Functionalized Graphene Oxide Shields Tooth Dentin from Decalcification.

This in vitro study assessed the efficacy of functionalized graphene oxide (f-GO) nanocomposites on the decalcification of dentin, because dental caries of the root surface is becoming one of the new problems in aged society. Hydroxyapatite plates (HAP) and dentin slices were coated with f-GO nanocomposites by comparing them to silver diamine fluoride as a positive control, then treated with decalcification solutions such as ethylenediaminetetraacetic acid and citrate at 37°C for 24 h. Scanning electron microscopy (SEM) revealed significant protection of the surface morphology of HAP and dentin. On the other hand, a cariogenic Streptococcus mutans growth was inhibited by f-GO nanocomposites. In addition, cytotoxicity of them to epithelial cells was much less than that of povidone-iodine, which is commonly used for oral disinfectant. We synthesized 5 different f-GO nanocomposites such as GO-silver (Ag), GO-Ag-calcium fluoride (CaF2), GO-CaF2, GO-zinc, and GO-tricalcium phosphate (Ca3(PO4)2). They were standardized by evaluating under SEM, transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetry analysis (TGA), and Raman spectra after being synthesized in an aseptic technique. The abilities of GO-Ag, GO-Ag-CaF2, and GO-CaF2 nanocomposites were most preventive for decalcification. In addition, GO-Ag and GO-Ag-CaF2 almost completely inhibited S. mutans growth. However, they did not exhibit cytotoxicity to epithelial cells except at the highest concentration (0.1 w/v%) of GO-Ag and GO-Ag-CaF2. Furthermore, these f-GO nanocomposites exhibited less or no discoloration of dentin, although commonly used silver diamine fluoride causes discoloration of dentin to black. Thus, these f-GO nanocomposites are useful to protect dental caries on the tooth root that becomes a social problem in aged society.

Tuning the redox potential of vitamin K3 derivatives by oxidative functionalization using a Ag(i)/GO catalyst.

We propose herein initial results to develop optimum redox mediators by the combination of computational simulation and catalytic functionalization of the core structure of vitamin K3. We aim to correlate the calculated energy value of the LUMO of different vitamin K3 derivatives with their actual redox potential. For this, we optimized the catalytic alkylation of 1,4-naphthoquinones with a designed Ag(i)/GO catalyst and synthesized a series of molecules.

On the ligands in charge-transfer complexes of porcine kidney flavoenzyme D-amino acid oxidase in three redox states: a resonance Raman study.

To investigate the structural modulation of ligands and their interaction in the active-site nanospace when they form charge-transfer (CT) complexes with D-amino acid oxidase (DAO) in three redox states, we compared Raman bands of the ligands in complex with DAO with those of ligands free in solution. Isotope-labeled ligands were synthesized for assignments of observed bands. The COO(-) stretching of ligands observed around, 1,370 cm(-1) downshifted by about 17 cm(-1) upon complexation with oxidized, semiquinoid and reduced DAO, except for the case of reduced DAO-N-methylisonicotinate complex (8 cm(-1) downward shift); the interaction mode of the carboxylate group with the guanidino group of Arg283 and the hydroxy moiety of Tyr228 of DAO is similar in the three redox states. The C=N stretching mode (1,704 cm(-1)) of Delta(1)-piperideine-2-carboxylate (D1PC) downshifted to 1,675 and 1,681 cm(-1) upon complexation with reduced and semiquinoid DAO, respectively. The downward shifts indicate that the C=N bond is weakened upon the complexation. This is probably due mainly to charge-transfer (CT) interaction between D1PC and semiquinoid or reduced flavin, i.e., the partial electron donation from the highest occupied molecular orbital (HOMO) of reduced flavin or a singly occupied molecular orbital (SOMO) of semiquinoid flavin to the lowest unoccupied molecular orbital (LUMO), an antibonding orbital, of D1PC. This speculation was supported by the finding that the magnitude of the shift is smaller by 5 cm(-1) (observed at 1,680 cm(-1)) in the case of reduced DAO reconstituted with 7,8-Cl(2)-FAD, whose reduced form has lower electron-donating ability than natural reduced FAD. The amount of electron flow was estimated by applying the theory of Friedrich and Person [(1966) J. Chem. Phys. 44, 2166-2170] to these complexes; the amounts of charge transfer from reduced FAD and reduced 7,8-Cl(2)-FAD to D1PC were estimated to be about 10 and 8% of one electron, respectively, in the CT complexes of reduced DAO with D1PC.

