Yasuzo Nishina

A resonance Raman study on the nature of charge-transfer interactions in butyryl CoA dehydrogenase.

Butyryl-CoA dehydrogenase (BCD) (EC 1.3.99.2) is a flavoprotein that catalyses the first step in fatty acid p-oxidation. When isolated from various sources, the pure enzyme has a characteristic green colour [l-3], due to a long-wavelength absorption band centred at 710 nm. We have proposed that a new chemical species, CoA persulphide, tightly-bound at the enzyme’s active site, is the donor in a chargetransfer interaction with the FAD prosthetic group [4,5]. Complexes with long-wavelength absorption are also formed between the enzyme and various acyl-CoA compounds, including acetoacetyl-CoA, which gives a grey-green complex with an absorbance maximum at 580 nm [6]. Butyryl-CoA dehydrogenase was purified from Megasphaera elsdenii as in [ 121. Coenzyme A was purchased from Sigma. Other reagents were from BDH.

Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.

Porcine kidney d-amino acid oxidase: the three-dimensional structure and its catalytic mechanism based on the enzyme–substrate complex model

Abstract The three-dimensional structure of porcine kidney d -amino acid oxidase (DAO), an FAD-dependent oxidase, has been solved by X-ray crystallography. The overall structure is a dimer, subunits of which are correlated by a non-crystallographic two-fold axis. Each subunit comprises two domains, ‘αβ domain’ and ‘pseudo-barrel domain’. The coenzyme FAD is in an elongated conformation and is bound at the N -terminal βαβ dinucleotide binding motif. The active site is located in the boundary region between the two domains. The crystal structure of DAO in complex with a substrate analog, o -aminobenzoate, was also solved and is used for modeling the DAO- d -leucine complex, i.e. Michaelis complex, by means of molecular mechanics simulation. The Michaelis-complex model provided structural information leading to two alternative hypothetical mechanisms for the reductive half-reaction of DAO. These two hypotheses characterize themselves by electron transfer from the lone-pair orbital of the substrate amino nitrogen to flavin C(4a) and by proton transfer from the substrate α-position to flavin N(5) which acts as a catalytic base.

Resonance Raman study on the purple intermediates of the flavoenzyme D-amino acid oxidase

Abstract Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm −1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm −1 in the RR spectrum with D-alanine was shifted to 1675 cm −1 upon [ 15 N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm −1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FADs, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.

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.

A STUDY OF THE INTERACTION BETWEEN CHLORPROMAZINE AND RIBOFLAVIN BINDING PROTEIN

The interaction between chlorpromazine (CPZ) and riboflavin binding protein (RBP) was studied by the spectrophotometric and spectrofluorometric methods.

Coenzyme-induced subunit association of the flavoenzyme D-amino acid oxidase: a kinetic light scattering study

Hog kidney D-amino acid oxidase [EC I .4.3.3] contains a single FAD per 35 000-40 000 MW monomer [l-4], and undergoes self-association [S-12]. Many ligands, such as competitive inhibitor, product, and substrate, influence the association process of the oxidase f 11 ,13-161. The holoenzyme has a higher apparent MW than the apoenzyme at a given enzyme concentration [6,9-l 11. Recently, Yagi et al, [ 17,181 analyzed the binding property of FAD in D-amino acid oxidase according to monomerdimer equilibrium and demonstrated that the affinity of the dimer for the coenzyme FAD is about 70 times higher than that of the mono~~er. These thermodynamic studies indicate that the coenzyme induces association of the subunits in the equilibrium state of the oxidase. However, there is no direct evidence yet available concerning the reaction mechanism of FADinduced subunit association. In this communication, we demonstrate the subunit association subsequent to FAD binding to the apoenzyme and explore the ratelimiting step of this reaction by the kinetic light scattering method.

Hydrogen Bonding Environment of the N3–H Group of Flavin Mononucleotide in the Light Oxygen Voltage Domains of Phototropins

The light oxygen voltage (LOV) domain is a flavin-binding blue-light receptor domain, originally found in a plant photoreceptor phototropin (phot). Recently, LOV domains have been used in optogenetics as the photosensory domain of fusion proteins. Therefore, it is important to understand how LOV domains exhibit light-induced structural changes for the kinase domain regulation, which enables the design of LOV-containing optogenetics tools with higher photoactivation efficiency. In this study, the hydrogen bonding environment of the N3-H group of flavin mononucleotide (FMN) of the LOV2 domain from Adiantum neochrome (neo) 1 was investigated by low-temperature Fourier transform infrared spectroscopy. Using specifically 15N-labeled FMN, [1,3-15N2]FMN, the N3-H stretch was identified at 2831 cm-1 for the unphotolyzed state at 150 K, indicating that the N3-H group forms a fairly strong hydrogen bond. The N3-H stretch showed temperature dependence, with a shift to lower frequencies at ≤200 K and to higher frequencies at ≥250 K from the unphotolyzed to the intermediate states. Similar trends were observed in the LOV2 domains from Arabidopsis phot1 and phot2. By contrast, the N3-H stretch of the Q1029L mutant of neo1-LOV2 and neo1-LOV1 was not temperature dependent in the intermediate state. These results seemed correlated with our previous finding that the LOV2 domains show the structural changes in the β-sheet region and/or the adjacent Jα helix of LOV2 domain, but that such structural changes do not take place in the Q1029L mutant or neo1-LOV1 domain. The environment around the N3-H group was also investigated.

Decomposition of the fluorescence spectra of two FAD molecules in electron-transferring flavoprotein from Megasphaera elsdenii

Electron-transferring flavoprotein (ETF) from Megasphaera elsdenii contains two FAD molecules, FAD-1 and FAD-2. FAD-2 shows an unusual absorption spectrum with a 400-nm peak. In contrast, ETFs from other sources such as pig contain one FAD and one AMP with the FAD showing a typical flavin absorption spectrum with 380- and 440-nm peaks. It is presumed that FAD-2 is the counterpart of the FAD in other ETFs. In this study, the FAD-1 and FAD-2 fluorescence spectra were determined by titration of FAD-1-bound ETF with FAD using excitation-emission matrix (EEM) fluorescence spectroscopy. The EEM data were globally analysed, and the FAD fluorescence spectra were calculated from the principal components using their respective absorption spectra. The FAD-2 fluorescence spectrum was different from that of pig ETF, which is more intense and blue-shifted. AMP-free pig ETF in acidic solution, which has a comparable absorption spectrum to FAD-2, also had a similar fluorescence spectrum. This result suggests that FAD-2 in M. elsdenii ETF and the FAD in acidic AMP-free pig ETF share a common microenvironment. A review of published ETF fluorescence spectra led to the speculation that the majority of ETF molecules in solution are in the conformation depicted by the crystal structure.