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R Hille

Publications and source records attributed to R Hille.

At least 73 records · Page 4Linked to original sources
PubMed1994

The reaction of trimethylamine dehydrogenase with diethylmethylamine.

The reductive half-reaction of trimethylamine dehydrogenase has been studied using the substrate diethylmethylamine over the pH range 6-10. It is found that the reaction occurs with three distinct and, under most conditions, fully resolved kinetic phases. The hyperbolic substrate concentration dependence of the observed rate constant for the fast kinetic phase is consistent with a two-step scheme in which free enzyme and substrate are in rapid equilibrium with an enzyme-substrate Michaelis complex, which then reacts to produce reduced flavin. The pH dependence of the limiting rate constant for the fast phase of the reaction (klim) exhibits a pKa value of 7.9, whereas klim/Kd exhibits a pKa value of 8.7. The rate constant for the intermediate kinetic phase, which reflects intramolecular electron transfer, is controlled by an ionizable group exhibiting a pKa value of 7.3. The equilibrium distribution of reducing equivalents between the flavin and iron-sulfur center of the enzyme at the end of the intermediate phase is controlled by a different ionizable group exhibiting a pKa value of approximately 6. An overall reductive half-reaction kinetic mechanism is proposed involving formation and decay of a covalent substrate-flavin intermediate, with intrinsically rapid intramolecular electron transfer limited by the rate of decay of the covalent adduct. Under conditions of excess substrate, product release is followed by binding of a second substrate molecule, which results in full development of the spectral properties diagnostic of the spin-interacting state.

Diethylamines↗
PubMed1994

Crystallization and preliminary crystallographic investigation of electron-transfer flavoprotein from the bacterium Methylophilus W3A1.

Electron-transfer flavoprotein from Methylophilus W3A1 has been crystallized using the sitting drop vapour diffusion technique. Hexagonal crystals, suitable for high-resolution structure determination grow to approximately 0.7 mm x 0.7 mm x 0.5 mm in size and diffract to at least 2.2 A. The space group is either P6(1) or P6(5) with unit cell dimensions a,b = 119.1 A and c = 85.6 A.

Crystallography, X-Ray↗
PubMed1994

Electron spin echo envelope modulation spectroscopy of the molybdenum center of xanthine oxidase.

The pulsed EPR technique of electron spin echo envelope modulation (ESEEM) has been utilized to examined both the 'very rapid' and 'desulfo inhibited' Mo(V) signals of xanthine oxidase in order to probe for magnetic interactions with nitrogen, phosphorus and hydrogen nuclei. No 14N modulation is observed in the 'desulfo inhibited' EPR signal, indicating that histidine is unlikely to be a ligand to molybdenum. Strong 14N modulation is observed in the 'very rapid' EPR signal formed with 2-hydroxy-6-methylpurine substrate bound to molybdenum. We interpret this modulation as arising from nitrogens of the bound purine substrate. This interpretation is consistent with the present evidence indicating that the purine ring present in the species giving rise to the 'very rapid' EPR signal is coordinated to the molybdenum center through the catalytically introduced hydroxyl group. No modulation is observed from non-exchangeable deuterons in experiments performed with deuterated 2-hydroxy-6-methylpurine. Given the signal-to-noise level of the spectra, the lack of modulation indicates that each of the substrate methyl group deuterons is greater than 4.9 A from the Mo(V). The deuteron removed from the C8 position in the binding of the substrate is also exchanged to a site or sites greater than 4.9 A from the Mo(V) in the time-course of sample preparation. Moderately deep deuteron modulation arises from exchangeable sites. A large portion of this modulation can be accounted for by the exchangeable N7 deuteron of the 2-hydroxy-6-methylpurine substrate, which we estimate to be approximately 3.2 A from the molybdenum. Additional exchangeable deuterons on the protein or within the buffer must be present within 5 A of the molybdenum to account for the remaining modulation. No modulation from weakly-coupled 31P nuclei is observed in either the 'desulfo inhibited' or 'very rapid' EPR signal.