Crystallization and preliminary X-ray characterization of rat liver acyl-CoA oxidase.

A recombinant form of the flavoenzyme acyl-CoA oxidase from rat liver has been crystallized by the hanging-drop vapour-diffusion technique using PEG 20 000 as a precipitating agent. The crystals grew as yellow prisms, with unit-cell parameters a = 71.05, b = 87.29, c = 213.05 A, alpha = beta = gamma = 90 degrees. The crystals exhibit the symmetry of space group P2(1)2(1)2(1) and are most likely to contain a dimer in the asymmetric unit, with a V(M) value of 2.21 A(3) Da(-1). The crystals diffract to a resolution of 2.5 A at beamline BL6A of the Photon Factory. Two heavy-atom derivatives have been identified.

Cytoplasmic gamma actin as a Z-disc protein.

To investigate the precise localization of cytoplasmic gamma actin in skeletal muscle and the relationship to dystrophin molecules, we designed an antibody against the N-terminal peptide of cytoplasmic gamma actin. Western blot analysis using SDS-PAGE and isoelectric focusing (IEF) gel revealed that the antibody reacted only with the actin isoforms having gamma motility, confirming that the antibody is specific to the cytoplasmic (nonmuscle) gamma actin. Immunohistochemical analysis of the skeletal muscle of the adult mouse revealed a dot-like staining pattern of the antibody in transverse sections and a striated staining pattern in longitudinal sections. The double immunostaining technique revealed the colocalization of cytoplasmic gamma actin with alpha-actinin, implying the localization of the actin on the Z-disc. Contrary to previous findings (1), we did not detect the colocalization of cytochrome oxidase, a mitochondria marker, with this actin.

Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1.

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K(m) values for NADH and FMN were 208 and 10.8 microM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35 degrees C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80 degrees C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705-1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.

Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X.

We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation.

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.

Three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase. Optimization of the oxidative half-reaction through alignment of the product with reduced flavin.

The three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase (DAO) was solved by cryo-X-ray crystallography; the purple intermediate is known to comprise a complex between the dehydrogenated product, an imino acid, and the reduced form of DAO. The crystalline purple intermediate was obtained by anaerobically soaking crystals of oxidized DAO in a buffer containing excess D-proline as the substrate. The dehydrogenated product, delta(1)-pyrrolidine-2-carboxylate (DPC), is found sandwiched between the phenol ring of Tyr 224 and the planar reduced flavin ring. The cationic protonated imino nitrogen is within hydrogen-bonding distance of the backbone carbonyl oxygen of Gly 313. The carboxyl group of DPC is recognized by the Arg 283 guanidino and Tyr 228 hydroxyl groups through ion-pairing and hydrogen-bonding, respectively. The (+)HN=C double bond of DPC overlaps the N(5)-C(4a) bond of reduced flavin. The electrostatic effect of the cationic nitrogen of DPC is suggested to shift the resonance hybridization of anionic reduced flavin toward a canonical form with a negative charge at C(4a), thereby augmenting the electron density at C(4a), from which electrons are transferred to molecular oxygen during reoxidation of reduced flavin. The reactivity of reduced flavin in the purple intermediate, therefore, is enhanced through the alignment of DPC with respect to reduced flavin.

Molecular characterization of a mouse heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its tissue-specific expression.

The human DNA- and RNA-binding protein JKTBP is a new member of heterogeneous nuclear ribonucleoproteins (hnRNPs) that are involved in mRNA biogenesis. We cloned and characterized a mouse homolog and studied its expression in mouse tissues. The cDNA encoded a 301-residue polypeptide. There is only a single amino acid difference between the mouse and human sequences. Northern blotting indicated ubiquitous but varied expressions of approximately 1.4 and 2.8kb mRNAs in various tissues. Immunoblotting indicated that the amounts of protein of about 38kDa were higher in the brain and testis than in other tissues. An additional protein of about 53kDa was found in the brain and testis. Germ cell-deficient W/W(v) mutant mice and aged mice had the reduced amounts of JKTBP in the testes. Immunohistochemical staining indicated cell type-specific expression of JKTBP in tissues: neurons and spermatocytes displayed strong signal intensities. The signals were confined to the nucleus. The amount of 38kDa JKTBP was estimated to be approximately 1.3x10(7) molecules per HL-60 cell. These results indicate that JKTBP is an abundant, highly conserved nuclear protein.

