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PubMed1978

Production of a covalent flavin linkage in lipoamide dehydrogenase. Reaction with 8-Cl-FAD.

A method is described for preparation of apolipoamide dehydrogenase which gives quantitative removal of FAD. Active holoenzyme can be reconstituted by incubation with FAD. Reconstitution of apoenzyme with 8-Cl-FAD results in the fixation of most of the flavin to the protein in a covalently bound form. The portion noncovalently bound was shown to be unmodified 8-Cl-FAD. The covalently bound flavin has an absorption spectrum quite different from that of 8-Cl-FAD. It has a single band in the visible with a maximum at 459 nm (extinction coefficient of 22 mM-1 cm-1) and a shoulder at 480 nm. Model reactions between 8-Cl-Flavin (riboflavin or FAD) and organic thiols (thiophenol, beta-mercaptoethanol, or N-acetylcysteine) give products with spectra which are similar to that of FAD covalently bound to lipoamide dehydrogenase. The products of the model reactions have a single visible band with a maximum at 480 nm (extinction coefficient of 23.6 mM-1 cm-1 to 28.4 mM-1 cm-1) and a shoulder at 460 nm. The products of the model reaction and the covalently bound FAD of lipoamide dehydrogenase appear to be the result of a nucleophilic attack on the carbon at position 8 of the flavin ring by a thiolate anion, displacing the chloride. Thus, the product of the model reaction is 8-(RS)-flavin, and the product of the reaction between 8-Cl-FAD and protein probably has a cysteinyl residue covalently attacked at position 8 of FAD. Reconstitution of apoliopoamide dehydrogenase with 8-Cl-FAD gives two enzyme products which are fractionated by ammonium sulfate. Enzyme fractionating between 20% and 45% ammonium sulfate is monomeric and contains covanently bound FAD. Enzyme fractionating between 55% and 75% ammonium sulfate is dimeric and contains both covalently bound FAD and noncovalently bound 8-Cl-FAD. Both protein fractions contain one FAD per protein subunit and both are active with physiological substrates with Km values for NAD and dihydrolipoamide similar to those of native lipoamide dehydrogenase. The maximum turnover rates differ dramatically. Enzyme fractionating between 55% and 75% ammonium sulfate has a Vmax which is 61 times slower than native enzyme. Enzyme fractionating between 20% and 45% ammonium sulfate has a Vmax which is 7400 times slower than native enzyme. These slower rates are partially explainable by the oxidation-reduction potentials of the modified enzymes. Both covalently bound FAD and noncovalently bound FAD appear to reside in the native flavin binding site of the enzyme. However, once dimerization of the protien has taken place, the noncovalently bound 8-Cl-FAD cannot be induced to form a covalent bond with the protein except under protein denaturing conditions. The implications of these findings are discussed.

Apoenzymes↗
PubMed1983

Reaction of 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase with hydrogen peroxide. Formation of a covalent flavin-protein linkage.

Hydrogen peroxide reacts with 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase to yield a long wavelength intermediate (lambda max = 360, 620 nm) which can be isolated in stable form on removal of excess H2O2. The blue flavin derivative slowly decays in a second peroxide-dependent reaction to yield a new flavin product lacking long wavelength absorbance (lambda max = 408, 472 nm). This final peroxide-modified enzyme binds p-hydroxybenzoate with a 10-fold lower affinity than does the native enzyme; furthermore, substrate binding leads to the inhibition of enzyme reduction by NADPH. Trichloroacetic acid treatment of the final peroxide-modified enzyme results in the quantitative conversion of the bound flavin to free FAD. However, gel filtration of the modified enzyme in guanidine hydrochloride at neutral pH leads to the co-elution of protein and modified flavin. The nondenatured peroxide product reacts rapidly with hydroxylamine to yield 2-NHOH-substituted FAD. These observations indicate that the secondary reaction of peroxide with the blue intermediate from 2-thio-FAD p-hydroxybenzoate hydroxylase results in the formation of an acid-labile covalent flavin-protein linkage within the enzyme active site, involving the flavin C-2 position.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2000

Covalently bound flavin in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus.

Na(+)-translocating NADH-quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS-PAGE of the purified complex, NqrB and NqrC subunits were found to give yellow-green fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N-terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed-phase high performance liquid chromatography. N-Terminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr-235 in the NqrB and Thr-223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin-linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress.

Amino Acid Sequence↗
PubMed2001

Determination of the midpoint potential of the FAD and FMN flavin cofactors and of the 3Fe-4S cluster of glutamate synthase.

