Intramolecular and intermolecular perturbation on electronic state of FAD free in solution and bound to flavoproteins: FTIR spectroscopic study by using the C = O stretching vibrations as probes

The intramolecular and intermolecular perturbation on the electronic state of FAD was investigated by FTIR spectroscopy by using the C=O stretching vibrations as probes in D(2)O solution. Natural and artificial FADs, i.e. 8-CN-, 8-Cl-, 8-H-, 8-OCH(3)-, and 8-NH(2)-FAD labelled by 2-(13)C, (18)O=C(2), or 4,10a-(13)C(2) were used for band assignments. The C(2)=O and C(4)=O stretching vibrations of oxidized FAD were shifted systematically by the substitution at the 8-position, i.e. the stronger the electron-donating ability (NH(2) > OCH(3) > CH(3) > H > Cl > CN) of the substituent, the lower the wavenumber region where both the C(2)=O and C(4)=O bands appear. In contrast, the C(4)=O band of anionic reduced FAD scarcely shifted. The 1,645-cm(-1) band containing C(2)=O stretching vibration shifted to 1,630 cm(-1) in the medium-chain acyl-CoA dehydrogenase (MCAD)-bound state, which can be explained by hydrogen bonds at C(2)=O of the flavin ring. The band was observed at 1,607 cm(-1) in the complex of MCAD with 3-thiaoctanoyl-CoA. The 23 cm(-1) shift was explained by the charge-transfer interaction between oxidized flavin and the anionic acyl-CoA. In the case of electron-transferring flavoprotein, two bands associated with the C(4)=O stretching vibration were obtained at 1,712 and 1,686 cm(-1), providing evidence for the multiple conformations of the protein.     

Substrate recognition and activation mechanism of D-amino acid oxidase: a study using substrate analogs

We investigated the mechanism of recognition and activation of substrate by D-amino acid oxidase (DAO) by thermodynamical and spectrophotometric methods using zwitterionic ligands [N-methylisonicotinate (NMIN), trigonelline, and homarine] and monoanionic ligands as model compounds of the substrate and the product. In terms of the charge within the substrate D-amino acid, monoanionic (e.g., benzoate), zwitterionic (e.g., NMIN), and dianionic (e.g., terephthalate) ligands are thought to be good models for neutral, basic, and acidic amino acids, respectively, because when a substrate binds to DAO, as previously reported, the a-ammonium group (-NH(3)(+)) probably loses a proton to become neutral (-NH(2)) before the oxidation. Zwitterionic ligands can also be good model compounds of product in the purple complex (the complex of reduced DAO with the product imino acid), because the imino nitrogen of the imino acid is in a protonated cationic form. We also discuss electrostatic interaction, steric effect, and charge-transfer interaction as factors which affect the affinity of substrate/ligand for DAO. Monoanionic ligands have high affinity for neutral forms of oxidized and semiquinoid DAO, while zwitterionic ligands have high affinity for anionic forms of oxidized, semiquinoid, and reduced DAO; this difference was explained by the electrostatic interaction in the active site. The low affinity of homarine (N-methylpicolinate) for oxidized DAO, as in the case of o-methylbenzoate, is due to steric hindrance: one of the ortho carbons of benzoate is near the phenol carbons of Tyr228 and the other ortho carbon is near the carbonyl oxygen of Gly313. The correlation of the affinity of meta- and para-substituted benzoates for oxidized DAO with their Hammet's s values are explained by the HOMO-LUMO interaction between the phenol group of Tyr224 and the benzene ring of benzoate derivative. The pK(a) of neutral flavin [N(3)-H of oxidized flavin, N(5)-H of semiquinoid flavin, and N(1)-H of reduced flavin] decreases by its binding to the apoenzyme. The magnitude of the decrement is oxidized flavin < semiquinoid flavin < reduced flavin. The largest factor in the substantially low pK(a) of reduced flavin in DAO is probably the steric hindrance between the hydrogen atom of H-N(1)(flavin) and the hydrogen atom of H-N of Gly315, which becomes significant when a hydrogen is bound to N(1) of flavin.     

