative than that for the wild type for all the mutants, increasing by as much as 90 mV for the M56I mutant. For the M56A mutant, the loss of approximately 0.5 kcal/mol in the binding energy for oxidized FMN and an increase of 1. 6 kcal/mol for the flavin hydroquinone, relative to that of the wild type, are responsible for the observed changes in the midpoint potentials. The stability of the semiquinone complex of this mutant was not affected. The one-election reduction potentials for the M56L, M56I, and M56V mutants are also influenced by the differential stabilization of the three redox states; however, the semiquinone complex was significantly less stable in these proteins. These differences are likely the consequence of the introduction of additional steric factors and an apparent structural preference for a smaller or more flexible side chain at this position in the semiquinone complex. While the other factors may contribute, it is argued that the results obtained for the entire group of mutants are consistent with the elimination of important sulfur-flavin interactions that contribute in part to the stabilization of the oxidized and destabilization of the hydroquinone states of the cofactor in this flavodoxin. The results of this study also demonstrate unequivocally the functional importance of this methionine residue and that it is unique among the aliphatic amino acids in its capacity to generate the physiologically relevant low reduction potential exhibited by the C. beijerinckii flavodoxin.

Amino Acid Substitution↗
PubMedDate not supplied

[The release of flavin adenine dinucleotide upon local conformational transition in electron-transferring flavoprotein induced by trimethylamine dehydrogenase].

The electron-transferring proteins, trimethylamine dehydrogenase (TMAD) and electron-transferring flavoprotein (ETF) from the bacterium Methylophilius methylotrophus, were studied in vitro by fluorescence spectroscopy. Flavin adenine dinucleotide (FAD) was found to be capable of a slow and spontaneous release from ETF, which is accompanied by an increase in flavin fluorescence. At a rather high ionic strength (0.1 M NaCl or 50 mM phosphate), the FAD release is sharply activated by TMAD preparations that induce a local conformational transition in ETF. The values of tryptophan fluorescence polarization and lifetime and the use of the Levshin-Perrin equation helped show that the size of protein particles remain unchanged upon the TMAD and ETF mixing; i.e., these proteins themselves do not form a stable complex with each other. The protein mixture did not release flavin from ETF in the presence of trimethylamine and formaldehyde. In this case, a stable complex between the proteins appeared to be formed under the action of formaldehyde. Upon a short-term incubation of ETF with ferricyanide, FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP. This fact explains the previous detection of AMP in ETF preparations by some researches. A fluorescence method was proposed for distinguishing FAD from FMN in solution using ethylene glycol. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Electron-Transferring Flavoproteins↗
PubMed2000

Alkyl-dihydroxyacetonephosphate synthase. Presence and role of flavin adenine dinucleotide.

Alkyl-dihydroxyacetonephosphate synthase is a peroxisomal enzyme involved in ether lipid synthesis. It catalyzes the exchange of the acyl chain in acyl-dihydroxyacetonephosphate for a long chain fatty alcohol, yielding the first ether linked intermediate, i.e. alkyl-dihydroxyacetonephosphate, in the pathway of ether lipid biosynthesis. Although this reaction is not a net redox reaction, the amino acid sequence of the enzyme suggested the presence of a flavin adenine dinucleotide (FAD)-binding domain. In this study we show that alkyl-dihydroxyacetonephosphate synthase contains an essential FAD molecule as cofactor, which is evidenced by fluorescence properties, UV-visible absorption spectra and the observation that the enzyme activity is dependent on the presence of this cofactor in a coupled in vitro transcription/translation assay. Furthermore, we could demonstrate that the FAD cofactor directly participates in catalysis. Upon incubation of the enzyme with the substrate palmitoyl-dihydroxyacetonephosphate, the flavin moiety is reduced, indicating that in this initial step the substrate is oxidized. Stopped flow experiments show that the reduction of the flavin moiety is a monophasic process yielding a oxygen stable, reduced enzyme species. Upon addition of hexadecanol to the reduced enzyme species, the flavin moiety is efficiently reoxidized. A hypothetical reaction mechanism is proposed that is consistent with the data in this paper and with previous studies.

Alkyl and Aryl Transferases↗
PubMed1976

Nicotinamide-dependent one-electron and two-electron (flavin) oxidoreduction: thermodynamics, kinetics, and mechanism.

1. Biological nicotinamide-dependent oxidoreduction consists of reversible 2e- oxidoreduction of substrates. A mechanism involving subsequent 1e- steps is shown to be very unfavourable due to the high energy of the nicotinamide radical. 2. Free energy relationships provide a convenient tool, allowing one to differentiate between hydride transfer and hydrogen atom transfer. It is concluded that biological nicotinamide-dependent, as well as flavin-nicotinamide oxidoreduction, proceed via hydride transfer but not via hydrogen atom transfer. 3. In flavin-nicotinamide oxidoreduction, flavin-nicotinamide charge transfer complexes are very likely the catalytic intermediates, preceding transfer of hydride ion. The energy of the long-wavelength charge transfer transition of zwitterionic oxidized-nicotinamide/reduced-flavin complexes is strongly dependent on polarity. It is maximal in a highly polar environment. 4. 5-Deazaflavins show the high thermodynamic radical instability of nicotinamides. They have to be considered as nicotinamide analog 2e- oxidoreductants rather than flavin analogs, therefore, lacking the ability to catalyze reversible 1e- oxidoreduction, essential for many flavoenzymes.

Binding Sites↗
PubMed1976

The dissociation of flavin coenzymes from trypsin-solubilized NADPH/Cytochrome c (P-450) reductase of pig-liver microsomes.

The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P-450) reductase. In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first-order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4-3.8 (0.02 M acetate buffer) where the rate constants increased 80-fold. Modification of the enzyme by treatment with p-chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first-order dissociations. It was concluded that p-chloromercuribenzoate treatment modified the protein so that the two flavin cofactors dissociated at different rates. Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, an analogue computer simulation of the dissociation as well as a manual curve-fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein.

Animals↗
PubMed1993

Flavin dynamics in reduced flavodoxins. A time-resolved polarized fluorescence study.

The time-resolved fluorescence and fluorescence anisotropy characteristics of reduced flavin mononucleotide in solution as well as bound in flavodoxins isolated from the bacteria Desulfovibrio gigas, Desulfovibrio vulgaris, Clostridium beijerinckii MP and Megasphaera elsdenii have been examined. All fluorescence and fluorescence anisotropy decays were analyzed by two different methods: (a) least-squares fitting with a sum of exponentials and (b) the maximum entropy method to yield distributed lifetimes and correlation times. The results of both approaches are in excellent agreement. The fluorescence decay of the free as well as protein-bound reduced flavin chromophore is made up of three components. The shortest component proves to be relatively sensitive to the environment and can therefore be used as a diagnostic tool to probe the microenvironment of the reduced isoalloxazine ring system. The other two longer fluorescence lifetime components are insensitive to the chromophore environment and seem therefore to be related to intrinsic, photophysical properties of the reduced chromophore. Fluorescence anisotropy decays show that the flavin mononucleotide in all four reduced flavodoxins is immobilized within the protein matrix, as indicated by the recovery of a single rotational correlation time, reflecting the rotation of the whole protein. No indications are found that rapid structural fluctuations occur in reduced flavodoxins, and the mechanism of electron transfer from flavodoxin to other redox proteins seems to involve immobilized reduced flavin.

Clostridium↗