Daisuke Seo

Four-electron Reduction of Dioxygen by a Multicopper Oxidase, CueO, and Roles of Asp112 and Glu506 Located Adjacent to the Trinuclear Copper Center

The mechanism of the four-electron reduction of dioxygen by a multicopper oxidase, CueO, was studied based on reactions of single and double mutants with Cys500, a type I copper ligand, and the noncoordinating Asp112 and Glu506, which form hydrogen bonds with the trinuclear copper center directly and indirectly via a water molecule. The reaction of C500S containing a vacant type I copper center produced intermediate I in an EPR-silent peroxide-bound form. The formation of intermediate I from C500S/D112N was restricted due to a reduction in the affinity of the trinuclear copper center for dioxygen. The state of intermediate I was realized to be the resting form of C500S/E506Q and C500S of the truncated mutant Δα5–7CueO, in which the 50 amino acids covering the substrate-binding site were removed. Reactions of the recombinant CueO and E506Q afforded intermediate II, a fully oxidized form different from the resting one, with a very broad EPR signal, g < 2, detectable only at cryogenic temperatures and unsaturated with high power microwaves. The lifetime of intermediate II was prolonged by the mutation at Glu506 involved in the donation of protons. The structure of intermediates I and II and the mechanism of the four-electron reduction of dioxygen driven by Asp112 and Glu506 are discussed.

Asymmetric dimeric structure of ferredoxin-NAD(P)+ oxidoreductase from the green sulfur bacterium Chlorobaculum tepidum: implications for binding ferredoxin and NADP+.

Ferredoxin-NAD(P)(+) oxidoreductase (FNR) catalyzes the reduction of NAD(P)(+) to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 A resolution, which reveals a unique structure-function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by pi-pi stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.

Crystal structure analysis of Bacillus subtilis ferredoxin-NADP(+) oxidoreductase and the structural basis for its substrate selectivity

Bacillus subtilis yumC encodes a novel type of ferredoxin‐NADP+ oxidoreductase (FNR) with a primary sequence and oligomeric conformation distinct from those of previously known FNRs. In this study, the crystal structure of B. subtilis FNR (BsFNR) complexed with NADP+ has been determined. BsFNR features two distinct binding domains for FAD and NADPH in accordance with its structural similarity to Escherichia coli NADPH‐thioredoxin reductase (TdR) and TdR‐like protein from Thermus thermophilus HB8 (PDB code: 2ZBW). The deduced mode of NADP+ binding to the BsFNR molecule is nonproductive in that the nicotinamide and isoalloxazine rings are over 15 Å apart. A unique C‐terminal extension, not found in E. coli TdR but in TdR‐like protein from T. thermophilus HB8, covers the re‐face of the isoalloxazine moiety of FAD. In particular, Tyr50 in the FAD‐binding region and His324 in the C‐terminal extension stack on the si‐ and re‐faces of the isoalloxazine ring of FAD, respectively. Aromatic residues corresponding to Tyr50 and His324 are also found in the plastid‐type FNR superfamily of enzymes, and the residue corresponding to His324 has been reported to be responsible for nucleotide specificity. In contrast to the plastid‐type FNRs, replacement of His324 with Phe or Ser had little effect on the specificity or reactivity of BsFNR with NAD(P)H, whereas replacement of Arg190, which interacts with the 2′‐phosphate of NADP+, drastically decreased its affinity toward NADPH. This implies that BsFNR adopts the same nucleotide binding mode as the TdR enzyme family and that aromatic residue on the re‐face of FAD is hardly relevant to the nucleotide selectivity.

Studies of interaction of homo-dimeric ferredoxin-NAD(P)+ oxidoreductases of Bacillus subtilis and Rhodopseudomonas palustris, that are closely related to thioredoxin reductases in amino acid sequence, with ferredoxins and pyridine nucleotide coenzymes

Ferredoxin-NADP(+) oxidoreductases (FNRs) of Bacillus subtilis (YumC) and Rhodopseudomonas palustris CGA009 (RPA3954) belong to a novel homo-dimeric type of FNR with high amino acid sequence homology to NADPH-thioredoxin reductases. These FNRs were purified from expression constructs in Escherichia coli cells, and their steady-state reactions with [2Fe-2S] type ferredoxins (Fds) from spinach and R. palustris, [4Fe-4S] type Fd from B. subtilis, NAD(P)(+)/NAD(P)H and ferricyanide were studied. From the K(m) and k(cat) values for the diaphorase activity with ferricyanide, it is demonstrated that both FNRs are far more specific for NADPH than for NADH. The UV-visible spectral changes induced by NADP(+) and B. subtilis Fd indicated that both FNRs form a ternary complex with NADP(+) and Fd, and that each of the two ligands decreases the affinities of the others. The steady-state kinetics of NADPH-cytochrome c reduction activity of YumC is consistent with formation of a ternary complex of NADPH and Fd during catalysis. These results indicate that despite their low sequence homology to other FNRs, these enzymes possess high FNR activity but with measurable differences in affinity for different types of Fds as compared to other more conventional FNRs.