Effect of pH on oxidation-reduction potentials of 8 alpha-N-imidazole-substituted flavins

The pKa values for the various ionic forms of 8 alpha-N-imidazolylriboflavin were determined in its oxidized and hydroquinone forms and estimated for its semiquinone form. The pH dependence of the absorption and fluorescence spectral properties and potentiometric titration data show the pKa values for the oxidized form to be 6.02 +/- 0.03 for the 8 alpha-imidazole nitrogen and 9.67 +/- 0.05 for the N(3) position of the flavin ring. The pH dependence of the oxidation-reduction potential was determined by spectrocoulometric titrations, and the data points were compared with computer-simulated plots. Two pKa values for the hydroquinone form of the flavin were determined and assigned. The pKa for the imidazole ring is found to be 6.9 +/- 0.1 and for the N(1) position of the flavin hydroquinone is found to be 5.5 +/- 0.1. Analysis of the pH dependence of the one-electron couples E2 (flavoquinone/flavin semiquinone, Flox/Fl.) and E1 (flavin semiquinone/flavin hydroquinone, Fl./Flred) resulted in an estimated pKa of 6.5 for the 8 alpha-imidazole ring in the flavin semiquinone form. These data show the possible involvement of the ionization of the 8 alpha-imidazole substituent in the redox chemistry of flavoenzymes containing either an 8 alpha-N1- or an 8 alpha-N3-histidyl-linked covalent flavin coenzyme. Future work on oxidation-reduction potentials of this class of enzymes must take into consideration the influence of the 8 alpha-histidyl substituent.     

Medium-chain acyl-coenzyme A dehydrogenase bound to a product analogue, hexadienoyl-coenzyme A: effects on reduction potential, pK(a), and polarization

2,4-Hexadienoyl-coenzyme A (HD-CoA) has been used to investigate the redox and ionization properties of medium-chain acyl-CoA dehydrogenase (MCAD) from pig kidney. HD-CoA is a thermodynamically stabilized product analogue that binds tightly to oxidized MCAD (K(dox) = 3.5 +/- 0.1 microM, pH 7.6) and elicits a redox potential shift that is 78% of that observed with the natural substrate/product couple [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715]. The midpoint potential of the MCAD.HD-CoA complex exhibits a pH dependence that is consistent with the redox-linked ionization of two key glutamic acids as well as the flavin adenine dinucleotide (FAD) cofactor. The estimated ionization constants for Glu376-COOH (pK(a,ox) approximately 9.3) and Glu99-COOH (pK(a,ox) approximately 7.4) in the oxidized MCAD.HD-CoA complex indicate that while binding of the C(6) analogue makes Glu376 a stronger catalytic base (pK(a,ox) approximately 6.5, free MCAD), it has little effect on the pK of Glu99 (pK(a,ox) approximately 7.5, free MCAD) [Mancini-Samuelson, G. J., Kieweg, V., Sabaj, K. M., Ghisla, S., and Stankovich, M. T. (1998) Biochemistry 37, 14605-14612]. This finding is in agreement with the apparent pK of 9.2 determined for Glu376 in the human MCAD.4-thia-octenoyl-CoA complex [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. The pK(a)s estimated for Glu376 and Glu99 in the reduced pig kidney MCAD.HD-CoA complex, 9.8 and 8.6, respectively, suggest that both of these residues remain protonated in the charge-transfer complex under physiological conditions. Polarization of HD-CoA in the enzyme active site may contribute to the observed pK(a) and redox potential shifts. Consequently, the electronic structures of the product analogue in its free and MCAD-bound forms have been characterized by Raman difference spectroscopy. Binding to either the oxidized or reduced enzyme results in localized pi-electron polarization of the hexadienoyl C(1)=O and C(2)=C(3) bonds. The C(4)=C(5) bond, in contrast, is relatively unaffected by binding. These results suggest that, upon binding to MCAD, HD-CoA is selectively polarized such that partial positive charge develops at the C(3)-H region of the ligand, regardless of the oxidation state of the enzyme.     

