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PubMed1999

The effects of pH and semiquinone formation on the oxidation-reduction potentials of flavin mononucleotide. A reappraisal.

Calculation shows that there is poor agreement between frequently cited values for the midpoint redox potentials of the two one-electron steps in the reduction of flavin mononucleotide and equations for the lines that relate these potentials to pH and that use the published pKa values for the three redox states of the flavin [Draper, R. & Ingraham, L.L. (1969) Arch. Biochem. Biophys. 125, 802-808]. Equilibrium data for the first step in the reduction obtained by pulse radiolysis [Anderson, R.F. (1983) Biochim. Biophys. Acta 722, 158-162] show much closer agreement with theory and lead to values for the semiquinone formation constant of flavin mononucleotide that are close to those derived from measurements of the radical concentration using ESR spectroscopy. It is concluded that the data from the second method are more reliable. The redox potentials for flavin mononucleotide at pH 7.0 and 20 degrees C are calculated to be -0.207 V for the overall two-electron reduction (Em), -0.313 V for reduction of the oxidized flavin to the semiquinone (E2) and -0.101 V for the reduction of the semiquinone to the hydroquinone (E1). Information is provided to allow calculation of the three redox potentials at other pH values in the physiological range.

Benzoquinones↗
PubMed1995

Active site studies of DT-diaphorase employing artificial flavins.

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD-containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK alpha of the enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones.

Binding Sites↗
PubMed1990

Dithionite treatment of flavins: spectral evidence for covalent adduct formation and effect on in vitro bacterial bioluminescence.

Intrigued by the apparent requirement of dithionite for FMN reduction (as opposed to photoreduction or catalytic hydrogenation) in the H2O2-initiated bacterial bioluminescence reaction, we chose 5-ethyl-3-methyllumiflavinium cation I as a model to investigate possible flavin adduct formation by treatment with dithionite or (bi)sulfite. In the range of pH 5-8, the reaction of dithionite with 5-ethyl-3-methyllumiflavinium cation, which is in equilibrium with the 5-ethyl-4a-hydroxy-3-methyl-4a, 5-dihydrolumiflavin pseudobase II (X = OH), is not limited to the formation of flavosemiquinone and dihydroflavin following two one-electron steps. Several parallel and sequential reactions may take place involving the intermediacy of covalent flavin adducts. Addition of (bi)sulfite gave a 4a-sulfiteflavin adduct II (X = SO3-). Consistent with the S2O4(2-) in equilibrium with 2 SO2-. equilibrium, the reaction of dithionite and II (X = OH; SO3-) gave rise to two flavin adducts in competitive nucleophilic displacements: a 4a-sulfoxylate-flavin radical (II, X = SO2.) and a 4a-dithioniteflavin adduct (II, X = S2O4-), respectively. On increasing the (S2O4(2-), SO2.-)/flavin ratio under N2, the formation of the 4a-sulfoxylate-flavin radical became predominant. The II (X = SO2.) so formed was in equilibrium with the flavosemiquinone and bisulfate and can be trapped by reacting with hydroxylamine. In the initial presence of oxygen, II (X = SO2.) was highly reactive toward O2, giving a fast oxidation to II (X = SO3-) and effectively suppressing the formation of the flavosemiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)

Dithionite↗
PubMed1994

The mobile flavin of 4-OH benzoate hydroxylase.

Para-hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para-hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.

Benzoate 4-Monooxygenase↗
PubMed1972

Purification and properties of the flavine-stimulated anaerobic L- -glycerophosphate dehydrogenase of Escherichia coli.

The anaerobic l-alpha-glycerophosphate (l-alpha-GP) dehydrogenase of Escherichia coli was purified approximately 40-fold. The activity of the dehydrogenase, although not affected by the addition of pyridine nucleotides, was stimulated three- to fourfold by flavine adenine dinucleotide (K(m) about 10(-7)m) and up to 10-fold by flavine mononucleotide (K(m) about 10(-4)m). Maximal activity of the enzyme was found only in the combined presence of saturating concentrations of both flavines (stimulation by a factor of 10 to 15). The dependence of the rate of the reaction on the concentration of l-alpha-GP was complex in the presence of both flavines, but in the presence of flavine adenine dinucleotide alone the kinetics were of the Michaelis-Menten type with the K(m) for l-alpha-GP being about 10(-4)m. The product of the reaction was identified as dihydroxyacetone phosphate, and the molecular weight of the dehydrogenase was estimated to be 80,000 +/- 10,000. Phenazine methosulfate, menadione and ferricyanide served as artificial acceptors for the dehydrogenase. The enzyme was sensitive to iodoacetate, p-chloromercuribenzoate, and N-ethymaleimide.

