Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria

The fluorescence signal of flavoproteins of rat liver mitochondria was investigated to determine the respective contributions of the various flavoenzymes. About 50% of the overall signal were found to be NAD-linked and caused by alpha-lipoamide dehydrogenase flavin (Em7.4 = -283 mV). Roughly 25% were due to a flavoprotein reducible in a non-NAD-linked reaction. This fluorescent flavoenzyme (Em7.4 = -52 mV) has been tentatively identified as a flavoprotein of the fatty-acid-oxidizing system, most probably the electron transfer flavoprotein. The remaining 25% of the signal are accounted for by flavoenzymes which are reducible by dithionite only. These flavoenzymes were not involved in the flavoprotein fluorescence alterations accompanying changes in electron flow through the respiratory chain. Contributions of other mitochondrial flavoproteins such as succinate dehydrogenase, NADH dehydrogenase, alpha-glycerophosphate dehydrogenase, proline dehydrogenase, and choline oxidase, to the overall flavin fluorescence signal of isolated rat liver mitochondria can be neglected.     

The Respiratory Chain of Plant Mitochondria: VI. Flavoprotein Components of the Respiratory Chain of Mung Bean Mitochondria

Redox changes of the flavoproteins of mung bean (Phaseolus aureus) mitochondria were measured by differential absorbance at 468 to 493 nanometers and by fluorescence emission above 500 nanometers excited at 436 nanometers. Four flavoproteins are distinguishable by the ratio of their fluorescence to absorbance changes, and by their requirement, or lack of it, for energy-linked reverse electron transport for reduction by succinate. Two flavoproteins are reduced by succinate in fully depleted mitochondria which lack the capacity for reverse electron transport. These are designated Fp_ha and Fp_hf and have fluorescence to absorbance ratios of 0 and 1.4, respectively. The two flavoproteins have the same half-time for oxidation, but Fp_hf is reduced more slowly than Fp_ha by substrate in the presence of cyanide. One flavoprotein with a fluorescence to absorbance ratio of 0 is not reduced by succinate in anaerobic, fully depleted mitochondria, but is rapidly reduced on subsequent addition of malate; it is designated Fp_m. The fourth distinguishable flavoprotein component is reducible by succinate in an energy-linked reaction, even in partially depleted mitochondria. This component has a fluorescence to absorbance ratio of 3.8 and is designated Fp_1f. In addition to these four flavoproteins reducible by substrates, there is a highly fluorescent flavin-containing component in or associated with these mitochondria, which is rapidly reduced by dithionite.     

Fluorescence properties of reduced flavins and flavoproteins

Fluorescence lifetimes and polarized emission properties of reduced flavin were measured using several model compounds and flavoproteins. Depending on the conditions of solvent and temperature or reduction method the lifetimes vary between 1 and 15 ns. The longer lifetime values are found in several forms of reduced lactate oxidase, in which a good correlation exists between fluorescence intensity and lifetime. In practically all flavoproteins the fluorescence is heterogeneous. Several mechanisms are proposed to explain the observed heterogeneity in lifetimes. The reduced models in glycerol at subzero temperature exhibit high degrees of polarization of the fluorescence, whereas distinct depolarization is encountered in several reduced flavoproteins suggesting a certain mobility of the flavin chromophor.     

A comparative analysis of the action of oxidative metabolic inhibitors on the luminescence parameters of normal and tumorous cells]

Comparative investigation of different mitochondrial oxidative metabolism inhibitors action on NAD(P)H and flavoproteins fluorescence intensity of minimal transformed 3T3 NIH mouse fibroblasts and rat HTC hepatoma cells was made. Principle differences were shown between these cells in oxidized flavoproteins fluorescence intensity changes under the action of used inhibitors. It is suggested that the unusual HTC hepatoma cells flavin fluorescence intensity increase is connected with the oxidation of unidentified flavin-containing component functionally attached to mitochondrial respiratory chain.     

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.     