Activation of substrate/product couples by medium-chain acyl-CoA dehydrogenase

Natural substrate/product binding activates medium-chain acyl-CoA dehydrogenase (MCAD) to accept electrons from its substrate by inducing a positive flavin midpoint potential shift. The energy source for this activation has never been fully elucidated. If ground-state alterations of the ligand, such as polarization, are entirely responsible for enzyme activation, the ligand potential should shift equally to that of the flavin but in the opposite direction. Ligand polarization is likely responsible for only a small portion of this activation. Here, thiophenepropionoyl- and furylpropionoyl-CoA analogs were used to directly measure the redox modulations of several ligand couples upon binding to MCAD. These measurements identified the thermodynamic contribution of ligand polarization to enzyme activation. Because the ligand potential alterations are significantly smaller than modulations in the flavin potential due to binding, other phenomena such as pK(a) changes, desolvation, and charge alterations are likely responsible for the thermodynamic modulations required for MCAD's activity.     

Structure of the transition state analog of medium-chain acyl-CoA dehydrogenase. Crystallographic and molecular orbital studies on the charge-transfer complex of medium-chain acyl-CoA dehydrogenase with 3-thiaoctanoyl-CoA

The flavoenzyme medium-chain acyl-CoA dehydrogenase (MCAD) eliminates the alpha-proton of the substrate analog, 3-thiaoctanoyl-CoA (3S-C8-CoA), to form a charge-transfer complex with deprotonated 3S-C8-CoA. This complex can simulate the metastable reaction intermediate immediately after the alpha-proton elimination of a substrate and before the beta-hydrogen transfer as a hydride, and is therefore regarded as a transition-state analog. The crystalline complex was obtained by co-crystallizing MCAD in the oxidized form with 3S-C8-CoA. The three-dimensional structure of the complex was solved by X-ray crystallography. The deprotonated 3S-C8-CoA was clearly located within the active-site cleft of the enzyme. The arrangement between the flavin ring and deprotonated 3S-C8-CoA is consistent with a charge transfer interaction with the negatively charged acyl-chain of 3S-C8-CoA as an electron donor stacking on the pyrimidine moiety of the flavin ring as an electron acceptor. The structure of the model complex between lumiflavin and the deprotonated ethylthioester of 3-thiabutanoic acid was optimized by molecular orbital calculations. The obtained theoretical structure was essentially the same as that of the corresponding region of the X-ray structure. A considerable amount of negative charge is transferred to the flavin ring system to stabilize the complex by 9.2 kcal/mol. The large stabilization energy by charge transfer probably plays an important role in determining the alignment of the flavin ring with 3S-C8-CoA. The structure of the highest occupied molecular orbital of the complex revealed the electron flow pathway from a substrate to the flavin ring.     

Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.     

Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase

Acyl-CoA oxidase (ACO) catalyzes the first and rate-determining step of the peroxisomal beta-oxidation of fatty acids. The crystal structure of ACO-II, which is one of two forms of rat liver ACO (ACO-I and ACO-II), has been solved and refined to an R-factor of 20.6% at 2.2-A resolution. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into the N-terminal alpha-domain, beta-domain, and C-terminal alpha-domain. The X-ray analysis showed that the overall folding of ACO-II less C-terminal 221 residues is similar to that of medium-chain acyl-CoA dehydrogenase (MCAD). However, the N-terminal alpha- and beta-domains rotate by 13 with respect to the C-terminal alpha-domain compared with those in MCAD to give a long and large crevice that accommodates the cofactor FAD and the substrate acyl-CoA. FAD is bound to the crevice between the beta- and C-terminal domains with its adenosine diphosphate portion interacting extensively with the other subunit of the molecule. The flavin ring of FAD resides at the active site with its si-face attached to the beta-domain, and is surrounded by active-site residues in a mode similar to that found in MCAD. However, the residues have weak interactions with the flavin ring due to the loss of some of the important hydrogen bonds with the flavin ring found in MCAD. The catalytic residue Glu421 in the C-terminal alpha-domain seems to be too far away from the flavin ring to abstract the alpha-proton of the substrate acyl-CoA, suggesting that the C-terminal domain moves to close the active site upon substrate binding. The pyrimidine moiety of flavin is exposed to the solvent and can readily be attacked by molecular oxygen, while that in MCAD is protected from the solvent. The crevice for binding the fatty acyl chain is 28 A long and 6 A wide, large enough to accommodate the C23 acyl chain.     