Unusually high standard redox potential of acrylyl-CoA/propionyl-CoA couple among enoyl-CoA/acyl-CoA couples: a reason for the distinct metabolic pathway of propionyl-CoA from longer acyl-CoAs.

The standard redox potential of acrylyl-CoA/propionyl-CoA couple (C(3)) was determined to be 69 mV (vs. standard hydrogen electrode) at pH 7 and 25 degrees C. This value implies that the 2, 3-dehydrogenation of propionyl-CoA is thermodynamically much more unfavorable than that of longer acyl-CoAs because the standard redox potentials of crotonyl-CoA/butyryl-CoA (C(4)), octenoyl-CoA/octanoyl-CoA (C(8)), and hexadecenoyl-CoA/palmitoyl-CoA (C(16)) are all about -10 mV. The unusually high standard redox potential of the acrylyl-CoA/propionyl-CoA couple is thought to be one of the reasons that in mammals propionyl-CoA is not metabolized by beta-oxidation as in the case of longer acyl-CoAs, but by a methylmalonyl-CoA pathway. The obvious structural difference between C(3) and C(4) (and longer) is whether an H or the C(4) atom is connected to -C(3)H=C(2)H-C(1)O-S-CoA. The molecular orbital calculations (MOPAC) for the enoyl and acyl forms of C(3) and C(4) revealed that this structural feature is the main cause for the higher standard redox potential of the C(3) couple. That is, the C(4)-C(3) bond is stabilized by the dehydrogenation to a greater degree than the H-C(3) bond.

Identification of a novel Sry-related gene and its germ cell-specific expression.

Sox family proteins are characterized by a unique DNA-binding domain, a HMG box which shows at least 50% sequence similarity with mouse Sry, the sex-determining factor. At present almost 30 Sox genes have been identified. Members of this family have been shown to be conserved during evolution and to play key roles during animal development. Some are involved in human diseases, including sex reversal. Here we report the isolation of a novel member of the Sox gene family, Sox30, which may constitute a distinct subgroup of this family. Using a bacterially expressed DNA-binding domain of Sox30, we show that it is able to specifically recognize the ACAAT motif. Furthermore, Sox30 is capable of activating transcription from a synthetic promoter containing the ACAAT motif. The specific expression of Sox30 in normal testes, but not in maturing germ cell-deficient testes, suggests the involvement of Sox30 in differentiation of male germ cells. Mapping analyses revealed that the Sox30 gene is located on human chromosome 5 (5q33) and on mouse chromosome 11.

Mechanism for the recognition and activation of substrate in medium-chain acyl-CoA dehydrogenase.