Ammonium Sulfate↗
PubMed2004

Biochemical characterization of StyAB from Pseudomonas sp. strain VLB120 as a two-component flavin-diffusible monooxygenase.

Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg(-1) (k(cat) = 1.6 s(-1)) and K(m) values for styrene of 0.45 +/- 0.05 mM (wild type) and 0.38 +/- 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg(-1) (k(cat) [NADH] = 60 s(-1)). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.

Cloning, Molecular↗
PubMed1981

Active site probes of flavoproteins. Determination of the solvent accessibility of the flavin position 8 for a series of flavoproteins.

The chemical reactivity of 8-chloroflavins and 8-mercaptoflavins has been exploited in order to examine the orientation of protein-bound flavins relative to solvent. The apoprotein form of a series of flavoproteins was prepared and the native flavin was replaced by either 8-Cl-flavin or 8-mercaptoflavin (FAD, FMN, or riboflavin form as was appropriate). The reconstituted proteins were exposed to reagents capable of reacting with the group at position 8. The 8-Cl-proteins were challenged with sodium sulfide and thiophenol, while the 8-mercaptoproteins were faced with iodoacetamide and iodoacetic acid. The kinetics of the ensuing reactions served as a measure of the solvent availability of position 8 for the protein-bound flavin. These studies indicated that position 8 of flavin bound to melilotate hydroxylase, D-amino acid oxidase, old yellow enzyme, p-OH-benzoate hydroxylase, and flavodoxin is accessible to solvent, while position 8 on L-lactate oxidase, glucose oxidase, putrescine oxidase, and riboflavin-binding protein appears to be inaccessible. For luciferase, D-lactate dehydrogenase, and xanthine oxidase, the data suggest that position 8 is exposed but the results are inconclusive. The effect of ligand binding on the accessibility of position 8 was also studied. NADPH binding to 8-mercapto old yellow enzyme and benzoate binding to 8-Cl-D-amino acid oxidase results in complete blockage of previously available position 8. On the other hand, p-OH-benzoate hydroxylase and melilotate hydroxylase bind their respective substrates (p-OH-benzoate and melilotate) without significantly altering the reactivity of position 8.

Apoproteins↗
PubMed1993

Resonance Raman spectroscopic evidence for an anionic flavin semiquinone in bovine liver monoamine oxidase.

The flavoprotein monoamine oxidase B (MAO B) from bovine liver, as isolated, has an unusual additional absorption band at 412 nm, which is similar to the absorption of its anionic flavin semiquinone form, (Fl.-), and other typical (Fl.-) flavoproteins. Denaturation of the enzyme results in the elimination of this anomalous absorption. The resonance Raman (RR) spectrum of MAO B as isolated is virtually identical to that of its dithionite-reduced (Fl.-) form. Both spectra show features similar to those of the RR spectrum of the (Fl.-) form of Aspergillus niger glucose oxidase (GO) in the region between 300 and 1700 cm-1 with 406.7 nm excitation. These features are readily distinguishable from those of oxidized flavin, neutral flavin semiquinone, and hemoprotein, strongly suggesting the presence of an (Fl.-) form in MAO B as isolated, even with preparations isolated in the absence of light. There are significant differences between the RR spectra of the (Fl.-) form of MAO B and those of GO or the published RR spectra of the (Fl.-) form of D-amino acid oxidase with excess substrate analog. At least some of these differences can be attributed to the different binding of flavin in the three enzymes. No EPR signals due to (Fl.-) are observed in MAO B as isolated. The dithionite-reduced (Fl.-) form exhibits approximately 50% less EPR signal than that expected from the absorption spectrum, which suggests a possible coupling of the (Fl.-) flavin with a paramagnetic center of unknown identity in the protein. The implications of these observations on MAO B with the current view of its catalytic mechanism are discussed.

Animals↗
PubMed2003

A major integral protein of the plant plasma membrane binds flavin.

