The acceptor specificity of flavins and flavoproteins. II. Free flavins

1. For comparison with flavoprotein oxidases, a study has been made of free flavins in the reduced form with respect to the specificity and stoichiometry of their oxidation by a series of acceptors.

2. Reduced flavins uncombined with proteins show very little acceptor specificity and react very rapidly with nearly all the commonly used acceptors. Their behaviour resembles that of dithionite very closely indeed, and it differs considerably from that of flavoproteins. Like dithionite, free reduced flavins reduce O₂ quantitatively to H₂O₂; this oxidizes a further molecule of flavin.

3. H₂O₂ and cytochrome c react more slowly than most acceptors with reduced flavins. Nitrate and NDA⁺ do not act at all and require special activation.

4. Catalase can act as a catalyst for the aerobic oxidation of flavins by converting slowly-reacting H₂O₂ into rapidly-reacting O₂.

5. In the absence of catalytic metals ascorbate reacts with acceptors much more slowly than reduced flavins do.     

The acceptor specificity of flavins and flavoproteins. I. Techniques for anaerobic spectrophotometry

1. Easily constructed apparatus is described for spectrophotometry under strictly anaerobic conditions without requiring special cuvettes. It permits the addition of several reagents successively without opening the system to the air.

2. The absorption spectrum of dithionite shows a strong peak at 314 nm, the molar absorbance of which has been determined. This gives a convenient method for the titration of acceptors with dithionite.

3. One molecule of dithionite reacts very rapidly with one molecule of O₂ in solution. The O₂ is reduced quantitatively to H₂O₂. With excess of dithionite another, much slower, reaction follows, in which a second molecule of dithionite is oxidized by the peroxide.

4. A study has been made of the reduction by dithionite of a variety of acceptors commonly used in the study of flavoproteins. The majority react very rapidly, but a few are reduced relatively slowly or not at all.

5. The majority of acceptors do not react significantly with sulphite, the oxidation product of dithionite. One molecule of dithionite then provides two reduction equivalents. A few acceptors, however, react with the sulphite formed, giving a second reaction involving two more equivalents.     

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.     

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.     

Covalent bonding of riboflavin homologs by hepatic mitochondrial membrane enzymes

Radioactive riboflavin and homologs of riboflavin possessing an ethyl group in positions 7- or 8-, or both were used to investigate the covalent bonding of the flavins. No strict correlation could be found between the location of the ethyl group and the activities of the enzymes known to be covalently-bound flavoproteins. It is proposed that the widely varying activities of these enzymes when utilizing certain homologs is the result of a combination of the abnormal flavin coenzyme and the differences in the specificity of the apoenzymes in the various tissues.     

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.     

4-Thioflavins as active site probes of flavoproteins. General properties

4-Thioflavins (oxygen at position 4 replaced by sulfur) have been studied as potential active site probes of flavoproteins. They react readily with thiol reagents, with large spectral changes, which should be useful for testing the accessibility of the flavin 4-position in flavoproteins. They have an oxidation-reduction potential at pH 7 of -0.055 V, approximately 0.15 V higher than that of native flavins. The spectral characteristics in the fully reduced state show two clear absorption bands, dependent on the ionization state (pK = 4.5). The lowest energy band of the neutral dihydroflavin has a maximum at approximately 485 nm while that of the anion is approximately 425 nm. This should be useful in defining the ionization state of the reduced flavin in flavoproteins. The spectral characteristics of the semiquinoid forms of 4-thioflavins have been determined bound to the apoproteins of flavodoxin and D-amino acid oxidase. The neutral radical has an absorption maximum at 730 nm, while the anion radical has an unusually sharp peak at 415 nm. The reduced forms of 4-thioflavins, free and enzyme bound, react with O2 to regenerate oxidized 4-thioflavin. Reduced 4-thio-FAD p-hydroxybenzoate hydroxylase, however, in its reaction with O2, undergoes a substantial conversion to the native FAD-enzyme. 4-Thioflavins are unusually susceptible to attack by nucleophiles such as hydroxylamine and amines to form the respective 4-hydroxyimino- and 4-aminoflavins, offering the possibility of forming stable covalent flavin-protein linkages with suitably positioned protein residues. Thiols also react with 4-thioflavins, promoting their conversion to the normal (4-oxo) flavin coenzymes. Such reactivity has been found with the apoenzymes of glucose oxidase and lactate oxidase, providing evidence for a thiol residue in the active site of these enzymes.     

Proteolysis of L-(+)-lactate cytochrome c oxidoreductase (cytochrome b2) extracted from Saccharomyces cerevisiae and Hansenula anomala yeasts.

