Toshiharu Hase

Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe-2S] cluster in poplar glutaredoxin C1

When expressed in Escherichia coli, cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron–sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe–2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron–sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or iron–sulfur biosynthesis is discussed with respect to their intracellular localization.

Atomic Structure of Plant Glutamine Synthetase: A KEY ENZYME FOR PLANT PRODUCTIVITY

Plants provide nourishment for animals and other heterotrophs as the sole primary producer in the food chain. Glutamine synthetase (GS), one of the essential enzymes for plant autotrophy catalyzes the incorporation of ammonia into glutamate to generate glutamine with concomitant hydrolysis of ATP, and plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Elucidation of the atomic structure of higher plant GS is important to understand its detailed reaction mechanism and to obtain further insight into plant productivity and agronomical utility. Here we report the first crystal structures of maize (Zea mays L.) GS. The structure reveals a unique decameric structure that differs significantly from the bacterial GS structure. Higher plants have several isoenzymes of GS differing in heat stability and catalytic properties for efficient responses to variation in the environment and nutrition. A key residue responsible for the heat stability was found to be Ile-161 in GS1a. The three structures in complex with substrate analogues, including phosphinothricin, a widely used herbicide, lead us to propose a mechanism for the transfer of phosphate from ATP to glutamate and to interpret the inhibitory action of phosphinothricin as a guide for the development of new potential herbicides.

The Interaction of Ferredoxin with Ferredoxin-Dependent Enzymes

Summary Ferredoxin, reduced by Photosystem I (PS I) in the light, serves as the electron donor for the reduction of NADP + to NADPH, of sulfite to sulfide, of nitrite to ammonia and for the reductant-requiring of glutamate and 2-oxoglutarate to glutamate in all oxygenic photosynthetic organisms. Reduced ferredoxin also serves as the electron donor for the reduction of nitrate to nitrite in cyanobacteria. In addition to its role in supplying a source of electrons for the net reduction of oxidized species in reductant-requiring assimilatory pathways, reduced ferredoxin plays an important role, via the ferredoxin/thioredoxin system, in the regulation of carbon assimilation and other pathways. This chapter focuses on the interactions between ferredoxin and six enzymes that utilize reduced ferredoxin as an electron donor (NADP + reductase, nitrate reductase, nitrite reductase, glutamate synthase, sulfite reductase, and thioredoxin reductase). The mechanisms of several of these enzymes will also be discussed.

DNA-binding and partial nucleoid localization of the chloroplast stromal enzyme ferredoxin : sulfite reductase

Sulfite reductase (SiR) is an important enzyme catalyzing the reduction of sulfite to sulfide during sulfur assimilation in plants. This enzyme is localized in plastids, including chloroplasts, and uses ferredoxinu2003as an electron donor. Ferredoxin‐dependent SiR has been found in isolated chloroplast nucleoids, but its localization inu2003vivo or in intact plastids has not been examined. Here, we report the DNA‐binding properties of SiRs from pea (PsSiR) and maize (ZmSiR) using an enzymatically active holoenzyme with prosthetic groups. PsSiR binds to both double‐stranded and single‐stranded DNA without significant sequence specificity. DNA binding did not affect the enzymatic activity of PsSiR, suggesting that ferredoxin and sulfite are accessible to SiR molecules within the nucleoids. Comparison of PsSiR and ZmSiR suggests that ZmSiR does indeed have DNA‐binding activity, as was reported previously, but the DNA affinity and DNA‐compacting ability are higher in PsSiR than in ZmSiR. The tight compaction of nucleoids by PsSiR led to severe repression of transcription activity in pea nucleoids. Indirect immunofluorescence microscopy showed that the majority of SiR molecules colocalized with nucleoids in pea chloroplasts, whereas no particular localization to nucleoids was detected in maize chloroplasts. These results suggest that SiR plays an essential role in compacting nucleoids in plastids, but that the extent of association of SiR with nucleoids varies among plant species.

NMR study of the electron transfer complex of plant ferredoxin and sulfite reductase: mapping the interaction sites of ferredoxin.