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.

Amino Acid Sequence↗
PubMed1990

High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase.

Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 mumols NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b5 reductase.

Amino Acid Sequence↗
PubMed1985

Trapping of reactive intermediates in enzymology. Exogenous flavin reduction during catalytic turnover of substrate by glyoxalase I.

Although but weak inhibitors of glyoxalase I under steady-state conditions, flavins are reduced by yeast glyoxalase I (lactoyl-glutathione lyase, EC 4.4.1.5) plus its substrate (the hemithiolacetal from glutathione and phenylglyoxal) during catalytic turnover. Studies with 10-ethylisoalloxazine showed that this flavin reduction was peculiar not merely to glyoxalase I's substrate, but was characteristic of the complete system, enzyme plus substrate undergoing catalytic turnover. Flavins are poor hydride-ion acceptors and the reduction observed most likely represents an oxidative trap of a transient carbanion formed in the glyoxalase I mechanism of action. Hydrophobic flavins were more efficient traps than the hydrophilic ones, and values of the Km for the phenylglyoxal: glutathione hemithiolacetal adduct measured by the flavin-reaction and by normal steady-state kinetics were closely similar. This argues that trapping has occurred of an enediolate ion (an enzyme-generated carbanion) still bound to glyoxalase I.

Animals↗
PubMed1982

On the environment and the rotational motion of amphiphilic flavins in artificial membrane vesicles as studied by electron paramagnetic resonance spectrometry.

This paper continues the studies of vesicle-bound flavins ('anisotropic flavin chemistry'). It is possible to anchor the flavin nucleus in various modes within the lipids/water interface by means of long aliphatic chains and using different saturated lipids, thereby mimicking the specific binding of the coenzyme to the apoprotein in flavoproteins. Based on absorption spectroscopy and EPR spectroscopy studies we explored the rotational mobility and the microenvironment of membrane-bound amphiflavin radicals. N(5)-unsubstituted amphiflavin radicals exhibit a similarly high disproportionation constant as known from isotropic flavin chemistry. However, reasonable stabilization of the radical was achieved by introduction of an alkyl group in position 5 in the reduced state prior to the one-electron oxidation. Adopting the fine structure of the corresponding EPR spectra as assay for the mobility of the semiquinone, we determined rotational relaxation times ranging from 60 ns in the crystalline state down to 10 or 15 ns in the liquid-crystalline state of the membrane. The solvatochromic effect shown by absorption spectra of the membrane-bound flavin radicals reflects a dielectric constant of the microenvironment of c = 30-40, corresponding to the lipid/water interface region. The results obtained in this study are consistent with those obtained previously, from fluorescence analyses, supporting our former conclusions.

Flavins↗
PubMed1995

Tryptophan 250 on the alpha subunit plays an important role in flavin and aldehyde binding to bacterial luciferase. Effects of W-->Y mutations on catalytic function.

Bacterial luciferase is a heterodimer (alpha beta) that catalyzes the oxidation of FMNH2 and a fatty aldehyde, resulting in light emission. To explore the nature of the flavin binding site with respect to the role of tryptophan residues, the catalytic and binding properties of single-point mutants of Xenorhabdus luminescens luciferase with one of the eight tryptophans converted to a tyrosine residue were investigated by luminescence and fluorescence measurements. Conversion of tryptophans 194 and 250 on the alpha subunit to tyrosine had relatively large effects on the properties of luciferase with only minor changes in the properties on mutation of the other four tryptophans on alpha and the two on the beta subunit. Mutation of alpha W250 decreased the binding to FMNH2, FMN, aldehyde, and fatty acid, causing major changes in luminescence emission and decay. The results are consistent with alpha W250 interacting with flavin which in turn affects aldehyde binding. Mutation of alpha W194 did not affect the interaction with flavin or aldehyde but did change the relative rate of decay of light emission with aldehydes of different chain lengths as well as the activation energy for this process. Moreover, these results provide evidence for alpha W250, and to a lesser extent alpha W194, being in contact with the isoalloxazine ring of flavin, a proposal that has been recently made based on a model with flavin bound to the alpha subunit and anchored at a binding site for the phosphate moiety of FMN(H2) identified in the crystal structure of Vibrio harveyi luciferase [Fisher, A. J. Raushel, F. M., Baldwin, T. O., & Rayment, I. (1995) Biochemistry 34, 6581-6586].