Glutamate synthase is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of the L-glutamine amide group to C(2) of 2-oxoglutarate, forming two molecules of L-glutamate. The bacterial enzyme is an alphabeta protomer, which contains one FAD (on the beta subunit, approximately 50 kDa), one FMN (on the alpha subunit, approximately 150 kDa), and three different Fe-S clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters at an unknown location). To address the problem of the intramolecular electron pathway, we have measured the midpoint potential values of the flavin cofactors and of the 3Fe-4S cluster of glutamate synthase in the isolated alpha and beta subunits and in the alphabeta holoenzyme. No detectable amounts of flavin semiquinones were observed during reductive titrations of the enzyme, indicating that the midpoint potential value of each flavin(ox)/flavin(sq) couple is, in all cases, significantly more negative than that of the corresponding flavin(sq)/flavin(hq) couple. Association of the two subunits to form the alphabeta protomer does not alter significantly the midpoint potential value of the FMN cofactor and of the 3Fe-4S cluster (approximately -240 and -270 mV, respectively), but it makes that of FAD some 40 mV less negative (approximately -340 mV for the beta subunit and -300 mV for FAD bound to the holoenzyme). Binding of the nonreducible NADP(+) analogue, 3-aminopyridine adenine dinucleotide phosphate, made the measured midpoint potential value of the FAD cofactor approximately 30-40 mV less negative in the isolated beta subunit, but had no effect on the redox properties of the alphabeta holoenzyme. This result correlates with the formation of a stable charge-transfer complex between the reduced flavin and the oxidized pyridine nucleotide in the isolated beta subunit, but not in the alphabeta holoenzyme. Binding of L-methionine sulfone, a glutamine analogue, had no significant effect on the redox properties of the enzyme cofactors. On the contrary, 2-oxoglutarate made the measured midpoint potential value of the 3Fe-4S cluster approximately 20 mV more negative in the isolated alpha subunit, but up to 100 mV less negative in the alphabeta holoenzyme as compared to the values of the corresponding free enzyme forms. These findings are consistent with electron transfer from the entry site (FAD) to the exit site (FMN) through the 3Fe-4S center of the enzyme and the involvement of at least one of the two low-potential 4Fe-4S centers, which are present in the glutamate synthase holoenzyme, but not in the isolated subunits. Furthermore, the data demonstrate a specific role of 2-oxoglutarate in promoting electron transfer from FAD to the 3Fe-4S cluster of the glutamate synthase holoenzyme. The modulatory role of 2-oxoglutarate is indeed consistent with the recently determined three-dimensional structure of the glutamate synthase alpha subunit, in which several polypeptide stretches are suitably positioned to mediate communication between substrate binding sites and the enzyme redox centers (FMN and the 3Fe-4S cluster) to tightly control and coordinate the individual reaction steps [Binda, C., et al. (2000) Structure 8, 1299-1308].

Azospirillum brasilense↗
PubMed2004

The recognition of glycolate oxidase apoprotein with flavin analogs in higher plants.

The dependence of glycolate oxidase apoprotein (apoGO) activity on flavin analogs was surveyed in 9 higher plants from 7 families. Activities of all apoGOs depended not only on flavin mononucleotide (FMN) but also on flavin adenine dinucleotide (FAD), but not on riboflavin. The kinetic analysis showed that FMN was the optimum cofactor for apoGO from leaves of Brassica campestris. In plant kingdom, FMN, FAD and riboflavin are three flavin analogs with very similar structure, and they could coexist and be inter-converted from each other, so the question is how the apoprotein of glycolate oxidase (GO) recognized these flavin analogs. No inhibition effect of riboflavin on the activity of apoGO with FMN or FAD was found and no obvious quenching of riboflavin or apoGO protein fluorescence was detected with the addition of apoGO or riboflavin, respectively. These results indicated that riboflavin did not bind to apoGO tightly like FMN and FAD. Inorganic phosphate (Pi) did inhibit the activity of GO, and kinetic analysis revealed that this inhibition was caused by the competitive binding to apoGO between Pi and FMN. This competitive binding was further confirmed by the inhibition of Pi to the quenching of FMN and apoGO protein fluorescence with apoGO and FMN, respectively. It was suggested that the 5'-phosphate group of FMN or FAD may play a key role in the recognition and binding of riboflavin analog cofactors with apoGO.

Alcohol Oxidoreductases↗
PubMed1999

[Chemical and functional properties of flavin coenzymes].

