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Shun Hirota

Publications and source records attributed to Shun Hirota.

30 records · Page 2Linked to original sources
PubMed2004

Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor.

The topa quinone (TPQ) cofactor of copper amine oxidase is produced by posttranslational modification of a specific tyrosine residue through the copper-dependent, self-catalytic process. We have site-specifically mutated three histidine residues (His431, His433, and His592) involved in binding of the copper ion in the recombinant phenylethylamine oxidase from Arthrobacter globiformis. The mutant enzymes, in which each histidine was replaced by alanine, were purified in the Cu/TPQ-free precursor form and analyzed for their Cu-binding and TPQ-generating activities by UV-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. Among the three histidine-to-alanine mutants, only H592A was found to show a weak activity to form TPQ upon aerobic incubation with Cu(2+) ions. Also for H592A, exogenous imidazole rescued binding of copper and markedly promoted the TPQ formation. Accommodation of a free imidazole molecule within the cavity created in the active site of H592A was suggested by X-ray crystallography. Although the TPQ cofactor in H592A mutant was readily reduced with substrate, its catalytic activity was very low even in the presence of imidazole. Combined with the crystal structures of the mutant enzymes, these results demonstrate the importance of the three copper-binding histidine residues for both TPQ biogenesis and catalytic activity, fine-tuning the position of the essential metal.

Alanine↗
PubMed2004

Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c.

Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82+/-9s(-1)) corresponding to the transition from the His/H(2)O to the His/Met coordinated species, whereas the slow phase (24+/-3s(-1)) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.

Amino Acids↗
PubMed2004

Interaction of plastocyanin with oligopeptides: effect of lysine distribution within the peptide.

We synthesized and purified four oligopeptides containing four lysines (KKKK, GKKGGKK, KKGGGKK, and KGKGKGK) as models for the plastocyanin (PC) interacting site of cytochrome f. These peptides competitively inhibited electron transfer between cytochrome c and PC. The inhibitory effect increased as the peptide concentrations were increased. The association constants between PC and the peptides did not differ significantly (3500-5100 M(-1)), although the association constant of PC-KGKGKGK was a little larger than the constants between PC and other peptides. Changes in the absorption spectrum of PC were observed when the peptides were added to the PC solution: peaks and troughs were detected at about 460 and 630 nm and at about 560 and 700 nm, respectively, in the difference absorption spectra between the spectra with and without peptides. These changes were attributed to the structural change at the copper site of PC by interaction with the peptides. The structural change was most significant when tetralysine was used. These results show that binding of the oligopeptide to PC is slightly more efficient when lysines are distributed uniformly within the peptide, whereas the structural change of PC becomes larger when the lysines are close to each other within the peptide.

Amino Acid Sequence↗
PubMed2004

Structure and ethanol complexation of cyclic tetrasaccharide in aqueous solution studied by NMR and molecular mechanics.

The structure and ethanol complexation of a cyclic tetrasaccharide (CTS) in aqueous solution were investigated by proton NMR spectroscopy and molecular mechanics calculations. Two glucose units, A and B, of CTS are alternatively bonded by alpha-1,3 and alpha-1,6 linkages. The overlapped signals of protons A5, A6S, A6R, B3, B6S and B6R were resolved by spectral simulations to determine their chemical shifts and vicinal coupling constants. All vicinal coupling constants except for the A5-A6 spin system are consistent with the dihedral angles in the X-ray crystal structure. Each of protons A5, A6S, and A6R in the two units of A is equivalent with respect to the chemical shift. The vicinal coupling constants of (3)J(5-6S) and (3)J(5-6R) for unit A are close to the average of two rotamers that are present in crystals. The intensities of cross-peaks in the rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectrum were rather well correlated with the effective distances calculated for the X-ray structure and molecular mechanics structures calculated in vacuo and water, although they are slightly better correlated with molecular mechanics structure in vacuo than with the other structures. From the changes of the chemical shifts of several CTS protons with increasing ethanol concentration, it was suggested that adsorption sites of ethanol on the plate structure of CTS are protons B2 and B4 (site B) in the concave face side and protons A1 and A2 (site A) in the convex back side. The binding constants for sites A and B are 0.0061 and 0.0176 M(-1), respectively. These binding constants are much smaller than a value of 4.1 M(-1) for the ethanol-alpha-cyclodextrin complex.