Deuterium↗
PubMed1994

Assembly of redox centers in the trimethylamine dehydrogenase of bacterium W3A1. Properties of the wild-type enzyme and a C30A mutant expressed from a cloned gene in Escherichia coli.

In trimethylamine dehydrogenase, the enzyme-bound FMN is covalently linked to Cys-30 by a 6-S-cysteinyl FMN bond. The role played by this bond in catalysis has been investigated using a recombinant wild-type trimethylamine dehydrogenase and a Cys-30 to Ala-30 mutant, both expressed from a cloned gene (tmd) in the heterologous host Escherichia coli. The recombinant wild-type and C30A enzymes were found to be quantitatively associated with the 4Fe-4S center and ADP which are both present in the enzyme isolated from bacterium W3A1. In contrast to the enzyme isolated from bacterium W3A1, however, both recombinant proteins contained less than stoichiometric amounts of flavin and were refractory to reconstitution by FMN. The FMN in the recombinant wild-type enzyme was shown to be covalently linked to the protein, and the enzyme possessed catalytic properties similar to its counterpart isolated from bacterium W3A1. It is envisaged that flavinylation proceeds via a nucleophilic attack by the thiolate of Cys-30 at C-6 of the isoalloxazine ring of enzyme-bound FMN. The C30A mutant was found to bind FMN noncovalently and to also catalyze the demethylation of trimethylamine. The major effect of removing the 6-S-cysteinyl FMN bond is to raise the apparent Km for trimethylamine by 2 orders of magnitude and to diminish the apparent kcat for the reaction by only a factor of 2. Therefore, the 6-S-cysteinyl FMN bond is not essential for catalysis, but it is required for efficient functioning of the enzyme at micromolar concentrations of substrate.

Adenosine Diphosphate↗
PubMed1994

Studies of the substrate binding to xanthine oxidase using a spin-labeled analog.

A spin-labeled adenine derivative [N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine; SLAD] is found to be a very slow substrate of xanthine oxidase based on the observed reduction of enzyme by SLAD under anaerobic conditions. A room-temperature EPR spectrum of SLAD in the presence of oxidized xanthine oxidase shows the appearance of "wings" on the three-line spectrum of the free spin-label, indicating formation of an E.SLAD complex. This spectrum can be obtained on a timescale that is short compared to catalysis. Using this spectral change as an experimental probe, the room-temperature Kd's of SLAD binding to oxidized xanthine oxidase at various pH's have been determined. Obtained Kd values are 1.5 +/- 0.3 mM, 1.6 +/- 0.3 mM, and 1.5 +/- 0.3 mM at pH 10.0, 8.5, and 7.0, respectively, indicating no significant difference in the equilibrium dissociation of SLAD from enzyme upon pH change. These results are consistent with the calculated equilibrium dissociation constant for substrate binding to oxidized molybdenum center based on Kd to reduced enzyme and the perturbation of MoVI/MoV and MoV/MoIV reduction potentials by product and substrate analogs.

Adenine↗
PubMed1994

Electron-transferring flavoprotein from pig and the methylotrophic bacterium W3A1 contains AMP as well as FAD.

Electron-transferring flavoproteins from pig kidney and from the methylotrophic bacterium W3A1 have been found to contain one molecule of AMP in addition to the single FAD molecule bound to these heterodimers. The nucleotide was identified by spectral and chromatographic methods and via its behavior toward adenylate kinase and alkaline phosphatase. The role of this additional non-redox active prosthetic group in electron transferring-flavoprotein is at present unclear.

Adenosine Monophosphate↗
PubMed1993

Paradoxical stabilization of the neutral flavin semiquinone of xanthine dehydrogenase at high pH.