The yellow-colored compounds with the basic structural frame work of 7,8-dimethyl-10-alkylisoalloxazine are generally termed as flavins. The 10-ribityl derivative, riboflavin, is the most abundant flavin found in nature and is known as vitamin B2. Riboflavin is a precursor of the flavocoenzymes, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide) which function as prosthetic groups of flavocoenzymes. While flavocoenzymes are usually bound noncovalently to apoproteins of flavoenzymes, covalently-bound flavocoenzymes also occur in nature, though much less often. Flavin molecules can exist in three different redox states, i.e., oxidized, one-electron reduced and two-electron reduced states, and therefore can participate in redox reactions as either one- or two-electron mediator, making the flavoenzymes extremely versatile in terms of substrate and type of reactions catalyzed. We classified flavoenzymes according to the electron-transfer process in their reductive and oxidative half-reactions and the mechanism of each class of flavoenzymes is discussed in detail.

Coenzymes↗
PubMed1984

Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions.

A highly sensitive spectrophotometric assay was developed for measuring flavin-containing monooxygenase activity using methimazole (N-methyl-2-mercaptoimidazole) as the substrate. With the procedure described, flavin-containing monooxygenase activity can be accurately measured in whole cell homogenates without interference due to NADPH oxidase activities. The effects of detergents and octylamine on female mouse liver flavin-containing monooxygenase activity were characterized for whole homogenates and microsomes prepared under conditions which tend to cause or minimize microsomal aggregation. A small activation was observed with 0.2% (v/v) Emulgen 913 with nonaggregated microsomes; higher levels of detergents gave maximal activity with aggregated microsomes. Variations in the activity of the female mouse liver enzyme with nutritional state and time of day were evaluated. Higher specific activities were observed in homogenates and microsomes of livers from fed animals than from livers of 24-h starved animals, and higher specific activities were present in samples from livers of animals sacrificed in late afternoon than in the early morning. In the period where activity increased in fed animals (i.e., the AM to PM transition), a portion of flavin-containing monooxygenase was more resistant to thermal inactivation. Other properties are described which suggest structural differences for at least a portion of the flavin-containing monooxygenase. The possibility that these differences may be related to turnover of the flavin-containing monooxygenase is discussed.

Animals↗
PubMed1983

Proton nuclear magnetic resonance investigation of the mechanism of flavin C-4a adduct formation induced by oxidized nicotinamide adenine dinucleotide binding to monoalkylated pig heart lipoamide dehydrogenase.

The active center thiol of monoalkylated pig heart lipoamide dehydrogenase, EHR, is induced to form an adduct to the enzyme-bound flavin adenine dinucleotide (FAD) at the C-4a position upon binding oxidized nicotinamide adenine dinucleotide (NAD+) [Thorpe, C., & Williams, C. H., Jr. (1976) J. Biol. Chem. 251, 7726-7728]. In light of hypotheses on covalent electron transfer between pyridine nucleotide and flavin, the induction of the thiol-flavin C-4a adduct by NAD+ is reasonably envisioned as involving a covalent bond between the modified flavin and the NAD+. The double-resonance proton nuclear magnetic resonance technique of cross saturation was used to probe the existence of covalent bond formation between the modified flavin of EHR and its inducer molecule, NAD+. Cross-saturation of the free NAD+ signals was not observed even though the spin-lattice relaxation time of NAD+ and the rate of exchange between free NAD+ and NAD+ bound to EHR were well within the limits required for cross-saturation. We conclude that a noncovalent interaction between NAD+ and FAD induces the formation of the thiol-flavin C-4a covalent adduct in EHR. A model by which NAD+ binding induces nucleophilic attack by the nascent thiolate of EHR is discussed.

Animals↗
PubMed1987

Reduction of substituted flavins by .CO2- and cyclic disulphide anions.