Fluorescence spectroscopic detection of mitochondrial flavoprotein redox oscillations and transient reduction of the NADPH oxidase-associated flavoprotein in leukocytes

Steady-state and time-resolved fluorescence spectroscopy and fluorescence microscopy of leukocyte flavoproteins have been performed. Both living human peripheral blood monocytes and neutrophils have been utilized as experimental models, as the former relies much more heavily on mitochondrial metabolism for energy production than the latter. We confirm previous studies indicating that cellular flavoproteins absorb at 460 nm and emit at 530 nm, very similar to that of the FAD moiety. Furthermore, the emission properties of intracellular flavoproteins were altered by the metabolic inhibitors rotenone, antimycin A, azide, cyanide, DNP (2,4-dinitrophenol), and FCCP [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone]. Kinetic studies revealed flavoprotein emission oscillations in both monocytes and neutrophils. The flavoprotein intensity oscillations correlated with the physiological status of the cell and the nature of membrane receptor ligation. Microscopy revealed the presence of flavoprotein fluorescence in association with the plasma membrane, intracellular granules and distributed throughout the cytoplasm, presumably within mitochondria. Metabolic inhibitors such as cyanide suggest that the plasma membrane and granular components are cyanide-insensitive and therefore are likely associated with the flavoprotein component of the NADPH oxidase, which is located in these two compartments. This interpretation was found to be consistent with structural localization of the NADPH oxidase using an antibody molecule specific for this protein. Using peripheral blood neutrophils, which display less active mitochondria, and time-resolved emission spectroscopy, we show that the NADPH oxidase-associated flavoprotein undergoes a periodic transient reduction of about 54±2 ms in living cells. This finding is consistent with prior studies indicating that propagating substrate (NADPH) waves periodically promote electron transport across the NADPH oxidase.     

Biogenesis of flavoprotein and cytochrome components in hepatic mitochondria from riboflavin-deficient rats

Using difference spectrophotometry, measurements of succinate dehydrogenase activity, and SDS-polyacrylamide gels, the biochemical properties of hepatic mitochondria from riboflavin-deficient rats were monitored during recovery on riboflavin. [¹⁴C]Riboflavin was incorporated into four mitochondrial flavoproteins having covalently bound flavin coenzyme. Alterations in cytochromes, especially cytochrome oxidase, and the biosyntheses of succinate dehydrogenase, monoamine oxidase, sarcosine dehydrogenase, and an unknown flavoprotein were observed.     

Purification of electron-transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum

Electron-transferring flavoprotein (ETF), its redox partner flavoproteins, i.e., D-lactate dehydrogenase and butyryl-CoA dehydrogenase, and another well-known flavoprotein, flavodoxin, were purified from the same starting cell paste of an anaerobic bacterium, Megasphaera elsdenii. The purified ETF contained one mol FAD/mol ETF as the sole non-protein component and bound almost one mol of additional FAD. This preparation is a better subject for investigations of M. elsdenii ETF than the previously isolated ETF, which contains varying amounts of FAD and varying percentages of modified flavins such as 6-OH-FAD and 8-OH-FAD. The additionally bound FAD shows an anomalous absorption spectrum with strong absorption around 400 nm. This spectral change is not due to a chemical modification of the flavin ring because the flavin released by KBr or guanidine hydrochloride is normal FAD. It is also not due to unknown small molecules because the same spectrum appears when ETF is reconstituted from its guanidine-denatured subunits and FAD. A similar anomalous spectrum was observed for AMP-free pig ETF under acidic conditions, suggesting a common flavin environment between pig and M. elsdenii ETFs.     

Measurements of mitochondrial deficiencies in living cells by microspectrofluorometry.

Microspectrofluorometric methods were developed for detection of mitochondrial metabolites and marker molecules in living cells. After excitation in the near UV and blue spectral ranges, respiratory-deficient strains of Saccharomyces cerevisiae showed higher levels of intrinsic fluorescence than corresponding wild types. This may be attributed to an increased emission by NADH and flavin molecules of the mutants. After incubation with the mitochondrial marker rhodamine 123, there was a strong indication that an energy transfer from flavin to rhodamine molecules occurred, which was more pronounced for the respiratory-deficient yeast strains. Skin fibroblasts obtained from patients with mitochondrial diseases showed approximately the same levels of autofluorescence and energy transfer but higher variances than a control cell line. These higher variances may result from a coexistence of intact and defective mitochondria.     

