Form Of Oxidase Research Articles

Resonance Raman and FTIR Studies of Carbon Monoxide-Bound Cytochrome aa3-600 Oxidase of Bacillus subtilis

Resonance Raman and FTIR spectra are reported for the fully reduced carbon monoxy derivative of the quinol aa3-600 oxidase from Bacillus subtilis. The resonance Raman spectra display two isotope-dependent vibrational modes at 520 and 575 cm-1. The FTIR spectrum displays a single vibrational mode at 1963 cm-1. We assign the band at 520 cm-1 to the Fe−CO stretching mode, the band at 575 cm-1 to the Fe−C−O bending mode, and the band at 1963 cm-1 to the C−O stretching mode. The frequencies of these modes are similar to those that have been reported for the CO-bound mammalian cytochrome c oxidase. Despite the fact that two different heme−protein conformations that affect the iron−his bond strength are present in the ferrous ligand-free form of aa3-600, the CO-bound adduct has a single conformation in which the His−Fe−CO CuB moiety has the same structure as the α form found in the mammalian cytochrome c oxidase. The present and previous data on the vibrational frequencies of ferrous ligand-free and ferrous CO-bound forms of terminal oxidases show that an inverse linear relationship exists between the frequencies of the Fe−his and Fe−CO stretching modes. We suggest that the frequencies of both the Fe−CO and C−O modes found in heme−CuB oxidases are affected by the proximal His376, which is H-bonded to the peptide carbonyl of Gly351, and by distal effects on the heme a3-bound CO exerted by CuB.

Förster energy transfer from tryptophan to flavin in glucose oxidase enzyme

The difference in the properties of the tryptophan fluorescence of the hologlucose oxidase forms, as compared to those of the apoprotein, can unambiguously be interpreted as due to a Förster energy transfer from the tryptophan residues to the flavinic group. Indeed, the atomic absorption of a glucose oxidase solution shows that the enzyme is not associated to any metals which could be responsible for the tryptophan fluorescence quenching effect. The data show that only 7 Trp residues are partially coupled to the flavin group and that the strength of this coupling is dependent on the flavin redox state.

Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase.

Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism. A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide. Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction. The pH dependence of Vmax/Km(O2) reveals pKa's of 6.2 +/- 0.3 and 7.0 +/- 0.2, respectively. A pKa of 7.2 +/- 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV-vis spectrophotometry. The near identity of the pKa obtained from the reduced enzyme titration with the second pKa from steady-state kinetics suggests that this second pKa arises from the reduced cofactor. The assignment of pKa is supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions. To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined. The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step. Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step. The O-18 kinetic isotope effect is independent of pH with an average value of 18(V/K) = 1.0097 +/- 0. 0010. This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species [Tian and Klinman (1993) J. Am. Chem. Soc. 115, 8891], leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)-OOH complex could be the rate-limiting step. The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis of DV and D(V/K) for benzylamine oxidation. We conclude that the major species accumulating in the steady state are the oxidized cofactor-substrate Schiff base complex and the reduced, aminoquinol form of cofactor. These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction. This step is also estimated to be 40% rate-limiting in kcat. An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide. On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase from Hansenula polymorpha [Li et al. (1998) Structure 6, 293], a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site. Movement of superoxide from this site onto the Cu(II) at the active site may occur prior to further electron transfer from cofactor to superoxide.

Defense Mechanism of Rice Plant to Respiratory Inhibition by a Promising Candidate Blasticide, SSF126

A new candidate systemic rice blasticide, SSF126, dose-dependently inhibited NADH oxidation by submitochondrial particles from rice roots. However, oxidation by the root submitochondrial particles was much less susceptible to SSF126 compared to that by submitochondrial particles from mycelial cells ofPyricularia grisea, a pathogen causing rice blast. Interestingly, SSF126 did not completely suppress the respiration by intact rice roots, and the respiratory activity of the roots recovered from inhibition time-dependently even in the presence of SSF126 at concentrations sufficient to fully block oxidation by the submitochondrial particles. This recovery was not due to selective extrusion of SSF126 from the roots, but to switching from the cytochrome pathway to the alternative cyanide-resistant respiratory pathway. In immunoblots of the alternative oxidase, high molecular mass species were detected in the mitochondria from rice roots in addition to low molecular mass species. Quantification of high and low molecular mass species revealed an increase in the amount of a protein corresponding to a 36-kDa species equivalent to a decrease in the amount of a protein corresponding to a 72-kDa species following a 5-h incubation with SSF126. This conversion of the alternative oxidase to the low molecular mass species in the mitochondria was correlated with the respiratory recovery found in intact rice roots, suggesting that the low molecular mass species is the active form of the alternative oxidase and the high molecular mass species is the inactive form. These results suggest that rice plants can block the severe injury caused by limiting the cytochrome pathway by SSF126 through utilization of the alternative pathway promoted by the interconversion of the alternative oxidase protein.