5-Deazariboflavin was observed to undergo one electron-equivalent reduction on reaction with .CO2-, DS2-. or a mixture of .CO2- and eaq-. This is attributed to formation of .FlH, followed by dimerization: 2 5-DRFlH.----(5-DRFlH)2 a process not previously confirmed. In contrast, 3-deazariboflavin, 1-deazariboflavin 7-chlororiboflavin, 2-thioriboflavin, lumiflavin, flavin adenine dinucleotide and riboflavin all underwent two electron-equivalent reductions to FlH2 or FlH-species via the disproportionation: 2.FlH----FlH2+Fl Spectra of the reduced species are reported for 3-deazariboflavin, 1-deazarboflavin and 2-thioriboflavin. The anion radicals of dithiothreitol (DS2-.) and lipoamide (LS2-.) did not effect reduction beyond the two electron equivalence point. However, .CO2- was seen to effect further reactions with the dihydroforms of the higher redox potential flavins 7-chlororiboflavin and 2-thioriboflavin at pH 3 to 10, but for the other flavins only at or below pH 2. Except for the sulphur-containing 2-thioriboflavin, the protonated DS2.H and LS2.H radicals were unable to reduce any of the flavins at pH less than or equal to 3. This cause a 'falloff' in the yield of the reduced flavin which is in accord with the reaction rate constants and radical pK values. Despite their differences in absorbance, the reactions of DS2.H and LS2.H towards flavins were similar in all aspects examined. Several new rate constants are reported.

Anions↗
PubMed1994

Reactions of the Escherichia coli flavohaemoglobin (Hmp) with oxygen and reduced nicotinamide adenine dinucleotide: evidence for oxygen switching of flavin oxidoreduction and a mechanism for oxygen sensing.

The soluble flavohaemoglobin (Hmp) of Escherichia coli contains haem B and FAD in a single 44 kDa polypeptide, and shows NADH oxidase activity. The oxidized protein reacted rapidly with NADH in the presence of O2 to form an oxygenated species while the flavin remained largely oxidized. Spectral and kinetic analyses revealed rapid biphasic reduction and oxygenation of high-spin haem with apparent relaxation times of 6 and 64 ms at pH 8 and 25 degrees c, suggestive of a significant physiological role for the protein. This was followed by a monophasic reduction of the flavin with a relaxation time of 92 ms. On exhaustion of oxygen, the oxygenated haem was converted into the deoxy form biphasically with relaxation times of 43 and 170 s, followed by extensive reduction of the flavin with corresponding relaxation times of 70 and 256 s. Based on these observations, we propose that Hmp could act as an oxygen sensor in E. coli by combining with intracellular oxygen, thus limiting flavin reduction in the aerobic steady state. Lowering of the oxygen concentration causes dissociation of the oxy species and sustained flavin reduction. Because Hmp can reduce Fe(III), such a mechanism might control, for example, flavin-mediated Fe(III) reduction required for activation of the anaerobic gene regulator, Fnr.

Bacterial Proteins↗
PubMed1981

Effect of xenon on the excited States of phototropic receptor flavin in corn seedlings.

The chemically inert, water-soluble heavy atom gas, xenon, at millimolar concentrations specifically quenches the triplet excited state of flavin in solution without quenching the flavin singlet excited state. The preferential quenching of the flavin triplet over the singlet excited state by Xe has been established by showing that the flavin triplet-sensitized photooxidation of NADH is inhibited while the fluorescence intensity and lifetime of flavin are not affected by Xe.The phototropic response of corn seedlings in an atmosphere of 90% Xe and 10% O(2) was compared with that in the 90% N(2) plus 10% O(2) mixture. No significant inhibition of phototropism and geotropism by Xe was observed, suggesting that a flavin singlet state is more likely involved than the triplet state in the primary photoprocess of phototropism in corn.

Journal Article↗
PubMed1978

Photoejection of electrons from flavins in polar media.

Riboflavin and 12 of its derivatives have been shown to form solvated electrons under ultraviolet irradiation (253.7 nanometers) in various water-methanol solvent mixtures. The highest quantum yield of solvated electrons (about 0.03) was obtained for flavins containing tyrosine on a side chain in the isoalloxazine N-3 or N-10 position. The splitting of hydrogen atoms from excited flavin molecules was also observed. From the results presented here, it can be determined that the semiquinone transients are formed not only by way of the flavin triplet, as usually suggested, but also by the attack of the electrons and hydrogen atoms on flavin molecules in the ground state. This is important, because the flavin radicals remaining after the electron-ejection or hydrogen-splitting processes must also be considered in the subsequent reaction mechanisms. The electron-ejection process from electronically excited flavins has important implications in the photobiology of these compounds.

Electrons↗
PubMed2005

Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization.

Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.

Amino Acid Sequence↗
PubMed1990

Stereoselective S-oxygenation of 2-aryl-1,3-dithiolanes by the flavin-containing and cytochrome P-450 monooxygenases.