Characterization of the peptide-bound flavin of a bacterial sarcosine dehydrogenase.

As in the case of the succinate and sarcosine dehydrogenases of liver mitochondria, the flavin prosthetic group of the bacterial sarcosine dehydrogenase can be released from the enzyme by proteolytic digestion with trypsin and chymotrypsin. The flavin, isolated in the dinucleotide form and covalently bound to a peptide fragment, is converted to the mononucleotide and purified by sequential chromatography on Sephadex G-25, DEAE-Sephadex A-25, followed by preparative paper chromatography and high voltage electrophoresis.The absorption maxima of the purified flavin at pH 7 are found at 268, 350, and 447 nm, with 268:447 nm and 350:447 nm ratios of 3.08 and 0.79, respectively. The fluorescence excitation and emission maxima, 450 and 530 nm, respectively, are similar to those of flavin mononucleotide. The fluorescence of the flavin-peptide is maximal at pH 3.0–3.1.Amino acid analysis of the flavin-peptide (riboflavin form) gave the following molar ratios of amino acids to flavin: Lys(1), Asp(2), Thr(1), Ser(1), Glu(1), Gly(1), and Ala(1). Aspartic acid was the N-terminal amino acid. Upon more extensive hydrolysis, histidine was obtained in 71–84% yields. Employing aminopeptidase M, the partial sequence of amino acids in the flavin-peptide was determined to be as follows: Flavin

-Asp-Lys-Ser-Glu-Gly-His-(Asp,Ala,Thr)-Evidence is presented that the isoalloxazine ring is linked covalently via its 8 α-methyl group to N-3 of histidine.     

Ultraweak photoemission from dark-adapted leaves and isolated chloroplasts

Dark-adapted isolated spinach chloroplasts and leaves, unlike sub-chloroplast fractions, are capable of emitting ultraweak light spontaneously (50-125 counts/s per cm2). The emission of leaves is due to two processes with activation energies of 97 and 25 kJ/mol while in isolated chloroplasts, it is the result of a single process (98 kJ/mol), as indicated by the Arrhenius plots of the intensity. Emission spectra demonstrate that the terminal step of these reactions is the excitation of chlorophyll in both samples. We suggest that the additional component in the ultraweak light emission of leaves may be related to mitochondria.     

The respiratory chain of plant mitochondria: x. Oxidation-reduction potentials of the flavoproteins of skunk cabbage mitochondria

The oxidation-reduction potentials of the flavoproteins of skunk cabbage (Symplocarpus foetidus) mitochondria have been measured under anaerobic conditions by means of a combined spectrophotometric or fluorimetric-potentiometric method. Five components were resolved whose oxidation-reduction reactions corresponded to two-electron changes, as expected for flavoproteins. The midpoint potentials at pH 7.2 are as follows, listed in order of increasingly negative potential: +170 millivolts, +110 millivolts, +20 millivolts, −70 millivolts, and −155 millivolts. The most negative component was highly fluorescent; the other components could only be identified by their characteristic absorbance changes. In addition to these components, which are mitochondrial, variable amounts of a very highly fluorescent flavoprotein with a midpoint potential of −215 millivolts was found. This component appears to be extra-mitochondrial. The same midpoint potential values at pH 7.2 were obtained with mitochondria in the uncoupled state as in mitochondria energized with ATP in the absence of phosphate.     

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.     

Light-induced electron transfer in a cryptochrome blue-light photoreceptor

Cryptochromes are flavoproteins implicated in multiple blue light-dependent signaling pathways regulating, for example, photomorphogenesis in plants or circadian clocks in animals. Using transient absorption spectroscopy, it is demonstrated that the primary light reactions in isolated Arabidopsis thaliana cryptochrome-1 involve intraprotein electron transfer from tryptophan and tyrosine residues to the excited flavin adenine dinucleotide cofactor.     

Deflavination and reconstitution of flavoproteins.