Binding Sites↗
PubMed2003

Role of copper ion in bacterial copper amine oxidase: spectroscopic and crystallographic studies of metal-substituted enzymes.

The role of the active site Cu(2+) of phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been studied by substitution with other divalent cations, where we were able to remove >99.5% of Cu(2+) from the active site. The enzymes reconstituted with Co(2+) and Ni(2+) (Co- and Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original Cu(2+)-enzyme (Cu-AGAO), but their K(m) values for amine substrate and dioxygen were comparable. X-ray crystal structures of the Co- and Ni-AGAO were solved at 2.0-1.8 A resolution. These structures revealed changes in the metal coordination environment when compared to that of Cu-AGAO. However, the hydrogen-bonding network around the active site involving metal-coordinating and noncoordinating water molecules was preserved. Upon anaerobic mixing of the Cu-, Co-, and Ni-AGAO with amine substrate, the 480 nm absorption band characteristic of the oxidized form of the topaquinone cofactor (TPQ(ox)) disappeared rapidly (< 6 ms), yielding the aminoresorcinol form of the reduced cofactor (TPQ(amr)). In contrast to the substrate-reduced Cu-AGAO, the semiquinone radical (TPQ(sq)) was not detected in Co- and Ni-AGAO. Further, in the latter, TPQ(amr) reacted reversibly with the product aldehyde to form a species with a lambda(max) at around 350 nm that was assigned as the neutral form of the product Schiff base (TPQ(pim)). Introduction of dioxygen to the substrate-reduced Co- and Ni-AGAO resulted in the formation of a TPQ-related intermediate absorbing at around 360 nm, which was assigned to the neutral iminoquinone form of the 2e(-)-oxidized cofactor (TPQ(imq)) and which decayed concomitantly with the generation of TPQ(ox). The rate of TPQ(imq) formation and its subsequent decay in Co- and Ni-AGAO was slow when compared to those of the corresponding reactions in Cu-AGAO. The low catalytic activities of the metal-substituted enzymes are due to the impaired efficiencies of the oxidative half-reaction in the catalytic cycle of amine oxidation. On the basis of these results, we propose that the native Cu(2+) ion has essential roles such as catalyzing the electron transfer between TPQ(amr) and dioxygen, in part by providing a binding site for 1e(-)- and 2e(-)-reduced dioxygen species to be efficiently protonated and released and also preventing the back reaction between the product aldehyde and TPQ(amr).

Amine Oxidase (Copper-Containing)↗
PubMed2003

Type III Cu mutants of Myrothecium verrucaria bilirubin oxidase.

Type III Cu ligand, His456 and His458, of Myrothecium verrucaria (MT-1) bilirubin oxidases (BO) [EC 1.3.3.5] were doubly mutated as to Lys, Asp, and Val. In spite of perturbation of the type III Cu centers, these mutants were pale blue or colourless when isolated. However, they became intense blue on reaction with reducing agents such as dithionite, ascorbate, hexacyanoferrate(II), and octacyanotangstate(IV) under air, or with an oxidizing agent such as hexacyanoferrate(III), indicating that they are in mixed forms when expressed in Aspergillus oryzae. His456.458Lys and His456.458Asp mutated as to potential coordinating groups showed weak BO and ferroxidase activities, while His 456.458Val mutated as to non-coordinating groups showed no enzyme activity at all.

Amino Acid Sequence↗
PubMed2002

Structural studies of the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F: suggestion for the initial activation site for dihydrogen.