Heme-linked ionization of horseradish peroxidase compound II monitored by the resonance Raman Fe(IV)=O stretching vibration

Fe(IV)=O resonance Raman stretching vibrations were recently identified by this laboratory for horseradish peroxidase compound II and ferryl myoglobin. In the present report it is shown that Fe(IV)=O stretching frequency for horseradish peroxidase compound II will switch between two values depending on pH, with pKa values corresponding to the previously reported compound II heme-linked ionizations of pKa = 6.9 for isoenzyme A-2 and pKa = 8.5 for isoenzyme C. Similar pH-dependent shifts of the Fe(IV)=O frequency of ferryl myoglobin were not detected above pH 6. The Fe(IV)=O stretching frequencies of compound II of the horseradish peroxidase isoenzymes at pH values above the transition points were at a high value approaching the Fe(IV)=O stretching frequency of ferryl myoglobin. Below the transition points the horseradish peroxidase frequencies were found to be 10 cm-1 lower. Frequencies of the Fe(IV)=O stretching vibrations of horseradish peroxidase compound II for one set of isoenzymes were found to be sensitive to deuterium exchange below the transition point but not above. These results were interpreted to be indicative of an alkaline deprotonation of a distal amino acid group, probably histidine, which is hydrogen bonded to the oxyferryl group below the transition point. Deprotonation of this group at pH values above the pKa disrupts hydrogen bonding, raising the Fe(IV)=O stretching frequency, and is proposed to account for the lowering of compound II reactivity at alkaline pH. The high value of the Fe(IV)=O vibration of compound II above the transition point appears to be identical in frequency to what is believed to be the Fe(IV)=O vibration of compound X.     

Proton nuclear magnetic resonance studies of 8 alpha-N-imidazolylriboflavin in its oxidized and reduced forms

The oxidized and hydroquinone forms of synthetic 8 alpha-N-imidazolylriboflavin have been investigated by proton nuclear magnetic resonance spectroscopy at 360 MHz. Proton resonances due to the imidazole ring, isoalloxazine ring, and ribityl side chain have been assigned on the basis of two-dimensional 1H-1H correlated spectra (COSY), selective decoupling, and nuclear Overhauser effect difference spectra and by comparison of computer-simulated with experimental spectra. The effect of pH on the imidazolyl resonances shows a pKa for the unsubstituted imidazole nitrogen of 6.0 +/- 0.1 for the oxidized form and a value of 7.0 +/- 0.1 for the reduced form, in good agreement with the values obtained from oxidation-reduction potential data in a previous paper [Williamson, G., & Edmondson, D. E. (1985) Biochemistry 24, 7790-7797]. Slow exchange of the flavin 8 alpha-methylene and imidazolyl C(2) protons was observed at pH 6.1 but not at pH values below 4.0 for the oxidized form of the flavin. The reduced form, but not the oxidized form, of the flavin exhibits geminal coupling of the 8 alpha-methylene protons and of the C(1') methylene protons of the ribityl side chain. The magnetic nonequivalence of the protons of these two methylene groups is suggested to result from intermolecular association of the reduced flavin in aqueous solutions at the concentrations required for the spectral experiments.     