The pH dependence of the behavior of chicken liver xanthine dehydrogenase in the course of reductive titrations with sodium dithionite has been examined. Below pH 8.5, the behavior of xanthine dehydrogenase is similar to that of the much better understood milk xanthine oxidase, with the amount of neutral semiquinone accumulating transiently in the course of the titration increasing somewhat as the pH decreases. At pH 10, however, an anomalously large accumulation of the neutral semiquinone is observed by both UV/visible and EPR spectroscopy. Treatment of xanthine dehydrogenase with the thiol reagent iodoacetamide significantly diminishes the ability of the enzyme to stabilize the neutral flavin semiquinone at high pH. These data are consistent with the presence of a protein thiol in the immediate vicinity of the flavin, whose ionization above pH 8.5 results in thermodynamic stabilization of the neutral flavin semiquinone over the anionic form.

Animals↗
PubMed1993

Reductive half-reaction of xanthine oxidase: mechanistic role of the species giving rise to the "rapid type 1" molybdenum(V) electron paramagnetic resonance signal.

The reaction of xanthine oxidase with xanthine, 1-methylxanthine, and 2-hydroxy-6-methylpurine has been reinvestigated with the aim of elucidating the mechanistic role of the species giving rise to the "rapid" Mo(V) electron paramagnetic resonance (EPR) signal. It is found that addition of 2.0 mM 1-methylxanthine or 2-hydroxy-6-methylpurine to partially reduced enzyme generates substantial amounts of the Type 1 form of the "rapid" EPR signal, characterized by superhyperfine coupling to one strongly interacting (aav = 13 G) and one weakly interacting (aav = 3 G) proton. The "rapid" signals observed with both substrates are identical to those observed in the course of the anaerobic reaction of enzyme with a stoichiometric excess of substrate. With 2-hydroxy-6-methylpurine at pH 10, a burst phase in the formation of the species giving rise to the "rapid Type 1" signal is observed that is fast relative to the rate of formation of the species giving rise to the "very rapid" EPR signal. At pH 8.5, partial reduction of enzyme prior to reaction with xanthine, 1-methylxanthine, or 2-hydroxy-6-methylpurine reverses the relative amounts of "rapid" and "very rapid" EPR signal observed at the shortest reaction times. The substantial amounts of "rapid Type 1" signal formed by addition of substrates to partially reduced enzyme or by reaction of oxidized enzyme with a stoichiometric excess of substrate contrasts with previous work, which has shown that under single-turnover conditions none of the substrates investigated generates an appreciable amount of "rapid" EPR signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Reductive half-reaction of xanthine oxidase with xanthine. Observation of a spectral intermediate attributable to the molybdenum center in the reaction of enzyme with xanthine.

The reductive half-reaction of xanthine oxidase with substoichiometric concentrations of xanthine and 1-methylxanthine at pH 10 and 8.5 has been examined by UV-visible stopped-flow and rapid-quench electron paramagnetic resonance (EPR) kinetic experiments. A spectral intermediate is observed in stopped-flow experiments with xanthine which exhibits a difference absorbance maximum relative to oxidized enzyme at 480 nm and which decays at the same rate as the decay of the "very rapid" MoV EPR signal observed by freeze-quench EPR experiments both at 5 and 20 degrees C. The intermediate is observed in experiments using enzyme that has had its flavin removed, and most likely arises from the molybdenum center. With 40 microM xanthine oxidase and 10 microM xanthine, rate constants for the appearance and decay of this intermediate at pH 10 are 11 and 1.1 s-1, respectively; at pH 8.5 the corresponding values are 20 and 2.5 s-1. Based on the correlation of the stopped-flow kinetics with the appearance and decay of the MoV EPR signal designated very rapid as monitored in freeze-quench experiments, it is concluded that the spectral intermediate corresponds to the species exhibiting the very rapid EPR signal, the MoIV species that gives rise to it, or a combination of the two. None of the MoV EPR signals designated "rapid" is observed under single turnover conditions with either xanthine or 1-methylxanthine as substrate at pH 8.5, by contrast with the substantial amounts observed in both cases under conditions of excess substrate. These results call into question the prevailing view that the species giving rise to the rapid EPR signal lies downstream in the catalytic cycle from that exhibiting the very rapid signal.

Animals↗
PubMed1992

The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.

The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.

Alcaligenes↗
PubMed1991

The reductive half-reaction of xanthine oxidase. Identification of spectral intermediates in the hydroxylation of 2-hydroxy-6-methylpurine.