Xanthine oxidase activity associated with arterial blood pressure in spontaneously hypertensive rats.

Recent evidence in vivo indicates that spontaneously hypertensive rats (SHR) exhibit an increase in oxyradical production in and around microvascular endothelium. This study is aimed to examine whether xanthine oxidase plays a role in overproduction of oxidants and thereby may contribute to hypertensive states as a consequence of the increasing microvascular tone. The xanthine oxidase activity in SHR was inhibited by dietary supplement of tungsten (0.7 g/kg) that depletes molybdenum as a cofactor for the enzyme activity as well as by administration of (-)BOF4272 [(-)-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo(1,5-alpha)-1,3, 5-triazine-4-monohydrate], a synthetic inhibitor of the enzyme. The characteristic elevation of mean arterial pressure in SHR was normalized by the tungsten diet, whereas Wistar Koto (WKY) rats displayed no significant alteration in the pressure. Multifunctional intravital videomicroscopy in mesentery microvessels with hydroethidine, an oxidant-sensitive fluoroprobe, showed that SHR endothelium exhibited overproduction of oxyradicals that coincided with the elevated arteriolar tone as compared with WKY rats. The tungsten diet significantly repressed these changes toward the levels observed in WKY rats. The activity of oxyradical-producing form of xanthine oxidase in the mesenteric tissue of SHR was approximately 3-fold greater than that of WKY rats, and pretreatment with the tungsten diet eliminated detectable levels of the enzyme activity. The inhibitory effects of the tungsten diet on the increasing blood pressure and arteriolar tone in SHR were also reproducible by administration of (-)BOF4272. These results suggest that xanthine oxidase accounts for a putative source of oxyradical generation that is associated with an increasing arteriolar tone in this form of hypertension.

Why Does the Active Form of Galactose Oxidase Possess a Diamagnetic Ground State?

The relative orientation of the two magnetic orbitals, the CuII d x 2-y 2 orbital and the half-occupied π orbital of the tyrosyl radical, is the key to answering the question in the title. The arrangement shown (CuII -O-C bond angle of about 130° and a dihedral angle of about 90° between the x,y plane of the CuII polyhedron and the tyrosyl ring plane) leads to an overlap of the orbitals, which results in a singlet ground state.

Why Does the Active Form of Galactose Oxidase Possess a Diamagnetic Ground State?

Why Does the Active Form of Galactose Oxidase Possess a Diamagnetic Ground State?

Saguenay Lac Saint Jean cytochrome oxidase deficiency: sequence analysis of nuclear encoded COX subunits, chromosomal localization and a sequence anomaly in subunit VIc

A biochemically distinct form of cytochrome oxidase (COX) deficiency found in the Saguenay region of Quebec is an autosomal recessive trait. The cDNA sequences of all 10 nuclear-encoded subunits from a patient's fibroblasts showed normal coding sequence. Sequences for subunit VIc in two atypical patients showed a heterozygous base substitution. Subunit VIc was localized to chromosome 18.

Dynamic and structural properties of glucose oxidase enzyme.

The catalytic oxidation of beta-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I'band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH2 does not change the amount of alpha-helix present in the protein outer shell, but reorganizes a fraction of random coil to beta-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism.

A three-coordinate copper(i)-phenoxide complex that models the reduced form of galactose oxidase

A novel 3-coordinate Cu(I)-phenoxide complex with N-donor supporting ligation was structurally characterized and shown to be highly reactive towards dioxygen, similar to the reduced form of galactose oxidase.

The oxidation of dopamine and epinine by the two forms of monoamine oxidase from rat liver.

Information on the "in vitro" oxidation of epinine by monoamine oxidase (MAO) compared to dopamine is very poor. The aim of this work was to study the oxidative deamination of epinine and dopamine by rat liver MAO-A and MAO-B. The contributions of MAO-A and B to the metabolism of dopamine (55% and 45%, respectively) and epinine (70% and 30%, respectively) were similar. The results of this study show that epinine is a substrate for both forms of MAO in rat liver, although the contribution of MAO A to the deamination of this secondary amine appears to be slightly more important than that of MAO B.