The mechanism underlying the recognition and activation of the substrate for medium-chain acyl-CoA dehydrogenase (MCAD) was spectroscopically investigated using 3-thiaacyl-CoAs as substrate analogs. The complex of MCAD with 3-thiaoctanoyl-CoA (3-thia-C8-CoA) exhibited a charge-transfer (CT) band with a molar extinction coefficient of epsilon808 = 9.1 mM-1.cm-1. With increasing 3-thiaacyl-chain length, the CT-band intensity of the complex decreased concomitantly with changes in the FAD absorption at 416 and 482 nm, and no CT band was detected in complexes with chain-lengths longer than C15. Detailed analysis of the absorption spectra suggested that the complexed states represent a two-state equilibrium between the CT-inducing form and the CT-non-inducing form. 13C-NMR measurements with 13C-labeled ligand clarified that 3-thia-C8-CoA is complexed to MCAD in an anionic form with signals detected at 163.7 and 101.2 ppm for 13C(1) and 13C(2), respectively. In the MCAD complex with 13C(1)-labeled 3-thia-C12-CoA, two signals for the bound ligand were observed at 163.7 and 198.3 ppm, and assigned to the anionic and neutral forms, respectively. Only the neutral form signal was measured at 200.6 ppm in the complex with 13C(1)-labeled 3-thia-C17-CoA. These results indicate that the CT band can be explained in terms of an internal equilibrium between anionic (CT-inducing) and neutral (CT-non-inducing) forms of the bound ligand. Resonance Raman spectra of the MCAD.3-thia-C8-CoA complex, with excitation at the CT band, showed enhanced bands, among which the 854- and 1,368-cm-1 bands were assigned to the S-C(2) stretching mode of the ligand and to flavin band VII, respectively. Since the enhanced bands were observed at the same wave numbers in complexes with C8, C12, and C14-ligands, it appears that the CT-inducing form shares a common alignment relative to oxidized flavin irrespective of differences in the acyl-chain length. However, with longer ligands, the degree of resonance enhancement of the Raman bands decreased in parallel with the CT-band intensity; this is compatible with the increase in the CT-non-inducing form in complexes with longer ligands. Furthermore, the pH dependence of the CT band gave an apparent pKa = 5.6-5.7 for ligands with chain-lengths of C8-C12. The NMR measurements revealed that, like chain-length dependence, the pH dependence can be explained by a two-state equilibrium derived from the protonation/deprotonation of the CT-inducing form of the bound ligand. On the basis of these results we have established a novel model to explain the mechanism of recognition and activation of the substrates/ligands by MCAD.

Resonance Raman study on reduced flavin in purple intermediate of flavoenzyme: use of [4-carbonyl-18O]-enriched flavin.

4-Carbonyl-18O]-enriched lumiflavin, riboflavin, and FMN were prepared by incubating each corresponding non-labeled flavin in 1 M Na18OH (H218O) at 25 degrees C. [4-Carbonyl-18O]FAD was prepared from the corresponding riboflavin by using FAD synthetase. Isotope effects by [4-carbonyl-18O]-labeling confirmed that the 1,709-cm-1 band in the IR spectrum of lumiflavin and the 1,711-cm-1 band in the Raman spectrum of FAD are mainly derived from C(4)=O stretching vibrational mode. The 1,605-cm-1 Raman band of the anionic reduced flavin in the purple intermediate of D-amino acid oxidase (DAO) with D-proline or D-alanine does not shift in DAO reconstituted with [4-carbonyl-18O]FAD, although it shifts with [4,10a-13C2]- or [4a-13C]FAD. Thus the band is mainly due to the C(4a)=C(10a) stretching vibrational mode and includes no contribution from C(4)=O stretching vibration. The band frequencies cover a fairly wide range (1,602-1,620 cm-1) depending on the enzymes. The frequencies of the reduced flavin in the purple intermediates of the dehydrogenases (medium-chain acyl-CoA, short-chain acyl-CoA, and isovaleryl-CoA dehydrogenases) are higher than those of the oxidases (DAO and L-phenylalanine oxidase). This indicates that the C(4a)=C(10a) bond order of reduced flavin in the dehydrogenases with the low reactivity for molecular oxygen is stronger than that in the oxidases with high reactivity. Therefore, the band frequency of C(4a)=C(10a) stretching may serve as an indicator of the reactivity of flavoprotein with molecular oxygen. Furthermore, strong hydrogen bonding of flavin at the N(1) moiety with the hydroxyl group of Thr136 in MCAD is probably responsible for the strong bond of the C(4a)=C(10a) of reduced flavin in the dehydrogenase.

Molecular cloning and characterization of meichroacidin (male meiotic metaphase chromosome-associated acidic protein).

We have isolated a cDNA clone encoding a germ cell specific protein from an expression cDNA library prepared from the mouse testis, using testis-specific polyclonal antibodies. Sequence analysis of the cDNA revealed that the deduced amino acid sequence consisted of 284 residues, including a nominal repeat structure in the N-terminal region. Northern blot analysis revealed the presence of a transcript of 1.3 kb exclusively expressed in the testis and ovary, but at relatively low levels in the ovary. In contrast, no other tissues and organs expressed significant levels of the transcript. Expression of the mRNA in the testis was first detected on day 14 in postnatal development. Western blot analysis showed the presence of the protein with a molecular weight of approximately 40 kDa and an isoelectric point of 4.9. The protein was exclusively found in the testis and ovary, but in a far lesser amount in the ovary as was the case with the transcript. Immunohistochemical examination revealed that the protein was predominantly present in the cytoplasm in pachytene spermatocytes through to round spermatids. However, during the disappearance of the nuclear envelope at both the first and second meiotic divisions, the protein was localized around the metaphase chromosomes and spindles. Because of this, the name meichroacidin which stands for male meiotic metaphase chromosome-associated acidic protein is proposed for this antigen. The highly regulated stage-specific expression of meichroacidin and its specific association with the metaphase chromosomes and spindles suggest that the protein plays important roles in male meiosis.

