daisuke seo | Kanazawa University (original) (raw)

Papers by daisuke seo

The reaction center (RC) of green sulfur bacteria (PS-C) belongs to a Type 1 photosystem as PSI a... more The reaction center (RC) of green sulfur bacteria (PS-C) belongs to a Type 1 photosystem as PSI and RC of heliobacteria (PS-H). These RCs have Fe-S center(s) as electron acceptors, and their core polypeptides show amino acid sequence similarities among each other. However, the RC core polypeptides of PS-C and PS-H are assumed to be homodimeric in contrast with PSI. The sequence and kinetics of electron transfer in PSC as well as to and from PS-C are only partially elucidated (1,2). PS-C can directly photoreduce ferredoxin (Fd) in contrast with purple bacterial RC and we report here some of the results of kinetic studies. Kjwr and Scheller (3) found that purified PS-C from Chlorobium vibriofome photoreduced NADP+ in the presence of Fd from Clostridium and FNR from spinach. We purified several Fds from Chlorobium tepidum and studied their activities as electron mediators in NADP+ photoreduction. In PSI, phylloquinone functions as an electron mediator (A1) between A0 and Fx (A2). In heliobacterial membranes, procedures which were assumed to extract quinones did not result in significant changes in the yield of stable charge separation, suggesting that menaquinone is not an essential participant in the electron acceptor chain (4). More recently, Brettel et al. (5) concluded from time-resolved flash spectroscopy in ns time range and from photovoltage measurements of heliobacterial membranes that quinone is not functioning as an electron acceptor in PS-H. In PS-C, there have been controversies over the functioning of quinone as an electron acceptor (reviewed in (1, 2)). In order to answer these questions, we studied electron transfer kinetics in purified PS-C in ns-ms time range by flash absorption spectroscopy. We also measured the reduction rate of photooxidized P840 by Cyt c551 bound to PS-C.

Plant and Cell Physiology, May 1, 1998

Science Access, 2001

... Okkels JS, Kjær B, Hansson O, Svendsen I, Møller BL, Scheller HV (1992) The Journal of Biolog... more ... Okkels JS, Kjær B, Hansson O, Svendsen I, Møller BL, Scheller HV (1992) The Journal of Biological Chemistry 267, 21139-21145. Okumura N, Shimada K, Matsuura K (1994) Photosynthesis Research 41, 125-134. Ortega JM, Drepper F, Mathis P (1999) Photosynthesis ...

Journal of the American Chemical Society, Feb 16, 2021

Radicals of flavin adenine dinucleotide (FAD), as well as tyrosine and tryptophan, are widely inv... more Radicals of flavin adenine dinucleotide (FAD), as well as tyrosine and tryptophan, are widely involved as key reactive intermediates during electron transfer (ET) reactions in flavoproteins. Due to the high reactivity of these species, and their corresponding short lifetime, characterization of these intermediates in functional processes of flavoproteins is usually challenging, but can be achieved by ultrafast spectroscopic studies of light-activatable flavoproteins. In ferredoxin-NADP + oxidoreductase from Bacillus subtilis (BsFNR), fluorescence of the FAD cofactor that very closely interacts with a neighboring tyrosine residue (Tyr50), is strongly quenched. Here we study short-lived photoproducts of this enzyme and its variants with Tyr50 replaced by tryptophan or glycine. Using time-resolved fluorescence and absorption spectroscopies, we show that upon the excitation of WT BsFNR, ultrafast ET from Tyr50 to the excited FAD cofactor occurs in ~260 fs, an order of magnitude faster than the decay by charge recombination, facilitating the characterization of the reaction intermediates in the charge-separated state with respect to other recently studied systems. These studies are corroborated by experiments on the Y50W mutant protein, which yield photoproducts qualitatively similar to those observed in other tryptophan bearing flavoproteins. By combining the experimental results with molecular dynamics simulations and quantum mechanics calculations, we investigate in detail the effect of protein environment and relaxations on the spectral properties of those radical intermediates, and demonstrate that the spectral features of radical anionic FAD are highly sensitive to its environment, and in particular to the dynamics and nature of the counter-ions formed in the photoproducts. Altogether, comprehensive characterizations are provided for important radical intermediates that are generally involved in functional processes of flavoproteins.

Journal of Plant Research, Dec 1, 2006

Biochimica et Biophysica Acta, 2020

Biophysical Journal, Feb 1, 2017

and structural studies, using NMR spectroscopy and crystallography, indicate that these modular i... more and structural studies, using NMR spectroscopy and crystallography, indicate that these modular interactions act as a binding catalyst that accelerates the association between CrkII and cAbl kinase. We also present our recent finding of a potent inhibitor of the cAbl-CrkII interaction.

Archives of Microbiology, Jul 14, 2004

From Bacillus subtilis cell extracts, ferredoxin-NADP + reductase (FNR) was purified to homogenei... more From Bacillus subtilis cell extracts, ferredoxin-NADP + reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a ~94 kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as an electron acceptor, the affinity to NADPH was much higher than to NADH, with K m values of 0.57 vs. >200 µM. K cat values of YumC with NADPH were 22.7 and 35.4 s-1 in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in Gram-positive bacteria except for Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR which should be added to the already established plant type, bacteria type, and mitochondria type FNR groups.