Increased xanthine oxidase during labour—implications for oxidative stress

Xanthine dehydrogenase/oxidase (XDH/XO) produces uric acid. When in the oxidase form, this production is coupled with the generation of free radicals. Hypoxia-reperfusion enhances conversion of XDH to XO. Since the placenta is exposed to short periods of hypoxia reperfusion during labour, 17 placentae of pregnancy terminated by elective caesarean section and five placentae of pregnancies terminated by caesarean section during labour were examined for XDH/XO activity. It was found that XO activity was higher in the placentae of labouring women ( P=0.003), which suggests that labour enhances conversion of XDH to XO, facilitating free radical production.

The Conversion from the Dehydrogenase Type to the Oxidase Type of Rat Liver Xanthine Dehydrogenase by Modification of Cysteine Residues with Fluorodinitrobenzene

When rat liver xanthine dehydrogenase was incubated with fluorodinitrobenzene (FDNB) at pH 8.5, the total enzyme activity decreased gradually to a limited value of initial activity with modification of two lysine residues in a similar way to the modification of bovine milk xanthine oxidase with FDNB (Nishino, T., Tsushima, K., Hille, R. and Massey, V. (1982) J. Biol. Chem. 257, 7348-7353). After modification with FDNB, the two peptides containing dinitrophenyl-lysine were isolated from the molybdopterin domain after proteolytic digestion and were identified as Lys754 and Lys771 by sequencing the peptides. During the modification of these lysine residues, xanthine dehydrogenase was found to be converted to an oxidase form in the early stage of incubation. Incorporation of the 3H-dinitrophenyl group into enzyme cysteine residues was 0.96 mol per enzyme FAD for 68% conversion to the oxidase form. The modified enzyme was reconverted to the dehydrogenase form by incubation with dithiothreitol with concomitant release of 3H-dinitrophenyl compounds. After modification with 3H-FDNB followed by carboxymethylation under denaturating conditions, the enzyme was digested with proteases. Three 3H-dinitrophenyl-labeled peptides were isolated and sequenced. The modified residues were identified to be Cys535, Cys992 and Cys1324. These residues are conserved among the all known mammalian enzymes, but Cys992 and Cys1324 are not conserved in the chicken enzyme. Cys1324 of the rat enzyme was found not to be involved in the conversion from the dehydrogenase to the oxidase by limited proteolysis experiments, but Cys535 and Cys992 which seemed to be modified alternatively with FDNB appear to be involved in the conversion.

Synthetic Models of the Inactive Copper(II)−Tyrosinate and Active Copper(II)−Tyrosyl Radical Forms of Galactose and Glyoxal Oxidases

A series of CuII and ZnII complexes with new ligands having either one or two substituted phenolates appended to the 1,4,7-triazacyclononane frame were prepared and characterized by optical absorption, EPR, NMR, and/or resonance Raman spectroscopy, cyclic voltammetry, and, in eight cases, X-ray crystallography. Features of the active site geometries of the CuII−tyrosinate forms of galactose and glyoxal oxidases (GAO and GLO) were modeled by these complexes, including the binding of a redox-active phenolate and an exogenous ligand (Cl-, CH3CO2-, or CH3CN) in a cis-equatorial position of a square pyramidal metal ion. The role of the unique ortho S−C covalent bond between a cysteine (C228) and the equatorial tyrosinate (Y272) in the proteins was probed through an examination of the optical absorption and electrochemical properties of sets of similar complexes comprised of phenolate ligands with differing ortho substituents, including thioether groups. The o-alkylthio unit influences the PhO- → CuII LMCT transition and the MII−phenolate/MII−phenoxyl radical redox potential, but to a relatively small degree. Electrochemical and chemical one-electron oxidations of the CuII and ZnII complexes of ligands having tert-butyl protecting groups on the phenolates yielded new species that were identified as novel MII−phenoxyl radical compounds analogous to the active CuII−tyrosyl radical forms of GAO and GLO. The MII−phenoxyl radical species were characterized by optical absorption, EPR, and resonance Raman spectroscopy, as well as by their stoichiometry of formation and chemical reduction. Notable features of the CuII−phenoxyl radical compounds that are similar to their protein counterparts include EPR silence indicative of magnetic coupling between the CuII ion and the bound radical, a band with λmax ≈ 410 nm (ε ≈ 3900 M-1 cm-1) in UV−vis spectra diagnostic for the phenoxyl radical, and a feature attributable to the phenoxyl radical C−O vibration (ν7a) in resonance Raman spectra. Similar Raman spectra and electrochemical behavior for the ZnII analogs, as well as an isotropic signal at g = 2.00 in their X-band EPR spectra, further corroborate the formulations of the MII−phenoxyl radical species.