Crystallization and preliminary X-ray studies of ferredoxin-NAD(P)+ reductase from Chlorobium tepidum.

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 A resolution on the synchrotron-radiation beamline NW12 at the Photon Factory.

Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP+ oxidoreductase with NADP+/NADPH and ferredoxin

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 (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) 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 BsFd(red) with BsFNR(ox) 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 BsFd(red) increased from 40 to 120 μM. The reduction rate of BsFNR(ox) by BsFd(red) was markedly decreased by premixing BsFNR(ox) with BsFd(ox), indicating that the dissociation of BsFd(ox) from BsFNR(sq) is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs.

Crystallization and preliminary X-ray studies of ferredoxin-NADP+ oxidoreductase encoded by Bacillus subtilis yumC

Ferredoxin-NADP(+) oxidoreductase encoded by Bacillus subtilis yumC has been purified and successfully crystallized in complex with NADP(+) in two forms. Diffraction data from crystals of these two forms were collected at resolutions of 1.8 and 1.9 A. The former belonged to space group P2(1)2(1)2, with unit-cell parameters a = 63.90, b = 135.72, c = 39.19 A, and the latter to space group C2, with unit-cell parameters a = 207.47, b = 64.85, c = 61.12 A, beta = 105.82 degrees. The initial structure was determined by the molecular-replacement method using a thioredoxin reductase-like protein as a search model.

C-terminal residues of ferredoxin-NAD(P)+ reductase from Chlorobaculum tepidum are responsible for reaction dynamics in the hydride transfer and redox equilibria with NADP+/NADPH

Ferredoxin-NAD(P)+ reductase ([EC 1.18.1.2], [EC 1.18.1.3]) from Chlorobaculum tepidum (CtFNR) is structurally homologous to the bacterial NADPH-thioredoxin reductase (TrxR), but possesses a unique C-terminal extension relative to TrxR that interacts with the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group. In this study, we introduce truncations to the C-terminal residues to examine their role in the reactions of CtFNR with NADP+ and NADPH by spectroscopic and kinetic analyses. The truncation of the residues from Tyr326 to Glu360 (the whole C-terminal extension region), from Phe337 to Glu360 (omitting Phe337 on the re-face of the isoalloxazine ring) and from Ser338 to Glu360 (leaving Phe337 intact) resulted in a blue-shift of the flavin absorption bands. The truncations caused a slight increase in the dissociation constant toward NADP+ and a slight decrease in the Michaelis constant toward NADPH in steady-state assays. Pre-steady-state studies of the redox reaction with NADPH demonstrated that deletions of Tyr326–Glu360 decreased the hydride transfer rate, and the amount of reduced enzyme increased at equilibrium relative to wild-type CtFNR. In contrast, the deletions of Phe337–Glu360 and Ser338–Glu360 resulted in only slight changes in the reaction kinetics and redox equilibrium. These results suggest that the C-terminal region of CtFNR is responsible for the formation and stability of charge-transfer complexes, leading to changes in redox properties and reactivity toward NADP+/NADPH.

Crystal Structure of Ferredoxin-NAD(P)+ Reductase from the Green Sulfur Bacterium Chlorobaculum Tepidum

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.

Thiosulfate-Oxidizing Multi-component System in the Green Sulfur Bacterium Chlorobaculum tepidum

Green sulfur bacteria grow phototrophically using sulfur compounds such as sulfide, sulfur, or thiosulfate as electron donors. The components of the thiosulfate oxidoreductase system, and the functions of each component are controversial. The thiosulfate-dependent mammalian cytochrome c reducing activity of the cell extract from Chlorobaculum tepidum was resolved into four fractions (Fraction I, II, III and IV) by ammonium sulfate fractionation, anion-exchange chromatography and cation-exchange chromatography. Fraction I is a heterodimer of SoxY and SoxZ, Fraction II SoxB, Fraction III is composed of SoxA and SoxX, and Fraction IV is SoxF2. For reduction of mammalian cytochrome c by thiosulfate, all of Fraction I-III is indispensable. The optical spectrum of dithionite-reduced SoxAX showed characteristic of cyt c with an a peak at 551 nm. SoxYZ and SoxB are colorless. SoxF2 is yellow and binds flavin. We have also purified a soluble cyt c-554 (about 10 kDa). Addition of SoxF2 and cyt c-554 to Fraction I-III enhanced the thiosulfate oxidation.