Flavoproteins are ubiquitous redox proteins that are involved in many biological processes. In the majority of flavoproteins, the flavin cofactor is tightly but noncovalently bound. Reversible dissociation of flavoproteins into apoprotein and flavin prosthetic group yields valuable insights in flavoprotein folding, function and mechanism. Replacement of the natural cofactor with artificial flavins has proved to be especially useful for the determination of the solvent accessibility, polarity, reaction stereochemistry and dynamic behaviour of flavoprotein active sites. In this review we summarize the advances made in the field of flavoprotein deflavination and reconstitution. Several sophisticated chromatographic procedures to either deflavinate or reconstitute the flavoprotein on a large scale are discussed. In a subset of flavoproteins, the flavin cofactor is covalently attached to the polypeptide chain. Studies from riboflavin-deficient expression systems and site-directed mutagenesis suggest that the flavinylation reaction is a post-translational, rather than a cotranslational, process. These genetic approaches have also provided insight into the mechanism of covalent flavinylation and the rationale for this atypical protein modification.     

Partial purification of a plasma membrane flavoprotein and NAD(P)H-oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H-dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two-phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg^?1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non-covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H-oxidoreductase activity and suggests the presence of plasma membrane bound pterins.     

Light-mediated reduction of flavoproteins with flavins as catalysts

It has been found that small amounts of free flavins greatly accelerate the photochemical reduction of flavoproteins both to the radical and fully reduced oxidation states. This catalytic effect has been shown to be due to the rapid photochemical reduction of the free flavin to its fully reduced state, followed by its reaction with the flavoprotein to yield flavoprotein radical and by its reaction with flavoprotein radical to yield fully reduced flavoprotein. Evidence is presented that the same route may occur with flavoproteins in the absence of added flavins. In this case the photoreduction is mediated by the small equilibrium concentration of free flavin coenzyme present in a flavorprotein solution. Hence, it is suggested that flavoprotein reduction with EDTA as photosubstrate does not involve an excited state of the holoprotein, nor contact of EDTA with the enzyme, but exchange of electrons between enzyme flavin and free reduced flavin.     

The NADPH-dependent O-.2-generating oxidase from human neutrophils

A subcellular particulate fraction from normal neutrophils that was enriched in NADPH-dependent O-.2-generating activity (Gabig, T. G., Schervish, E. W., and Santinga, J. T. (1982) J. Biol. Chem. 257, 4114-4119) has been further characterized. This preparation contained 0.25 +/- 0.02 nmol of flavin adenine dinucleotide/mg of protein and 0.28 +/- 0.01 nmol of cytochrome b/mg of protein. Measurable amounts of riboflavin or flavin mononucleotide were not present. The flavoprotein was completely resolved from the cytochrome b by selective bile salt extraction of the particulate oxidase fraction. The identical subcellular particulate fraction was studied in the neutrophils from two male patients with chronic granulomatous disease. The neutrophil oxidase fraction from one of the chronic granulomatous disease patients had a cytochrome b component that was spectrally abnormal, but a normal content of flavin adenine dinucleotide. The fraction from this patient's neutrophils corresponding to the resolved flavoprotein from normal cells had fluorescence excitation and emission spectra that were identical to the normal flavoprotein. The neutrophil oxidase fraction from the second chronic granulomatous disease patient had a quantitatively and spectrally normal cytochrome b but less than 8% of the normal amount of flavin adenine dinucleotide. The fraction from the latter patient's neutrophils corresponding to the resolved flavoprotein from normal cells had no detectable flavoprotein by fluorescence excitation and emission spectroscopy. It is postulated that these two patients represent distinct mutants in two separate components of the neutrophil NADPH-dependent O-.2-generating oxidase system, flavoprotein and cytochrome b.     

A rapid micromethod for determination of FMN and FAD in mixtures

A rapid fluorometric method for the determination of FMN and FAD concentrations in mixtures of the two compounds is described. The method is applicable to measurement of flavin concentrations in the nanomolar range. It can be readily used for determination of the flavin composition of purified flavoproteins.     

Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.