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP+ reductase is the interaction partner

Fd : FNR Electron Transfer Complexes: Evolutionary Refinement of Structural Interactions

During the evolution of higher-plant root and leaf-type-specific Fd : FNR complexes from an original cyanobacterial type progenitor, rearrangement of molecular interaction has altered the relative orientation of prosthetic groups and there have been changes in complex induced conformational change. Selection has presumably worked on mutation of residues responsible for interaction between the two proteins, favoring optimized electron flow in a specific direction, and efficient dissociation following specific oxidation of leaf Fd and reduction of root Fd. Major changes appear to be: loss in both leaf and root complexes of a cyanobacterial mechanism that ensures Fd dissociation from the complex following change in Fd redox state, development of a structural rearrangement of Fd on binding to leaf FNR that results in a negative shift in Fd redox potential favorable to photosynthetic electron flow, creation of a vacant space in the root Fd:FNR complex that may allow access to the redox centers of other enzymes to ensure efficient channeling of heterotrophic reductant into bioassimilation. Further structural analysis is essential to establish how root type FNR distinguishes between Fd isoforms, and discover how residues not directly involved in intermolecular interactions may affect complex formation.

Arrest of chlorophyll synthesis and differential decrease of Photosystems I and II in a cyanobacterial mutant lacking light-independent protochlorophyllide reductase

The chlL gene encodes one subunit of the light-independent protochlorophyllide reductase. A chlL-lacking mutant of the cyanobacterium Plectonema boryanum is unable to synthesize chlorophyll (Chl) in the dark, causing Chl synthesis to become light-dependent as in angiosperms. When the mutant cells were cultivated heterotrophically in the dark, Chl synthesis was arrested and the Chl content decreased exponentially in reverse profile to cell propagation, indicating that most of the pre-existing Chl was recruited for daughter cells. During this `etiolating process the Chl content became less than 0.5% of the original level. In parallel to this there was a decrease in the activity of Photosystem I (PSI), the amount of its core Chl-binding subunits, PsaA/PsaB, and a peripheral subunit, PsaC. Levels of transcripts for these subunits were not significantly changed upon the arrest of Chl synthesis. In contrast, Photosystem II (PSII) was maintained to a significant extent in terms of activity and protein levels of D1 and CP47 until a late stage of the etiolation, implying that PSII is newly synthesized though Chl synthesis was arrested. Low-temperature (77xa0K) fluorescence spectral analysis supported a selective decrease in Chl associated with PSI. Taken together, it is suggested that the pre-existing Chl molecules in periphery of PSI could be released and re-distributed for PSII biosynthesis in the etiolating cyanobacterial cells.

FAD assembly and thylakoid membrane binding of ferredoxin:NADP+ oxidoreductase in chloroplasts

We investigated the process of flavin adenine dinucleotide (FAD) incorporation into the ferredoxin (Fd):NADP+ oxidoreductase (FNR) polypeptide during FNR biosynthesis, using pull‐down assay with resin‐immobilized Fd which bound strongly to FAD‐assembled holo‐FNR, but hardly to FAD‐deficient apo‐FNR. After FNR precursor was imported into isolated chloroplasts and processed to the mature size, the molecular form pulled down by Fd‐resin increasingly appeared. The mature‐sized FNR (mFNR) accumulated transiently in the stroma as the apo‐form, and subsequently bound on the thylakoid membranes as the holo‐form. Thus, FAD is incorporated into the mFNR inside chloroplasts, and this assembly process is followed by the thylakoid membrane localization of FNR.

A ferredoxin Arg-Glu pair important for efficient electron transfer between ferredoxin and ferredoxin-NADP(+) reductase.

In order to elucidate the importance of a ferredoxin (Fd) Arg‐Glu pair involved in dynamic exchange from intra‐ to intermolecular salt bridges upon complex formation with ferredoxin‐NADP+ oxidoreductase (FNR), Equisetum arvense FdI and FdII were investigated as normal and the pair‐lacking Fd, respectively. The FdI mutant lacking this pair was unstable and rapidly lost the [2Fe–2S] cluster. The catalytic constant (k cat) of the electron transfer for FdI is 5.5 times that for FdII and the introduction of this pair into FdII resulted in the increase of k cat to a level comparable to that for FdI, demonstrating directly that the Arg‐Glu pair is important for efficient electron transfer between Fd and FNR.