Oxidation of 2-thioflavins by peroxides. Formation of flavin 2-S-oxides

The reaction of 2-thioriboflavin (sulfur replacing the oxygen substituent at position 2 alpha) with hydrogen peroxide at pH approximately 10 leads to a blue flavin (lambda max = 565 nm) which was purified in stable, homogeneous form. Titrations of 2-thioflavins with m-chloroperoxybenzoic acid also yield the same blue flavins with consumption of 1 eq of peracid. Anaerobic reduction of the blue flavin by sodium dithionite requires 4e- eq, and leads to formation of 1,5-dihydro-2-thioflavin. Oxidation of the latter with O2 restores the original 2-thioflavin. pH titration of the blue flavin shows two pKa values of 2.4 and 6.6, with no apparent ionization in the pH range 8-11. These results suggest that the blue flavin is a flavin 2-S-oxide. The visible absorption spectra of flavin 2-S-oxides show a pronounced dependence on solvent polarity. This property suggests that these flavin analogs may be useful hydrophilic/hydrophobic probes of flavoprotein active sites. Flavin 2-S-oxides can be oxidized further to the 2-sulfinate and 2-sulfonate analogs, some properties of which are described.     

A 1H and 13C NMR study on the role of salt-bridges in the formation of a type I beta-turn in N-acetyl-L-Asp-L-Glu-L-Lys-L-Ser-NH2

The conformation of the tetrapeptide N-Acetyl-Asp7-Glu8-Lys9-Ser10-NH2, a fragment of the type I collagen alpha-1 chain N-telopeptide, has been studied by 1H and 13C NMR and circular dichroism spectroscopy. The spectroscopic evidence, based on two-dimensional, phase-sensitive NMR techniques such as COSY, ROESY, proton-carbon shift correlation and selective COLOC, indicates a strong dependence of the conformation on the experimental conditions. In CD3OH/H2O (60/40) at ca. neutral pH the tetrapeptide forms a beta-turn, stabilized by a hydrogen bond between NH(S10) and CO(D7) and a strong salt-bridge between COO-(E8) and NH3+(K9). The beta-turn is type I and appears to coexist with a non-hydrogen-bonded structure. The coexistence of these two conformers is proven by proton NMR data such as NH-NH ROEs, reduced NH-H alpha (E8) coupling constant, NH(E8) low-field shift and the temperature coefficient of NH(S10), whereas the conclusion regarding the salt-bridge is based on 13C results. In the same solvent, at a pH below the pKa of the carboxyl groups, no evidence for a conformation other than extended can be found. In aqueous solution at approximately neutral pH, evidence for the E8-K9 charge interaction is observed, but not for a hydrogen bond anywhere in the molecule.     

Nonresonance Raman study of the flavin cofactor and its interactions in the methylotrophic bacterium W3A1 electron-transfer flavoprotein

Nonresonance Raman spectroscopy has been used to investigate the protein-flavin interactions of the oxidized and anionic semiquinone states of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) in solution. Several unique features of oxidized wETF were revealed from the Raman data. The unusually high frequency of the Raman band for the C(4)=O of the flavin suggests that hydrogen-bonding interactions with the C(4)O are very weak or nonexistent in wETF. In contrast, hydrogen bonding with the C(2)=O is one of the strongest among the flavoproteins investigated thus far. According to the crystal structure, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized flavin cofactor in wETF. The replacement of alphaSer254 by cysteine by site-directed mutagenesis resulted in shifts in N(5)-relevant Raman bands in both the oxidized and anionic semiquinone states of the protein. These results confirm the presence of the hydrogen-bonding interaction at N(5) that is evident in the crystal structure of the oxidized protein and that it persists in the one-electron reduced state. The data suggest that these bands can serve as useful Raman markers for the N(5) interactions in both oxidation states of flavoproteins. The wETF displays unusually low frequencies of flavin ring I (o-xylene ring) relevant bands, which suggests a ring I microenvironment different from most of the other flavoproteins. As indicated by Raman data, the alphaS254C mutation changed the environment of ring I, perhaps as the consequence of changes in the mobility of the FAD domain of wETF. These unusual flavin-protein interactions may be associated with the unique redox properties of wETF.     

pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus

Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.     

Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.     