The L-(+)-Lactate:cytochrome c oxidoreductase or cytochrome b2 from the yeasts Saccharomyces cerevisiae and Hansenula anomala were partially hydrolysed in various concentrations of trypsin. Conditions were found which allowed the isolation from the Hansenula enzyme of a 140 000 +/- 10 000-dalton flavoprotein. The prosthetic flavin groups were still reducible by substrate (spectroscopic evidence) but the flavoprotein was unable to form a complex with cytochrome c, the physiological acceptor in the enzymatic reaction. No such flavoprotein units could be found during proteolysis of the Saccharomyces enzyme. The heme prosthetic group of the Hansenula enzyme remained bound to a 15 500 +/- 1000-dalton protein unit which was larger than, but very similar to, the well known 'cytochrome b2 core' of the Saccharomyces enzyme. Moreover, the degradation of different enzyme samples by contaminated proteases allowed the isolation of a particular form of Hansenula enzyme: each tetramer had, on the mean, four bound flavins and only two heme groups. These molecules completely retained their ability to form a complex with cytochrome c.     

The preparation and properties of the membranal DPNH dehydrogenase from Escherichia coli

1. The membrane bound DPNH oxidase of Escherichia coli can reduce the artificial electron acceptors: ferricyanide, dichlorophenolindophenol (DCIP) and menadione. All three are reduced by the flavoprotein of DPNH oxidase, but at different sites of the enzyme.

2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of K_m(K₃Fe(CN)₆) from 2.0 to 0.25 mM, while no change is detected in K_m(DPNH). This enzyme is not the DPNH diaphorase found in the bacteria.

3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD.

4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher K_m(DPNH) and slower v_max., while the K_m(K₃Fe(CN)₆) remains unchanged.

5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed.     

Interaction of phenols with old yellow enzyme. Physical evidence for charge-transfer complexes.

Evidence is presented that the changes in absorption spectrum obtained on complex formation between Old Yellow Enzyme and phenolic compounds are due to charge-transfer interactions. The positive correlation between the energy of the long wavelength transition and the Hammett para constant with a series of para-substituted phenols indicates that the phenol is the charge-transfer donor and the oxidized flavin of the enzyme is the charge-transfer acceptor. The same conclusion is drawn from studies in which the flavin of the native enzyme, flavin mononucleotide, was replaced by a variety of artificial flavins of different oxidation-reduction potential. The effect of pH on the dissociation constant for the enzyme-ligand binding also indicates that it is the phenolate anion, rather than the conjugate acid, which is responsible for the charge-transfer interaction. The significance of these results is discussed relative to long wavelength absorbing species detected with other flavoproteins.     

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.     

8-Mercaptoflavins as active site probes of flavoenzymes.

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

On the structure of flavin-oxygen intermediates involved in enzymatic reactions.

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421--505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C(4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the subsituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.     

NADPH-flavin reductase in human erythrocytes and the reduction of methemoglobin through flavin by the enzyme

A NADPH-dehydrogenase of human erythrocytes was exhaustively purified to a homogeneous protein judging from the electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulfate. Studies on the specificity for the electron acceptor of this enzyme suggest that flavins serve as the natural and direct electron acceptor. The enzyme showed a broad specificty for flavins and the Michaelis constants for flavins were estimated to be 5 × 10^?5 M for both FMN and riboflavin. Rapid reduction of methemoglobin by the enzyme in the presence of flavin was demonstrated, and the reduction was explained by the reduction of flavin by the enzyme, and subsequent non-enzymatic reduction of methemoglobin by the reduced flavin. The therapeutic significance of flavins was discussed with reference to the flavin reductase activity in hereditary methemoglobinemia.     

Understanding the FMN cofactor chemistry within the Anabaena Flavodoxin environment

The chemical versatility of flavin cofactors within the flavoprotein environment allows them to play main roles in the bioenergetics of all type of organisms, particularly in energy transformation processes such as photosynthesis or oxidative phosphorylation. Despite the large diversity of properties shown by flavoproteins and of the biological processes in which they are involved, only two flavin cofactors, FMN and FAD (both derived from the 7,8-dimethyl-10-(1′-D-ribityl)-isoalloxazine), are usually found in these proteins. Using theoretical and experimental approaches we have carried out an evaluation of the effects introduced upon substituting the 7- and/or 8-methyls of the isoalloxazine ring in the chemical and oxido-reduction properties of the different atoms of the ring on free flavins and on the photosynthetic Anabaena Flavodoxin (a flavoprotein that replaces Ferredoxin as electron carrier from Photosystem I to Ferredoxin-NADP⁺ reductase). In Anabaena Flavodoxin both the protein environment and the redox state contribute to modulate the chemical reactivity of the isoalloxazine ring. Anabaena apoflavodoxin is shown to be designed to stabilise/destabilise each one of the FMN redox states (but not of the analogues produced upon substitution of the 7- and/or 8-methyls groups) in the adequate proportions to provide Flavodoxin with the particular properties required for the functions in which it is involved in vivo. The 7- and/or 8-methyl groups of the ixoalloxazine can be discarded as the gate for electrons exchange in Anabaena Fld, but a key role in this process is envisaged for the C6 atom of the flavin and the backbone atoms of Asn58.     