The reaction of NalO4, highly purified flavin-containing monooxygenase (EC 1.14.13.8), and microsomes from hog liver with 2-aryl-1,3-dithiolanes and 2-aryl-1,3-dithiolane S-oxides was investigated. The initial rates determined for the microsome- and purified flavin-containing monooxygenase-catalyzed rate of S-oxidation of para-substituted 2-aryl-1,3-dithiolanes were similar, demonstrating that S-oxidation of these substrates occurred with similar velocities at saturating concentrations of substrate and, at least for the first S-oxidation, the reaction was insensitive to the nature of the para-substituent. The diastereoselectivity of S-oxygenation of 2-aryl-1,3-dithiolanes was determined and, in general, a marked preference for addition of oxygen to the sulfide sulfur atom was observed to occur trans to the aryl groups. In all cases examined, enantioselective enzymatic S-oxidation was observed. For S-oxide formation in microsomes, the data provided evidence for a minor role of cytochrome P-450 in S-oxide formation, but the flavin-containing monooxygenase was mainly responsible for production of S-oxide. In contrast to previous reports, the enantioselectivity of S-oxidation catalyzed by highly purified cytochrome P-450IIB-1 and cytochrome P-450IIB-10 was not always opposite to that catalyzed by hog liver flavin-containing monooxygenase activity. 2-Aryl-1,3-dithiolane S-oxides were also oxidized a second time by NalO4, microsomes, or highly purified flavin-containing monooxygenase from hog liver but not cytochrome P-450IIB-1 or P-450IIB-10. The rate of the second oxidation was 10-15-fold slower than the corresponding first S-oxidation and S,S'-dioxide formation was markedly dependent on the electronic nature of the para-substituent (Hammett correlation rho value of -1.3 and -1.1 for microsomes and highly purified flavin-containing monooxygenase from hog liver, respectively). The large dependence of the rate of S,S'-dioxide formation on the nature of the para-substituent demonstrates that velocity values at saturating concentrations of S-oxide were not the same for all 2-aryl-1,3-dithiolane S-oxides and suggests that the chemical nature of the 2-aryl-1,3-dithiolane S-oxide contributes to the rate-determining step of this enzymatic reaction.

Animals↗
PubMed1987

Tissue- and species-dependent expression of multiple forms of mammalian microsomal flavin-containing monooxygenase.

The immunochemical relatedness, multiplicity, and expression of microsomal flavin-containing monooxygenases were assessed in hepatic, pulmonary, and renal microsomal preparations from adult, male rabbits, mice, rats, guinea pigs, and hamsters. Preparations from adult female (pregnant and nonpregnant) and immature male and female rabbits were also examined. Microsomes were analyzed by immunoblotting with polyclonal antibodies to flavin-containing monooxygenases purified from pig, mouse, and rabbit liver, and rabbit lung. Pulmonary flavin-containing monooxygenases, which differ from the major forms of the enzyme present in liver, were detected in pulmonary samples from all species. The major hepatic enzymes, or very closely related proteins, were also detected in pulmonary samples from every species except the rabbit. Three forms of pulmonary flavin-containing monooxygenase are found in rabbit and guinea pig. Differences in the expression of these three forms were observed with rabbits; all three were detected with some individuals and only two with others. The hepatic form of the flavin-containing monooxygenase is present in kidney of all species examined, and the pulmonary form is detected in kidney of rabbits, mice, and hamsters, but not rats or guinea pigs. Kidneys and lungs of individual rabbits were of the same phenotype with respect to the expression of the pulmonary forms of the enzyme. The findings show that the expression of flavin monooxygenase isozymes is tissue and species dependent.

Animals↗
PubMed1993

Riboflavin 3'- and 5'-sulfate, two novel flavins accumulating in the roots of iron-deficient sugar beet (Beta vulgaris).

Roots from iron-deficient sugar beet grown in the presence of calcium carbonate exhibit a yellow color and autofluorescence typical of flavin-like compounds, whereas roots of control, iron-sufficient plants exhibited no yellow color and extremely low autofluorescence. The two major flavins whose accumulation is induced by iron deficiency have been shown to be different from riboflavin, FMN, and FAD by reversed-phase high performance liquid chromatography. These flavins, accounting for 82 and 15% of the total flavin concentration in deficient roots, have been shown unequivocally to be riboflavin 3'-sulfate and riboflavin 5'-sulfate, respectively, by electrospray-mass spectrometry, inductively coupled plasma emission spectroscopy, infrared spectrometry, and 1H nuclear magnetic resonance. These flavin sulfates have not been found previously in biological systems. The localization of riboflavin sulfates in deficient roots is similar, but not identical, to that of high iron reductase activity. The concentration of riboflavin sulfates has been estimated from root extracts to be at least 1 mM. We hypothesize, based on the similar localization of flavin and that of iron reduction, that the accumulation of riboflavin sulfates induced by iron deficiency may be an integral part of the turbo iron-reducing system in sugar beet roots.