The reaction of xanthine oxidase with 2-hydroxy-6-methylpurine (also called 2-oxo-6-methylpurine) has been studied under both anaerobic and aerobic conditions. Reaction of enzyme with substoichiometric concentrations of hydroxymethylpurine in aerobic 0.1 M 3-(cyclohexylamino)propanesulfonic acid, 0.1 N KCl, 0.3 mM EDTA, pH 10.0, exhibits two reaction intermediates detectable by UV-visible spectrophotometry. The rate constants for formation of the first intermediate, conversion of the first to the second, and the decay of the second to give oxidized enzyme are 18, 1.2, and 0.13 s-1, respectively. The difference spectra of these two intermediates relative to oxidized enzyme are characterized by absorbance maxima at 470 and 540 nm, respectively, with extinction changes (relative to oxidized enzyme) of approximately 410 M-1 cm-1. The 0.13 s-1 decay of the second intermediate agrees well with kcat of 0.11 s-1 determined under the same conditions. Based on a comparison of the kinetics of the reaction as monitored by UV-visible absorption and electron paramagnetic resonance spectrometry, it is concluded that these spectral intermediates arise from the molybdenum center of the enzyme in the MoIV and MoV valence states, respectively, the latter corresponding to the species exhibiting the "very rapid" MoV EPR signal known to be formed in the course of the reaction. This conclusion is supported by the results of experiments using cytochrome c reduction to follow the formation of superoxide production in the course of the aerobic reaction of xanthine oxidase with substoichiometric hydroxymethylpurine, which demonstrate unequivocally that the species exhibiting the very rapid EPR signal is formed by one-electron oxidation of a MoIV species rather than direct one-electron reduction of MoVI by substrate. No evidence is found for the formation of any of the MoV EPR signals designated "rapid" in the present studies, and it is concluded that this species is not a bona fide catalytic intermediate in the reductive half-reaction of xanthine oxidase.

Aerobiosis↗
PubMed1991

The kinetic behavior of xanthine oxidase containing chemically modified flavins.

The steady-state and rapid kinetic properties of xanthine oxidase containing a series of FAD analogs of varying reduction potential have been investigated. From steady-state analysis, Vmax is found to exhibit a sigmoidal dependence on the flavin midpoint potential in the homologous series. This dependence is accurately described by a model in which the rate of catalysis is attenuated by the amount of partially reduced enzyme generated during turnover possessing an unfavorable distribution of reducing equivalents among the several redox-active centers of the protein. The model assumes that reducing equivalents equilibrate among these centers rapidly compared to the limiting rates for the reductive and oxidative half-reactions. This assumption is borne out by a quantitative analysis of the reductive and oxidative half-reactions of the several enzyme forms investigated in detail. It is demonstrated in these studies that xanthine oxidase containing low potential flavin derivatives such as 1-deaza, 6-hydroxy, or 8-hydroxy FAD exhibits low turnover not because of inherently slow rates of reduction by xanthine or oxidation by molecular oxygen, but because in partially reduced enzyme generated in the course of turnover reducing equivalents are distributed within the enzyme in such a way that the enzyme can participate in neither the reductive nor oxidative half-reactions. These results provide confirmation of the operation of a thermodynamic control mechanism in a simple electron-transferring system.

Flavins↗
PubMed1991

Electron transfer within xanthine oxidase: a solvent kinetic isotope effect study.