Kinetic Isotope Effects and Electron Transfer in the Reduction of Xanthine Oxidoreductase with 4-Hydroxypyrimidine: A COMPARISON BETWEEN OXIDASE AND DEHYDROGENASE FORMS

Isolated from bovine milk, xanthine oxidase (XO) and xanthine dehydrogenase (XDH) are two interconvertible forms of the same protein, differing in the number of protein cysteines versus cystines. Most differences between XO and XDH are localized to the FAD center, the site at which the oxidizing substrates NAD and molecular oxygen react. A comparative study of the reduction of XO and XDH has been performed to assess differences in reactivity of the molybdopterin site, as well as subsequent electron-transfer events from molybdenum to 2Fe/2S and FAD centers. The compound 4-hydroxypyrimidine (4-OH-P) was chosen as reducing substrate because its higher Km value raised the possibility of binding weak enough to measure kinetically, and its high kcat value could allow detection of intramolecular electron-transfer reactions. As measured by stopped flow spectrophotometry, XO and XDH react with the first equivalent of 4-OH-P via similar mechanisms, differing in the magnitude of rate and dissociation constants. Using [2-2H]4-OH-P as substrate, a D(k/Kd) isotope effect of 1.9 to 2.3 suggests that movement of the hydrogen abstracted from substrate appreciably limits the rate of initial enzyme reduction from Mo(VI) to Mo(IV). Monitoring the visible spectrum of the enzymes, the first observed step is reduction of a single 2Fe/2S center and presumably re-oxidation of Mo(IV) to Mo(V). This suggests a common pathway for electron transfer involving reduction of a 2Fe/2S center prior to reduction of the second 2Fe/2S and FAD centers. Rates of the first electron transfer from molybdenum to the 2Fe/2S center are rapid, 290 s-1 with XO and 180 s-1 with XDH, and are consistent with rates measured by flash photolysis (Walker, M. C., Hazzard, J. T., Tollin, G., and Edmondson, D. E. (1991) Biochemistry 30, 5912-5917) allowing discrete observation of the electron-transfer reactions that occur during turnover. This step also exhibits a modest primary kinetic isotope effect of 1.5 to 1.6 when [2-2H]4-OH-P is used, possibly due to deprotonation of the molybdenum center prior to electron transfer. A second one-electron transfer, presumably oxidizing Mo(V) to Mo(VI), follows in a step coincident with product dissociation, consistent with a role for product release in controlling electron transfer events. The kinetics of this complex system are described and interpreted quantitatively in models that are consistent with all the data.

Effect of Metal on 2,4,5-Trihydroxyphenylalanine (Topa) Quinone Biogenesis in the Hansenula polymorpha Copper Amine Oxidase

Previous studies of wild-type and mutant forms of a recombinant copper amine oxidase from Hansenula polymorpha, expressed in Saccharomyces cerevisiae, have indicated a self-processing mechanism for 2,4,5-trihydroxyphenylalanine (topa) quinone biogenesis involving the active site copper (Cai, D., and Klinman, J. P. (1994) J. Biol. Chem. 269, 32039-32042). In contrast to prokaryotic copper amine oxidases, however, it has not been possible to initiate topa quinone formation by the addition of exogenous copper to precursor H. polymorpha amine oxidase lacking copper. Metal analysis of copper-depleted wild-type enzyme reveals 0.2-0.3 mol copper, together with 0.6 mol zinc. Despite changes in the zinc and copper levels in growth media, the level of zinc in purified enzyme remains fairly constant. Further, we have been unable to displace protein-bound zinc by exogenously added copper. The H. polymorpha amine oxidase gene was subsequently expressed in Escherichia coli and found to be almost completely free of copper and zinc. In vitro reconstitution of this apoprotein confirms that zinc binds to H. polymorpha amine oxidase and prevents reconstitution with copper. By contrast, addition of copper first to apoprotein leads to formation of topa quinone and stable activity in the presence of added zinc. These findings indicate efficient binding of either zinc or copper to a site that undergoes little or no exchange. The data confirm that topa quinone biogenesis in the H. polymorpha system is catalyzed by copper and occurs in the absence of added factors. We conclude that the mechanisms of cofactor biogenesis in pro- and eukaryotic systems are likely to be similar or identical. The results described herein imply different pathways for the in vivo assembly of heterologously expressed amine oxidases in S. cerevisiae and E. coli.