Fluorescence↗
PubMed1986

Kinetic studies of reduction of a 1:1 cytochrome c-flavodoxin complex by free flavin semiquinones and rubredoxin.

The kinetics of reduction by free flavin semiquinones and reduced rubredoxin of the individual components of the 1:1 complex formed between horse heart cytochrome c and Clostridium pasteurianum flavodoxin have been studied. Complex formation did not affect the rate constant for reduction of flavodoxin by 5-deazariboflavin semiquinone, indicating that the accessibility of the flavin mononucleotide (FMN) of complexed flavodoxin is the same as in the free protein. Reduction of the complexed cytochrome c by the neutral flavin semiquinones of lumiflavin and riboflavin was significantly affected by complex formation (2-3-fold rate constant decrease), indicating that there are steric constraints on the accessibility of the cytochrome heme to small exogenous reductants. Reduction of complexed cytochrome c by the negatively charged semiquinones of FMN and Cl2FMN was also characterized. A repulsive electrostatic interaction between the reductants and complexed cytochrome was observed, whereas with free cytochrome an attractive interaction had previously been found. This is consistent with the presence of negative electrostatic potential at the protein interface due to uncompensated flavodoxin carboxylates, as predicted by Matthew et al. [Matthew, J. B., Weber, P. C., Salemme, F. R., & Richards, F. M. (1983) Nature (London) 301, 169-171]. Further, pseudo-first-order rate constants for the reduction of complexed cytochrome by these flavins had a nonlinear concentration dependence, rather than obeying simple second-order kinetics. This is interpreted by using a mechanism involving a rate-determining structural isomerization of the protein complex prior to the second-order electron-transfer step. The magnitude of the decrease in the rate constant for reduction of complexed cytochrome c by the negatively charged reduced rubredoxin was approximately the same as observed for free flavins. Furthermore, simple second-order kinetics were obtained, and the apparent electrostatic interaction between rubredoxin and the complex was attractive. These results suggest that flavodoxin was partially displaced from its complex with cytochrome c by a collisional interaction with rubredoxin. The effects of complexation on the kinetics have been correlated with a solvent-accessible surface representation of the computer-generated model of the flavodoxin-cytochrome c complex [Simondsen, R. P., Weber, P. C., Salemme, F. R., & Tollin, G. (1982) Biochemistry 21, 6366-6375]. The experimental observations are generally consistent with the structural model but clearly require the invocation of dynamic motions at the protein-protein interface.

Clostridium↗
PubMed1979

Structure of the flavin adduct formed in the suicide reaction of alpha-hydroxybutynoate with D-lactate dehydrogenase.

The Zn-dependent flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii is irreversibly inactivated by the D form of the suicide substrate 2-hydroxy-3-butynoic acid. The process of inactivation involves formation of a new pink chromophore, which can be released in intact form from the protein and which was purified to homogeneity by affinity chromatography. Inactivation involves covalent addition of the suicide substrate to the flavin coenzyme. The optical spectra indicate an elongation of the flavin chromophore, and the chemical reactivity suggests a derivative of reduced flavin. The structure of this adduct was deduced from Fourier transform NMR, from the chemical properties, and from comparison with appropriate models, which were synthesized chemically. This structure involves the covalent linkage of the acetylenic inhibitor to positions N(5) and C(6) of the flavin coenzyme via carbon atoms 2 and 4 of the inhibitor to form an additional fused aromatic ring. The pink adduct can be reconverted to an isoalloxazine chromophore by reduction with borohydride and subsequent reoxidation with oxygen. This new isoalloxazine has the spectral properties of an isoflavin, and it is proposed to carry the moiety of the inactivator molecule as substituent at position C(6). The structure of the pink chromophore representing a cyclic adduct to the flavin positions N(5) and C(6) is compared to that of the adduct obtained from L-lactate oxidase from Mycobacterium smegmatis and the L form of the same inhibitor [C(4a)--N(5) cyclic adduct; Schonbrunn, A., Abeles, R. H., Walsh, C. T., Ghisla, S., Ogata, H., and Massey, V. (1976) Biochemistry 15, 1978]. This comparison allows deductions about the relative orientation of substrate, coenzyme, and active center functional groups in the two enzymes.

Alkynes↗