The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. The CO was not replaced with H(2) in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni-C distances and Ni-C-O angles were about 1.77 A and 160 degrees, respectively, except for one case (1.72 A and 135 degrees ), in which an additional electron density peak between the CO and Sgamma(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sgamma(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sgamma(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H(2)-binding process. Anaerobic addition of CO to dithionite-reduced [NiFe]hydrogenase led to a new absorption band at about 470 nm ( approximately 3000 M(-1)cm(-1)). Resonance Raman spectra (excitation at 476.5 nm) of the CO complex revealed CO-isotope-sensitive bands at 375/393 and 430 cm(-1) (368 and 413 cm(-1) for (13)C(18)O). The frequencies and relative intensities of the CO-related Raman bands indicated that the exogenous CO is bound to the Ni atom with a bent Ni-C-O structure in solution, in agreement with the refined structure determined by X-ray crystallography.

Binding Sites↗
PubMed2001

Kinetic studies on the oxidation of cytochrome b(5) Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes.

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.

Cytochrome c Group↗
PubMed2001

Observation of an isotope-sensitive low-frequency Raman band specific to metmyoglobin.

A resonance Raman band involving significantly the iron(III)-histidine stretching (upsilonFe-His) character is identified for metmyoglobin (metMb) through isotope sensitivity of its low-frequency resonance Raman bands, but the identification was not successful for methemoglobin (metHb) and its isolated alpha and beta subunits. A band at 218 cm-1 of natural abundance metMb exhibited a low-frequency shift for 15N-His-labeled metMb (-1.4 cm-1 shift), while the strong porphyrin bands at 248 and 271 cm-1 did not shift significantly. The frequency of the 218-cm-1 band of metMb decreased by 1.6 cm-1 in D2O, probably due to Ndelta-deuteration of the proximal His, in a similar manner to that of the upsilonFe-His band of deoxyMb in D2O. This 218-cm-1 band shifted slightly to a lower frequency in H2(18)O, whereas it did little upon 54Fe isotopic substitution (<0.3 cm-1), presumably because of the six-coordinate structure. The lack of the 54Fe-isotope shift shows that the 218-cm-1 band is specific to metMb and not due to the deoxy species. The intensity of this band decreased for hydroxymetMb and was indiscernible for cyanometMb. For metHb and its alpha and beta subunits, however, the frequencies of the band around 220 cm-1 were not D2O sensitive. These results suggest an assignment of the band around 220 cm-1 to a pyrrole tilting mode, which significantly contains the Fe-His stretching character for metMb but scarcely for metHb and its subunits. The differences in the isotope sensitivity of this band in different proteins are considered to reflect the heme distortion from the planarity and the Fe-His geometry specific to individual proteins.

Animals↗
PubMed1996

The Co-CH(3) Bond in Imine/Oxime B(12) Models. Influence of the Orientation and Donor Properties of the trans Ligand As Assessed by FT-Raman Spectroscopy.