Aldehydes↗
PubMed1992

Comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time-resolved polarized flavin fluorescence.

Time-resolved polarized fluorescence spectroscopy has been applied to the bound FAD in the structurally related flavoproteins lipoamide dehydrogenase from Azotobacter vinelandii (LipDH-AV) and glutathione reductase (GR) from human erythrocytes. The fluorescence parameters as obtained from the maximum entropy analysis differ considerably in both enzymes, reflecting the unique properties of the flavin microenvironment. Three conformational substates are revealed in LipDH-AV and five in GR. Almost 90% of the population of GR molecules has a fluorescence lifetime in the order of 30 ps which originates from efficient exciplex formation with Tyr197. Equilibrium fluctuations between conformational substates are observed for LipDH-AV on a nanosecond time scale in the temperature range 277-313 K. Interconversion between conformational substates in GR is slow, indicating that large activation barriers exist between the states. In agreement with these results, a model is postulated which ascribes a role in catalysis to equilibrium fluctuations between conformational substates in GR and LipDH-AV. From time-resolved fluorescence anisotropy as a function of temperature, distinction can be made between flavin reorientational motion and interflavin energy transfer. In both proteins intersubunit energy transfer between the prosthetic groups is observed. Furthermore, it is revealed that only the flavin in glutathione reductase exhibits rapid restricted reorientational motion. Geometric information concerning the relative orientation and distance of the flavins can be extracted from the parameters describing the energy-transfer process. The obtained spatial arrangement of the flavins is in excellent agreement with crystallographic data.

Azotobacter vinelandii↗
PubMed1994

Thermodynamic analysis of flavin in mitochondrial NADH:ubiquinone oxidoreductase (complex I).

This paper reports the first direct characterization of flavin (noncovalently bound FMN) in energy coupling site I of the mitochondrial respiratory chain. Thermodynamic parameters of its redox reactions were determined potentiometrically monitoring the g = 2.005 signal of its free radical form in isolated bovine heart NADH:ubiquinone oxidoreductase (complex I). The midpoint redox potentials of consecutive one-electron reduction steps are Em1/0 = -414 mV and Em2/1 = -336 mV at pH 7.5. This corresponds to a stability constant of the intermediate flavosemiquinone state of 4.5 x 10(-2). The pK values of the free radical (Fl.-<==>FlH.) and reduced flavin (FlH-<==>FlH2) were estimated as 7.7 and 7.1, respectively. The potentiometrically obtained g = 2.005 flavin free radical EPR signal revealed an unusually broad (2.4 mT) and pH-independent peak-to-peak line width. The spin relaxation of flavosemiquinone in complex I is much faster than that of flavodoxin due to strong dipole-dipole interaction with iron-sulfur cluster N3. Guanidine, an activator of NADH-ferricyanide reductase activity of complex I, was found to have a strong stabilizing effect on the flavin free radical generated both by equilibration with the NADH/NAD+ redox couple and by potentiometric redox titration. The addition of guanidine also leads to a slight modification of the EPR spectrum of iron-sulfur cluster N3. Anaerobic titration of flavosemiquinone free radical with the strictly n = 2 NADH/NAD+ and APADH/APAD+ redox couples revealed that nucleotide binding narrows the EPR signal line width of the flavin free radical to 1.7 mT and changes a shape of the titration curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Mechanism of light-induced reduction of biological redox centers by amino acids. A flash photolysis study of flavin photoreduction by ethylenediaminetetraacetate and nitrilotriacetate.

The mechanism of flavin photoreduction by the amino acids, EDTA, and nitrilotriacetate is shown to be due to light-induced charge separation, which is irreversible in the dark. The irreversibility originates from the decarboxylation of the amino acid radical. This fast process changes the redox properties of the radical and makes a further donation of an electron equivalent possible. In the case of EDTA the electron acceptor of the second electron is flavin, which was left unexcited by the flash or is formed by dismutation from the flavosemiquinone, generated in the primary one-electron transfer process. In contrast to this, a mechanism for the flavin photoreduction by nitrilotriacetate is proposed, in which the decarboxylated nitrilotriacetate radical adds to the flavosemiquinone to yield an alkylated flavohydroquinone. The latter decays to free reduced or oxidized flavin, depending on the position of addition at the flavin chromophore. The difference in reaction mechanism between the nitrite anion, EDTA, and nitrilotriacetate is discussed in terms of differences in molecular structure.