Phenoxyl-copper(II) complexes: models for the active site of galactose oxidase

The reaction of the macrocycles 1,4,7-tris (3,5-di-tert-butyl-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L1H3, or 1,4,7-tris(3-tert-butyl-5-methoxy-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L2H3, with Cu(ClO4)2·6H2O in methanol (in the presence of Et3N) affords the green complexes [CuII(L1H)] (1), [CuII(L2H)]·CH3OH (2) and (in the presence of HClO4) [CuII(L1H2)](ClO4) (3) and [CuII(L2H2)] (ClO4) (4). The CuII ions in these complexes are five-coordinate (square-base pyramidal), and each contains a dangling, uncoordinated pendent arm (phenol). Complexes 1 and 2 contain two equatorially coordinated phenolato ligands, whereas in 3 and 4 one of these is protonated, affording a coordinated phenol. Electrochemically, these complexes can be oxidized by one electron, generating the phenoxyl-copper(II) species [CuII(L1H)]+ ·, [Cu(L2H)]+ ·, [CuII(L1H2)]2+ ·, and [CuII(L2H2)]2+ ·, all of which are EPR-silent. These species are excellent models for the active form of the enzyme galactose oxidase (GO). Their spectroscopic features (UV-VIS, resonance Raman) are very similar to those reported for GO and unambiguously show that the complexes are phenoxyl-copper(II) rather than phenolato-copper(III) species.

Characterization and Application of Porcine Liver Aldehyde Oxidase in the Off-Flavor Reduction of Soy Proteins

Two enzyme forms of porcine liver aldehyde oxidase (PAO-I and PAO-II) (aldehyde:oxygen oxidoreductase, EC 1.2.3.1) were purified to homogeneity by using affinity chromatography. Both enzyme forms, PAO-I and PAO-II, have similar pI values of 5.8 and molecular masses of 262 000 and 25 000 Da, respectively. Compared with PAO-II, PAO-I showed greater affinity for the soy off-flavor-causing aldehydes n-pentanal and n-hexanal. Purified PAO-I was stable between pH 7.1 and 10.7 and at temperatures up to 45 °C. Energy of denaturation for PAO-I (158.1 kJ/mol·K-1) was more than 3-fold higher than the energy of activation (47 kJ/mol·K-1). Gas chromatography analysis showed that more headspace volatiles were present in the water extract of soy proteins at pH 7.0 than at pH 9.0. The incubation of a soy protein extract and PAO-I reduced the pentanal and hexanal contents by as much as 90%. The sensory panelists perceived lower beany flavor (p < 0.01) of the enzyme-treated soy protein extract than the control. Keywords: Porcine liver aldehyde oxidase; purification; characterization; off-flavor; aldehydes; carboxylic acids

Purification and characterization of diamine oxidase from porcine kidney and intestine

Diamine oxidase, the enzyme catalyzing the oxidative deamination of histamine and other diamines, was purified from porcine kidney and porcine intestine. During all purification steps the enzymes from both tissues showed identical binding and elution characteristics. The native enzymes are homodimeric glycoproteins with an apparent molecular weight of 186 kDa. Under reducing conditions the subunits migrate at 104 kDa on SDS polyacrylamide gels and the deglycosylated subunits migrate at 93 kDa which corresponds to a carbohydrate content of 11%. The native and deglycosylated forms of kidney and intestinal diamine oxidase migrate to the same positions, respectively, on two-dimensional isoelectric focussing/SDS polyacrylamide gels. The sequences of the 21 N-terminal amino acids of both proteins are identical. A polyclonal antibody raised against the kidney enzyme binds equally well to diamine oxidase from both kidney and intestine, inhibits the enzymatic activity, and precipitates all diamine oxidase activity from tissue homogenates. The kidney and intestinal enzymes have identical substrate specificities, efficiently converting aliphatic diamines, histamine, and spermidine. For both enzymes the K m values for histamine, putrescine, and spermidine are 0.02 mM, 0.35 mM, and 3.3 mM, respectively. Spermine, aliphatic monoamines, and aromatic mono- and diamines are poor substrates. In conclusion, the diamine oxidase proteins from porcine kidney and intestine are very likely identical and constitute the only diamine oxidase activity present in these tissues. The structural identity implies identical functions of the proteins in these organs, namely the protection of the organism against high concentrations of diamines.

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.