Fate determination of the flavin photoreceptions in the cyanobacterial blue light receptor TePixD (Tll0078)

PixD (Tll0078, Slr1694) is a BLUF (sensor of blue light using FAD)-type blue light receptor protein of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 and the mesophilic cyanobacterium Synechocystis sp. PCC 6803. BLUF protein is known to show light-induced approximately 10 nm red shift of flavin absorption that is coupled with strengthening of the hydrogen bond between the O(4) of the isoalloxazine ring and a certain amino acid residue. According to the 3D structure of TePixD we determined, O(4) of the ring is linked to Gln50 and Asn32. A survey of flavin-interacting residues by site-directed mutagenesis showed that Gln50 but not Asn32 is essential for the normal red-shifting photoreaction. Here, we further studied the role of Gln50 and its close neighbor Tyr8. All the mutated proteins of Gln50 and Tyr8 (Q50A, Q50N, Y8A and Y8F) lost the normal red-shifting photoreaction. Y8A, Y8F and Q50N, instead, showed a light-induced flavin triplet state and a low yield of subsequent flavin reduction that is analogous to the photocycle of the LOV (light-oxygen-voltage-sensing) domain of phototropins, while Q50A did not. Fourier-transform infrared (FT-IR) analysis of N32A showed that O(4) of the ring is hydrogen-bonded to Asn32 both in the light and dark. These results, together with the 3D structure, indicate that the hydrogen bond network of Tyr8-Gln50-O(4)/N(5) (flavin) is critical for the light reaction of the BLUF domain. Based on the structural and functional similarities of the BLUF and the LOV domain of phototropins, we propose that the interaction between apoprotein and N(5) of flavin determines the photoreaction of the flavin-binding sensors.     

Identification of the covalently bound flavin of thiamin dehydrogenase.

Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.     

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.     

Heme-linked proton dissociation of carbon monoxide complexes of myoglobin and peroxidase.

It was found from spectrophotometric titration and proton balance measurement that the pKa value of a heme-linked protonation group of horseradish ferro-peroxidase C (donor:H2O2 oxidoreductase, EC 1.11.1.7) shifted from 7.25 to 8.25 upon combination with CO. The spectrophotometric titration experiment with myoglobin also revealed the presence of a heme-linked protonation group, the pKa value being 5.57 in myoglobin and 5.67 in the CO-myoglobin complex. It was concluded that the distinct shift of the pKa value in the case of peroxidase was attributable to the presence of a hydrogen bond between the sixth ligand and the distal base. The difference in the strength of such hydrogen bonding between peroxidase and myoglobin was discussed.     

Cavitation as a mechanism of substrate discrimination by adenylosuccinate synthetases

Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, coupling L-aspartate and IMP to form adenylosuccinate. Km values of IMP and 2'-deoxy-IMP are nearly identical with each substrate supporting comparable maximal velocities. Nonetheless, the Km value for L-aspartate and the Ki value for hadacidin (a competitive inhibitor with respect to L-aspartate) are 29-57-fold lower in the presence of IMP than in the presence of 2'-deoxy-IMP. Crystal structures of the synthetase ligated with hadacidin, GDP, and either 6-phosphoryl-IMP or 2'-deoxy-6-phosphoryl-IMP are identical except for the presence of a cavity normally occupied by the 2'-hydroxyl group of IMP. In the presence of 6-phosphoryl-IMP and GDP (hadacidin absent), the L-aspartate pocket can retain its fully ligated conformation, forming hydrogen bonds between the 2'-hydroxyl group of IMP and sequence-invariant residues. In the presence of 2'-deoxy-6-phosphoryl-IMP and GDP, however, the L-aspartate pocket is poorly ordered. The absence of the 2'-hydroxyl group of the deoxyribonucleotide may destabilize binding of the ligand to the L-aspartate pocket by disrupting hydrogen bonds that maintain a favorable protein conformation and by the introduction of a cavity into the fully ligated active site. At an approximate energy cost of 2.2 kcal/mol, the unfavorable thermodynamics of cavity formation may be the major factor in destabilizing ligands at the L-aspartate pocket.