PQQ and quinoprotein enzymes in microbial oxidations

Abstract Pyrroloquinoline quinone (PQQ) is found in a wide range of microorganisms, and several bacteria even excrete this compound into their culture medium when grown on alcohols. The existence of different classes of quinoprotein (PQQ-containing) enzymes is now well established (alcohol dehydrogenases, aldose (glucose) dehydrogenases, amine dehydrogenases and amine oxidases) while several other enzymes are suspected to be quinoproteins. In addition, many bacteria produce a quinoprotein apoenzyme, e.g., Escherichia coli and Pseudomonas testosteroni , producing glucose and ethanol dehydrogenase apoenzyme, respectively. It is unclear why these bacteria do not produce the holoenzyme form, but the apoenzymes have the ability to become functional, as was shown when the organisms were provided with PQQ. With this approach it could be demonstrated that E. coli has a non-phosphorylative route of glucose dissimilation via gluconate. Also, results with mixed cultures indicate that PQQ is a growth factor for certain bacteria under certain conditions. Despite the relatively high redox potential of the PQQ/PQQH₂ couple, quinoproteins transfer electrons to a variety of natural electron acceptors. Depending on the type of quinoprotein enzyme, the following components of the respiratory chain appear to be active: cytochrome c (sometimes with a copper protein as an intermediate), cytochrome b , and NADH dehydrogenase. PQQ is not restricted to a particular group of organisms, and reactions catalysed by quinoproteins can also be performed by NAD(P)-dependent or flavoprotein enzymes. Thus, these observations do not provide arguments for the view that quinoproteins have a unique role in microbial oxidations. Further comparative studies on oxidoreductases are necessary to reveal the special features of this novel group of enzymes.     

Spectral properties of fluorescent flavoproteins of isolated rat liver mitochondria

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD-linked flavin component was identified as alpha-lipoamide dehydrogenase, while the non-NAD-linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.     

Catalytic properties of streptococcal NADH oxidase containing artificial flavins.

The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase.     

Regulation of fructose-1,6-bisphosphatase activity in intact chloroplasts. Studies of the mechanism of inactivation

1. The aim of this work was to investigate the mechanism of dark inactivation of fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) in isolated intact chloroplasts of Triticum aestivum.

2. Dark inactivation of the enzyme, which was rapid under aerobic conditions, was prevented under anaerobic conditions when chloroplasts were incubated in the absence of an electron acceptor. Electron acceptors such as oxaloacetate readily brought about inactivation under anaerobic conditions whether chloroplasts were illuminated or in the dark. Inactivation of the enzyme also occurred if illuminated or darkened anaerobic chloroplasts were exposed to oxygen.

3. Pyocyanine, which catalyses a cyclic electron flow around Photosystem I, also caused inactivation of the enzyme in illuminated, anaerobic chloroplasts.

4. It is proposed that the activity of fructose-1,6-bisphosphatase is regulated by the availability of electrons, and thus by electron acceptors, and that dark inactivation may occur by a direct reversal of the activation process.     

Mechanisms of reaction of some flavoprotein enzymes with oxygen

Biochemical investigations of the properties of free flavins and of flavoproteins have shown that reduction usually occurs in two stages, with the intermediate formation of semiquinones in the case of free flavins. Flavoproteins often show spectroscopically similar intermediates, when partially reduced with substrate. These may, however, be enzyme-product complexes. Detailed investigation of individual flavoprotein enzymes has shown examples in which catalysis involves transition of the enzyme between oxidized and fully reduced forms (glucose oxidase), between oxidized and intermediate forms (D-amino acid oxidase), and intermediate and fully reduced forms (TPNH—cytochrome c reductase). Further, examples are known in which both intermediate and reduced forms react with oxygen, in which only one reacts, while in TPNH—cytochrome c reductase neither the intermediate nor the reduced form reacts with molecular oxygen. The physiological significance of these complex findings is uncertain, partly because it is not known whether purified flavoproteins occur in the same form in the tissues. It seems unlikely, however, that flavoproteins make a major contribution to the respiratory exchange of mammals.