Structural and mechanistic studies on D-amino acid oxidase x substrate complex: implications of the crystal structure of enzyme x substrate analog complex.

As an extension of our recent X-ray crystallographic determination of the tertiary structure of D-amino acid oxidase (DAO) [Mizutani, H. et al. (1996) J. Biochem. 120, 14-17], we solved the crystal structure of the complex of DAO with a substrate analog, o-aminobenzoate (OAB). The alignment between flavin and OAB in the crystal structure of the complex is consistent with charge-transfer interaction through the overlap between the highest occupied molecular orbital of OAB and the lowest unoccupied molecular orbital of flavin. Starting with the atomic coordinates of this complex as the initial model, we carried out molecular mechanics simulation for the DAO-D-leucine complex and thus obtained a model for the enzyme-substrate complex. According to the enzyme-substrate complex model, the alpha-proton is pointed toward N(5) of flavin while the lone-pair of the substrate amino group can approach C(4a) of flavin within an interacting distance. This model as well as DAO-OAB complex enables the evaluation of the substrate-flavin interaction prior to electron transfer from the substrate to flavin and provides two possible mechanisms for the reductive-half reaction of DAO, i.e., the electron-proton-electron transfer mechanism and the ionic mechanism.

Mapping of eight testis-specific genes to mouse chromosomes.

We previously identified eight testis-specific genes using antibodies raised against testicular germ cells. They are expressed during spermatogenesis and are presumed to be involved in testicular germ cell differentiation and sperm formation. We have mapped the genomic loci for these testis-specific genes using restriction fragment length variants in interspecific backcross mice. The calmegin gene (Clgn) was mapped to Chr 8. The synaptonemal complex protein gene 1 (Sycp1) probe hybridized with two sequences on different chromosomes; Sycp1-rs2 was mapped to Chr 3, whereas Sycp1-rs3 was mapped to Chr 7. The relaxin-like factor gene (Rlnl) was mapped to Chr 8, and collapsin response mediator protein 1 (Crmp1) was mapped to Chr 5. Three novel genes encoding testis-specific proteins A2 (Tsga2), A8 (Tsga8), and A12 (Tsga12) were mapped to chromosomes 3, X, and 10, respectively.

Spectroscopic studies of rat liver acyl-CoA oxidase with reference to recognition and activation of substrate.

Two forms of rat peroxisomal acyl-CoA oxidase (ACO-I and -II) interact with the substrate analogs, 3-ketoacyl-CoAs, forming a complex characterized by the so-called charge-transfer (CT) band around 575 nm in the absorption spectra. The CT band of ACO-I exhibited a broad dependency on the acyl chain-length from C4 to C16, whereas that of ACO-II showed increased intensity with a longer acyl chain to reach a maximum with a chain-length of C12. These chain-length dependencies of the CT bands were compared with those of the enzymatic activities reported previously [Setoyama et al. (1995) Biochem. Biophys. Res. Commun. 217, 482-487]. The differences in spectroscopic and enzymatic properties between ACO-I and -II suggest that the amino acid stretch corresponding to the third exon in the ACO sequence affects the binding of the ligand and substrate, since the difference in the primary structure between ACO-I and -II lies in the short amino acid stretch corresponding to the third of the total of 14 exons. On the other hand, resonance Raman spectra of the complexes of ACO-I and -II with 3-ketoacyl-CoAs excited in the CT band showed similar features. The two prominent FAD bands II and III, associated with the C(4a)=N(5) moiety of FAD, were observed at 1,577 and 1,545 cm(-1), respectively. In contrast, the bands at 1,615 and 1,493 cm(-1) in the ACO-I x 3-keto-C8-CoA complex were assigned to the stretching modes of C=O at positions 3 and 1 of the ligand, respectively, by using the isotopically labeled ligands. Both C=O stretching bands were shifted to lower wave numbers upon complex formation with ACO-I, implying that the C=O bond involves the single bond (C-O-) character in the active site cavity. The downshift of the C(1)=O stretching band was larger than that of the C(3)=O stretching band. Therefore, the ligand lies in the active site as the anionic form with a major contribution from C(1)-O-. These observations demonstrate that the CT band around 575 nm arises from the charge-transfer interaction between the oxidized FAD and the enolate transformed after the elimination of the a-proton. The band II of FAD in the complexes reveals a significant decrease in the frequency in comparison with the complexes of medium-chain acyl-CoA dehydrogenase (MCAD) with 3-ketoacyl-CoA. This observation suggests a difference between ACO and MCAD in the hydrogen-bonding network associated with enzyme-bound FAD.