Science Journal of Kanagawa University, 2018

Acta crystallographica, Feb 23, 2008

Ferredoxin-NAD(P) + reductase (FNR) is a key enzyme that catalyzes the photoreduction of NAD(P) +... more Ferredoxin-NAD(P) + reductase (FNR) is a key enzyme that catalyzes the photoreduction of NAD(P) + to generate NAD(P)H during the final step of the photosynthetic electron-transport chain. FNR from the green sulfur bacterium Chlorobium tepidum is a homodimeric enzyme with a molecular weight of 90 kDa; it shares a high level of amino-acid sequence identity to thioredoxin reductase rather than to conventional plant-type FNRs. In order to understand the structural basis of the ferredoxin-dependency of this unique photosynthetic FNR, C. tepidum FNR has been heterologously expressed, purified and crystallized in two forms. Form I crystals belong to space group C222 1 and contain one dimer in the asymmetric unit, while form II crystals belong to space group P4 1 22 or P4 3 22. Diffraction data were collected from a form I crystal to 2.4 Å resolution on the synchrotron-radiation beamline NW12 at the Photon Factory.

Plant Physiology and Biochemistry, Aug 1, 2014

Ferredoxin-NADP + oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to th... more Ferredoxin-NADP + oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to the bacterial NADPHthioredoxin reductase, but possesses a unique C-terminal extension that covers the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide (FAD) prosthetic group. In this report, we utilize BsFNR mutants depleted of their C-terminal residues to examine the importance of the C-terminal extension in reactions with NADPH and ferredoxin (Fd) from B. subtilis by spectroscopic and steady-state reaction analyses. The depletions of residues Y313 to K332 (whole C-terminal extension region) and S325 to K332 (His324 intact) resulted in significant increases in the catalytic efficiency with NADPH in diaphorase assay with ferricyanide, whereas Km values for ferricyanide were increased. In the cytochrome c reduction assay in the presence of B. subtilis ferredoxin, the S325-K332 depleted mutant displayed a significant decrease in the turnover rate with an Fd concentration range of 1 to 10 μM. The Y313-K332 depleted mutant demonstrated an increase in the rate of the direct reduction of horse heart cytochrome c in the absence of Fd. These data indicated that depletion of the C-terminal extension plays an important role in the reaction of BsFNR with ferredoxin.

Bioscience, Biotechnology, and Biochemistry, Apr 23, 2010

In the green sulfur bacterium Chlorobaculum tepidum, three sulfur oxidizing enzyme system (Sox) p... more In the green sulfur bacterium Chlorobaculum tepidum, three sulfur oxidizing enzyme system (Sox) proteins, SoxAXK, SoxYZ, and SoxB (the core TOMES, thiosulfate oxidizing multi-enzyme system) are essential to in vitro thiosulfate oxidation. We purified monomeric flavoprotein SoxF from this bacterium, which had sulfide dehydrogenase activity. SoxF enhanced the thiosulfate oxidation activity of the purified core TOMES with various cytochromes as electron acceptors to different degrees without any change in the affinity for thiosulfate. The apparent reaction rates with 50 M C. tepidum cytochrome c-554 were slightly higher than with horse-heart cytochrome c, and the addition of 0.5 M SoxF increased the rate by 92%. The rates with 50 M horse-heart cytochrome c and 50 M horse-heart cytochrome c plus 0.5 M cytochrome c-554 were increased by SoxF by 31% and 120% respectively. We conclude that SoxF mediates electron transfer between the components of core TOMES and externally added cytochromes.

The Japanese Biochemical Society/The Molecular Biology Society of Japan, Oct 23, 2017

Springer eBooks, 2013

Green sulfur bacterium Chlorobaculum tepidum contains a novel type of ferredoxin-NAD(P)+ reductas... more Green sulfur bacterium Chlorobaculum tepidum contains a novel type of ferredoxin-NAD(P)+ reductase (FNR) with high amino acid sequence homology to the NADPH-thioredoxin reductase (TdR) from prokaryotes. In this study, we determine the crystal structure of C. tepidum FNR by X-ray crystallography. C. tepidum FNR retains its structural topology with E. coli TdR but possesses several characteristic features that is absent in TdR. Each protomer is composed of two nucleotide binding domains, FAD-binding and NAD(P)+-binding. The two domains are connected by a hinge region. Homo-dimeric C. tepidum FNR shows an asymmetric domain orientation between two protomers. The observed C-terminal sub-domain covers the re-face of the isoalloxazine ring of FAD prosthetic group. The C-terminal sub-domain includes the stacking Phe337 on the reface of the isoalloxazine ring of the FAD. On the si-face, Tyr57 residue is stacked on. The two stacking ring systems are positioned almost parallel with respect to isoalloxazine ring at a distance of 3.5 A. Such a configuration of stacking of two aromatic rings is absent in TdR but found in plastid-type FNRs, suggesting these structural characteristics are indispensable for the FNR reaction. To elucidate the function of these structural characteristics, mutational analysis was performed.

Biochimica Et Biophysica Acta - Bioenergetics, Jun 1, 2016

Ferredoxin-NADP + oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodime... more Ferredoxin-NADP + oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP + , NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP + and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNRox)-NADPH and reduced FNR (FNRred)-NADP + , both having CT absorption bands centered at approximately 600 nm. After mixing BsFNRox with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNRred was mixed with NADP + , the amount of BsFNRox increased with increasing NADP + concentration, but BsFNRred remained as the major species at equilibrium even with about 50-fold molar excess NADP +. In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s-1) was much higher than the reverse one (< 10 s-1). Mixing BsFdred with BsFNRox induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s −1 at 10°C, which increased as BsFdred increased from 40 to 120 μM. The reduction rate of BsFNRox by BsFdred was markedly decreased by premixing BsFNRox with BsFdox, indicating that the dissociation of BsFdox from BsFNRsq is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP + /NADPH were compared with those of other types of FNRs.

Biochimica Et Biophysica Acta - Proteins And Proteomics, May 1, 2002

Ferredoxin-NAD(P) + reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacteriu... more Ferredoxin-NAD(P) + reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin and reaction center complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP + and NAD +. When concentrations of NADP + exceeded 10 µM, NADP + photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD +. It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.