Solvent kinetic isotope effect studies of electron transfer within xanthine oxidase have been performed, using a stopped-flow pH-jump technique to perturb the distribution of reducing equivalents within partially reduced enzyme and follow the kinetics of reequilibration spectrophotometrically. It is found that the rate constant for electron transfer between the flavin and one of the iron-sulfur centers of the enzyme observed when the pH is jumped from 10 to 6 decreases from 173 to 25 s-1 on going from H2O to D2O, giving an observed solvent kinetic isotope effect of 6.9. An effect of comparable magnitude is observed for the pH jump in the opposite direction, the rate constant decreasing from 395 to 56 s-1. The solvent kinetic isotope effect on kobs is found to be directly proportional to the mole fraction of D2O in the reaction mix for the pH jump in each direction, consistent with the effect arising from a single exchangeable proton. Calculations of the microscopic rate constants for electron transfer between the flavin and the iron-sulfur center indicate that the intrinsic solvent kinetic isotope effect for electron transfer from the neutral flavin semiquinone to the iron-sulfur center designated Fe/S I is substantially greater than for electron transfer in the opposite direction and that the observed solvent kinetic isotope effect is a weighted averaged of the intrinsic isotope effects for the forward and reverse microscopic electron-transfer steps.(ABSTRACT TRUNCATED AT 250 WORDS)

Deuterium↗
PubMed1991

Intramolecular electron transfer in trimethylamine dehydrogenase from bacterium W3A1.

Reductive optical/EPR titrations of trimethylamine dehydrogenase with sodium dithionite have been performed, indicating that the equilibrium distribution of reducing equivalents between the covalently bound FMN and 4Fe/4S centers in partially reduced trimethylamine dehydrogenase is pH-dependent. In the case of two-electron reduced enzyme, formation of fully reduced flavin with oxidized iron-sulfur is favored below pH 7.5, whereas above pH 8 formation of flavin semiquinone with reduced iron-sulfur is preferred. The rates of electron transfer between the sites have been measured with the stopped-flow rapid mixing technique using a pH jump. The observed rate constants fall in the range of 200 s-1 to 1000 s-1 at 25 degrees C with the larger values occurring at higher values of final pH. The values of the rate constants depend on the final pH and are independent of observation wave-length. The temperature dependencies of these reactions give linear Arrhenius plots with activation energies in the range of 12 to 16 kcal/mol, consistent with prototropic equilibria being associated with electron transfer. The pH dependence of EPR spectral line widths for the flavin semiquinone and static optical spectra suggest that the semiquinone form of flavin present at pH 10 is anionic, whereas the neutral form is present at pH 7. The observed rate constants at 25 degrees C are greater than or equal to 100-fold larger than kcat for this enzyme and indicate that intramolecular electron transfer is not intrinsically rate-limiting in overall catalysis.

Bacteria↗
PubMed1991

Electron transfer in milk xanthine oxidase as studied by pulse radiolysis.

Electron transfer within milk xanthine oxidase has been examined by the technique of pulse radiolysis. Radiolytically generated N-methylnicotinamide radical or 5-deazalumiflavin radical has been used to rapidly and selectively introduce reducing equivalents into the enzyme so that subsequent equilibration among the four redox-active centers of the enzyme (a molybdenum center, two iron-sulfur centers, and FAD) could be monitored spectrophotometrically. Experiments have been performed at pH 6 and 8.5, and a comprehensive scheme describing electron equilibration within the enzyme at both pH values has been developed. All rate constants ascribed to equilibration between specific pairs of centers in the enzyme are found to be rapid relative to enzyme turnover under the same conditions. Electron equilibration between the molybdenum center and one of the iron-sulfur centers of the enzyme (tentatively assigned Fe/S I) is particularly rapid, with a pH-independent first-order rate constant of approximately 8.5 x 10(3) s-1. The results unambiguously demonstrate the role of the iron-sulfur centers of xanthine oxidase in mediating electron transfer between the molybdenum and flavin centers of the enzyme.

Alkylation↗
PubMed1990

Resonance-enhanced Raman scattering from the molybdenum center of xanthine oxidase.