A Raman study on the C(4)=O stretching mode of flavins in flavoenzymes: hydrogen bonding at the C(4)=O moiety.

Raman spectroscopy was used to investigate the hydrogen bonding at the C(4)=O moiety of the isoalloxazine nucleus in a series of flavins and flavoproteins. Isotope effects of Raman bands confirmed that the band observed around 1,710 cm(-1) is mainly derived from C(4)=O stretching vibrational mode. A linear correlation was observed between the frequency of C(4)=O stretching and the chemical shift of 13C(4), suggesting that the data from both Raman and NMR spectroscopies reflect a common perturbation, i.e., hydrogen bonding. The maximum difference of C(4)=O frequency among flavins and flavoproteins examined is 36 cm(-1) [1,723 cm(-1) for riboflavin-binding protein (Kim, M. and Carey, P.C. (1993) J. Am. Chem. Soc. 115, 7015-7016) and 1,687 cm(-1) for the complex of medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA]; the maximum difference of 40-70 kJ/mol in the hydrogen bonding strength at the C(4)=O exists among flavoproteins. By use of an empirical linear correlation between the frequency of C=O stretching and the bond length of the C=O, it is estimated that the maximum difference in the bond length among flavoproteins treated here is ca. 0.017 A. The hydrogen bonding at the C(4)=O in medium-chain and short-chain acyl-CoA dehydrogenases becomes stronger upon complexation with substrate analogs. Since the hydrogen bonding at the C(4)=O is expected to enhance the electron-accepting capacity of the N(5) position, substrate-binding itself probably raises the reactivity of flavin, through enhancing the hydrogen bonding.

In vitro assembly of FAD, AMP, and the two subunits of electron-transferring flavoprotein: an important role of AMP related with the conformational change of the apoprotein.

Electron-transferring flavoprotein from pig kidney is composed of four non-covalently bound components: alpha and beta subunits, flavin adenine dinucleotide (FAD), and adenosine monophosphate (AMP). This paper reveals the pathway of assembly of the electron-transferring flavoprotein. The holoprotein can be formed by two different pathways. (i) alpha + beta <==> (alpha-beta)*, (alpha-beta)* + AMP <==> (alpha-beta-AMP)*, (alpha-beta-AMP)* <==> alpha-beta-AMP, alpha-beta-AMP + FAD <==> holoprotein. (ii) alpha + beta <==> alpha-beta, alpha-beta + FAD <==> alpha-beta-FAD, alpha-beta-FAD + AMP <==> holoprotein. Here the presence and absence of asterisks distinguish different conformations with the same composition. The monomeric forms of alpha and beta showed no significant binding with FAD and AMP. AMP and FAD associated with different heterodimer forms which were formed as a result of weak binding between alpha and beta. The binding of alpha + beta + AMP was much faster than that of alpha + beta + FAD because the rate of alpha + beta --> (alpha-beta)* was much faster than that of alpha + beta --> alpha-beta. The alpha-beta-AMP complex associated with FAD rapidly. As a result, the binding of FAD with the subunits is promoted by AMP. The alpha-beta-FAD complex associated with AMP much more slowly than the mixture of alpha and beta. Thus the AMP binding with the subunits is inhibited by the preceding FAD binding.