Near-IR FT-Raman spectroscopy was used to probe the properties of three types of methyl imine/oxime B(12) model compounds in CHCl(3) solution. These types differ in the nature of the 1,3-propanediyl chain and were selected to test the influence of electronic and steric effects on the Co-CH(3) stretching (nu(Co)(-)(CH)()3) frequency, a parameter related to Co-C bond strength. For the first type studied, [LCo((DO)(DOH)pn)CH(3)](0/+) ((DO)(DOH)pn = N(2),N(2)(')-propane-1,3-diylbis(2,3-butanedione 2-imine 3-oxime)), nu(Co)(-)(CH)()3 decreased from 505 to 455 cm(-)(1) with stronger electron-donating character of the trans axial ligand, L, in the order Cl(-), MeImd, Me(3)Bzm, 4-Me(2)Npy, py, 3,5-Me(2)PhS(-), PMe(3), and CD(3)(-). This series thus allows the first assessment of the effect of negative axial ligands on nu(Co)(-)(CH)()3; these ligands (L = Cl(-), 3,5-Me(2)PhS(-), CD(3)(-)) span the range of trans influence. The CH(3) bending (delta(CH3)) bands were observed at 1171, 1159, and 1150/1105 cm(-)(1), respectively. The decrease in C-H stretching frequencies (nu(CH)) of the axial methyl suggests that the C-H bond strength decreases in the order Cl(-) > 3,5-Me(2)PhS(-) > CD(3)(-). This result is consistent with the order of decreasing (13)C-(1)H NMR coupling constants obtained for the axial methyl group. The trend of lower nu(Co)(-)(CH)()3 and nu(CH) frequencies and lower axial methyl C-H coupling constant for stronger electron-donating trans axial ligands can be explained by changes in the electronic character of the Co-C bond. The symmetric CH(3)-Co-CH(3) mode (nu(CH)()3(-)(Co)(-)(CH)()3) for (CH(3))(2)Co((DO)(DOH)pn) was determined to be 456 cm(-)(1) (421 cm(-)(1) for (CD(3))(2)Co((DO)(DOH)pn). The L-Co-CH(3) bending mode (delta(L)(-)(Co)(-)(CH)()3) was detected for the first time for organocobalt B(12) models; this mode, which is important for force field calculations, occurs at 194 cm(-)(1) for ClCo((DO)(DOH)pn)CH(3) and at 186 cm(-)(1) for (CH(3))(2)Co((DO)(DOH)pn. The nu(Co)(-)(CH)()3 frequencies were all lower than those reported for the corresponding cobaloxime type LCo(DH)(2)CH(3) (DH = monoanion of dimethylglyoxime) models for planar N-donor L. This relationship is attributed to a steric effect of L in [LCo((DO)(DOH)pn)CH(3)](+). The puckered 1,3-propanediyl chain in [LCo((DO)(DOH)pn)CH(3)](+) forces the planar L ligands to adopt a different orientation compared to that in the cobaloxime models. The consequent steric interaction bends the equatorial ligand toward the methyl group (butterfly bending); this distortion leads to a longer Co-C bond. In a second imine/oxime type, a pyridyl ligand is connected to the 1,3-propanediyl chain and oriented so as to minimize butterfly bending. The nu(Co)(-)(CH)()3 frequency for this new lariat model was close to that of pyCo(DH)(2)CH(3). In a third type, a bulkier 2,2-dimethyl-1,3-propanediyl group replaces the 1,3-propanediyl chain. The nu(Co)(-)(CH)()3 bands for two complexes with L = Me(3)Bzm and py were 2-5 cm(-)(1) lower in frequency than those of the corresponding [L(Co((DO)(DOH)pn)CH(3)](+) complexes. The decrease in the axial nu(Co)(-)(CH)()3 frequencies is probably due to the steric effect of the equatorial ligand. Thus, the nu(Co)(-)(CH)()3 frequency can be useful for investigating both steric and electronic influences on the Co-C bond.

Journal Article↗
PubMedDate not supplied

Gene organization and molecular modeling of copper amine oxidase from Aspergillus niger: re-evaluation of the cofactor structure.

Amine oxidase AO-I from Aspergillus niger AKU 3302 has been reported to contain topa quinone (TPQ) as a cofactor; however, analysis of the p-nitrophenylhydrazine-derivatized enzyme and purified active site peptides showed the presence of a carboxylate ester linkage of TPQ to a glutamate. The catalytic functionality of such a cross-linked cofactor has recently been shown unlikely by spectroscopic and voltammetric studies on synthesized model compounds. We have obtained resonance Raman spectra of native and substrate-reduced AO-I demonstrating that the catalytically active cofactor is unmodified TPQ. The primary structure of the enzyme (GenBank acc. no. U31869) has been reviewed and updated by repeated isolation and sequencing of AO-I cDNA. This allowed rectification of several errors that account for previously reported low homology to other amine oxidases in the regions around copper binding histididyl residues. The results were confirmed by cloning the ao-1 structural gene (GenBank acc. no. AF362473). Analysis of the gene 5'-upstream region of the gene revealed potential binding sites for an analog of NIT2, the nitrogen metabolism regulatory protein found in Neurospora crassa and other fungi. The molecular structure of AO-I was modeled by a comparative method using published crystal structures of amine oxidases as templates.

Amine Oxidase (Copper-Containing)↗