Acetates↗
PubMed1975

The kinetics of flavine oxidation--reduction. I. Dismutation in nonaqueous solvent.

The dismutation reactions of flavines in dimethylformanide have been investigated using the stopped-flow technique under anaerobic conditions. The ionization constants of fully reduced and oxidized tetraacetylriboflavine were measured spectrophotometrically in buffered dimethylformanide. The dismutation equilibrium of the flavine as a function of pH in dimethylformanide was roughly comparable to that reported in water and allowed the estimation of the pKa value of the flavosemiquinone. The dismutation kinetics of tetraacetylriboflavine in unbuffered dimethylformanide were investigated using the fully oxidized and reduced flavines in their neutral form at constant produce of concentrations and varying the reduction degree. The kinetics at very low reduction ratios (less than3%) were triphasic. The kinetic analysis of the initial and simultaneous formation of the anionic and neutral radicals revealed a second-order reaction. The electron transfer between the oxidized and reduced flavines was not directly coupled with prton exchange. The multiphasic time course of the reaction proceeded primarily from differences in the intrinsic rates of the direct and mixed backward dismutation reactions of the two radical species, and finally from a change in the equilibrium conditions resulting from the accumulation of anionic flavohydroquinone. An acidic-basic negative catalytic effect from the neutral flavohydroquinone appeared progressively as the reduction degree was increased. It was complete at reduction ratios higher than 30%, i.e. under conditions where the radical anion could not be observed at any reaction time. Acids with a pKa value lower than the second one of the flavosemiquinone exhibited a similar catalytic effect. These acidic-basic catalytic effects are associated with changes in the ionic state of labile intermediate dimers formed in the forward as well as in the backward direactions of the dismutation reaction. Such a transient complex revealed by the kinetic analysis could be observed directly by absorption spectroscopy in alkaline-buffered dimethylformanide. Its spectral characteristics, as well as the kinetic effects induced by substitution of the benzenoid part of the flavine, can hardly be taken into account by a quinhydrone-like structure for the intermediate dimers at any pH value. The experimental results favored a more specific interaction, possibly of covalent character, involving the benzenoid part of the isoalloxazine ring.

Chemical Phenomena↗
PubMed2001

Role of aromatic stacking interactions in the modulation of the two-electron reduction potentials of flavin and substrate/product in Megasphaera elsdenii short-chain acyl-coenzyme A dehydrogenase.

The effects of aromatic stacking interactions on the stabilization of reduced flavin adenine dinucleotide (FAD) and substrate/product have been investigated in short-chain acyl-coenzyme A dehydrogenase (SCAD) from Megasphaera elsdenii. Mutations were made at the aromatic residues Phe160 and Tyr366, which flank either face of the noncovalently bound flavin cofactor. The electrochemical properties of the mutants were then measured in the presence and absence of a butyryl-CoA/crotonyl-CoA mixture. Results from these redox studies suggest that the phenylalanine and tyrosine both engage in favorable pi-sigma interactions with the isoalloxazine ring of the flavin to help stabilize formation of the anionic flavin hydroquinone. Disruption of these interactions by replacing either residue with a leucine (F160L and Y366L) causes the midpoint potential for the oxidized/hydroquinone couple (E(ox/hq)) to shift negative by 44-54 mV. The E(ox/hq) value was also found to decrease when aromatic residues containing electron-donating heteroatoms were introduced at the 160 position. Potential shifts of -32 and -43 mV for the F160Y and F160W mutants, respectively, are attributed to increased pi-pi repulsive interactions between the ring systems. This study also provides evidence for thermodynamic regulation of the substrate/product couple in the active site of SCAD. Binding to the wild-type enzyme caused the midpoint potential for the butyryl-CoA/crotonyl-CoA couple (E(BCoA/CCoA)) to shift 14 mV negative, stabilizing the oxidized product. Formation of product was found to be even more favorable in complexes with the F160Y and F160W mutants, suggesting that the electrostatic environment around the flavin plays a role in substrate/product activation.

Acyl Coenzyme A↗