The molybdenum center of xanthine oxidase has been examined by resonance Raman spectroscopy. Making use of the long-wavelength absorption of the reduced molybdenum center in complex with violapterin (the product of enzymic action of lumazine), resonance Raman spectra were obtained using laser excitation at 676.4 nm. Several internal vibrational modes of violapterin were found to be resonance-enhanced, and a number of bands in the 250-1100 cm-1 range, presumably arising from vibrational modes of the molybdenum coordination sphere, were also observed. Upon substitution of 18O for 16O in the molybdenum coordination sphere, bands at 1469, 853, 517, 325, and 276 cm-1 exhibited shifts of 5-12 cm-1 to lower energy. By analogy to previous vibrational studies of Mo-O-Mo and Mo-O-R model compounds, the 853, 517, and 276 cm-1 frequencies were judged consistent with a labeled Mo-O-R linkage of the complexed violapterin. More importantly, the relatively small frequency shifts observed in these and other vibrations upon incorporation of 18O are very similar to those observed by others for 18O-labeled phenol and metal-phenolate complexes (Pinchas, S., Sadeh, D., and Samuel, D. (1965) J. Phys. Chem. 69, 2259-2264; Pyrz, W. J., Rue, L. A., Stern, L. J., and Que, L. J., Jr. (1985) J. Am. Chem. Soc. 107, 614-620) that model iron-tyrosinate proteins. The relatively small isotope-induced frequency shifts in multiple bands are thus interpreted as resulting from vibrational mixing of internal coordinates involving the oxygen atom with internal ring motions of the aromatic species. No oxygen isotope-sensitive bands were observed in the 900-1100 cm-1 region where Mo = O stretching modes typically occur. In agreement with the conclusions of previous workers (Davis, M.D., Olson, J. S., and Palmer, G. (1982) J. Biol. Chem. 257, 14730-14737) we interpret our results to indicate that the absorption band appearing upon complexation of violapterin with the molybdenum center of reduced xanthine oxidase is a molybdenum-to-violapterin charge-transfer band. These results, as well as several other lines of evidence, are consistent with direct coordination of violapterin to molybdenum in the charge-transfer complex via the 7-hydroxyl group (i.e. the hydroxyl group introduced into substrate by the enzyme). The Mo=O stretching mode of the complex is presumably not resonance enhanced because it is orthogonal to the charge-transfer electronic transition, suggesting that coordination of violapterin is cis to the oxo group.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Bacterial sarcosine oxidase: identification of novel substrates and a biradical reaction intermediate.

Corynebacterial sarcosine oxidase contains both covalently and noncovalently bound FAD and forms complexes with various heterocyclic carboxylic acids (D-proline and 2-furoic, 2-pyrrolecarboxylic, and 2-thiophenecarboxylic acids). 2-Furoic acid, a competitive inhibitor with respect to sarcosine, selectively perturbs the absorption spectrum of the noncovalent flavin, suggesting that the enzyme has a single sarcosine binding site near the noncovalent flavin. Several heterocyclic amines have been identified as new substrates for the enzyme. Similar reactivity is observed with L-proline and L-pipecolic acid whereas L-2-azetidine-carboxylic acid is less reactive. Turnover with L-proline is slow (TN = 4.4 min-1) as compared with sarcosine (TN = 1000 min-1). Anaerobic reduction of the enzyme with heterocyclic amine substrates at pH 8.0 occurs as a biphasic reaction. A similar long-wavelength intermediate is formed in the initial fast phase of each reaction and then decays in a slower second phase to yield 1,5-dihydroFAD. The slow phase is not kinetically significant during aerobic turnover at pH 8.0 and is absent when the anaerobic reactions are conducted at pH 7.0. EPR and other studies at pH 7.0 show that the long-wavelength species is a half-reduced form of the enzyme (1 electron/substrate-reducible flavin) containing 0.9 mol of flavin radical/mol of substrate-reducible flavin. This biradical intermediate exhibits an absorption spectrum similar to that expected for a 50:50 mixture of red anionic and blue neutral flavin radicals. A similar long-wavelength species is observed during titration of the enzyme with sarcosine and other reductants. Studies with L-proline suggest that reduction of the enzyme involves initial transfer of two electrons to the noncovalent flavin. The covalent flavin is not required and can be complexed with sulfite without affecting the rate of electron transfer. The initial half-reduced form of the enzyme appears to be rapidly converted to the biradical form via comproportionation of the reduced noncovalent flavin with the oxidized covalent flavin.

Azetidinecarboxylic Acid↗