Toru Hisabori

ATP synthase — a marvellous rotary engine of the cell

ATP synthase can be thought of as a complex of two motors — the ATP-driven F1 motor and the proton-driven Fo motor — that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

Comprehensive survey of proteins targeted by chloroplast thioredoxin

Possible target proteins of chloroplast thioredoxin (Trx) have been investigated in the stroma lysate of spinach chloroplasts. For that purpose, we immobilized a mutant of m-type Trx in which an internal cysteine at the active site was substituted with serine, on cyanogen bromide-activated resin. By using this resin, the target proteins in chloroplast were efficiently acquired when they formed the mixed-disulfide intermediates with the immobilized Trxs. We could acquire Rubisco activase (45 kDa) and 2-Cys-type peroxiredoxin (Prx), which were recently identified as targets of chloroplast Trxs. Glyceraldehyde-3-phosphate dehydrogenase and sedoheputulose 1,7-bisphosphatase, well-known thiol enzymes in the Calvin cycle, also were recognized among the collected proteins, suggesting the method is applicable for our purpose. Furthermore, four proteins were identified from a homology search of the NH2-terminal sequence of the acquired proteins: glutamine synthetase, a protein homologous to chloroplast cyclophilin, a homolog of Prx-Q, and the Rubisco small subunit. The Trx susceptibilities of the recombinant cyclophilin and Prx-Q of Arabidopsis thaliana were then examined. The method developed in the present study is thus applicable to investigate the various redox networks via Trxs and the related enzymes in the cell.

HCF164 Receives Reducing Equivalents from Stromal Thioredoxin across the Thylakoid Membrane and Mediates Reduction of Target Proteins in the Thylakoid Lumen

HCF164 is a membrane-anchored thioredoxin-like protein known to be indispensable for assembly of cytochrome b6 f in the thylakoid membranes. In this study, we report the finding that chloroplast stroma m-type thioredoxin is the source of reducing equivalents for reduction of HCF164 in the thylakoid lumen, providing strong evidence that higher plant chloroplasts possess a trans-membrane reducing equivalent transfer system similar to that found in bacteria. To probe the function of HCF164 in the lumen, a screen to identify the reducing equivalent acceptor proteins of HCF164 was carried out by using a resin-immobilized HCF164 single cysteine mutant, leading to the isolation of putative target thylakoid proteins. Among the newly identified target proteins, the reduction of the PSI-N subunit of photosystem I by HCF164 was confirmed both in vitro and in isolated thylakoids. Two components of the cytochrome b6 f complex, the cytochrome f and Rieske FeS proteins, were also identified as novel potential target proteins. The data presented here suggest that HCF164 serves as an important transducer of reducing equivalents to proteins in the thylakoid lumen.

The CHLI1 Subunit of Arabidopsis thaliana Magnesium Chelatase Is a Target Protein of the Chloroplast Thioredoxin

Insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway, which takes place in plant chloroplasts. ATP hydrolysis by the CHLI subunit of magnesium chelatase is an essential component of this reaction, and the activity of this enzyme is a primary determinant of the rate of magnesium insertion into the chlorophyll molecule (tetrapyrrole ring). Higher plant CHLI contains highly conserved cysteine residues and was recently identified as a candidate protein in a proteomic screen of thioredoxin target proteins (Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., and Buchanan, B. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 370–375). To study the thioredoxin-dependent regulation of magnesium chelatase, we first investigated the effect of thioredoxin on the ATPase activity of CHLI1, a major isoform of CHLI in Arabidopsis thaliana. The ATPase activity of recombinant CHLI1 was found to be fully inactivated by oxidation and easily recovered by thioredoxin-assisted reduction, suggesting that CHLI1 is a target protein of thioredoxin. Moreover, we identified one crucial disulfide bond located in the C-terminal helical domain of CHLI1 protein, which may regulate the binding of the nucleotide to the N-terminal catalytic domain. The redox state of CHLI was also found to alter in a light-dependent manner in vivo. Moreover, we successfully observed stimulation of the magnesium chelatase activity in isolated chloroplasts by reduction. Our findings strongly suggest that chlorophyll biosynthesis is subject to chloroplast biogenesis regulation networks to coordinate them with the photosynthetic pathways in chloroplasts.

ε Subunit, an Endogenous Inhibitor of Bacterial F1-ATPase, Also Inhibits F0F1-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980)Biochemistry 19, 526–531), the ε subunit, an endogenous inhibitor of bacterial F1-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F0F1-ATPase. However, we report here that the ε subunit is exerting inhibition in F0F1-ATPase. We prepared a C-terminal half-truncated ε subunit (εΔC) of the thermophilicBacillus PS3 F0F1-ATPase and reconstituted F1- and F0F1-ATPase containing εΔC. Compared with F1- and F0F1-ATPase containing intact ε, those containing εΔC showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F0F1-ATPase containing εΔC was capable of ATP-driven H+ pumping. The time-course of pumping at low ATP concentration was faster than that by the F0F1-ATPase containing intact ε. Thus, the comparison with noninhibitory εΔC mutant shed light on the inhibitory role of the intact ε subunit in F0F1-ATPase.

The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit.

In F1-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the β subunit moves and contacts the rotor γ subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous work, however, disproved the assumption (Hara, K. Y., Noji, H., Bald, D., Yasuda, R., Kinosita, K., Jr., and Yoshida, M. (2000) J. Biol. Chem. 275, 14260–14263), and the role of this motif remained unknown. Here, we found that the ε subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F1-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the ε subunit in the mutant F1-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the ε subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the β subunit or of the basic residues in the C-terminal α-helix of the ε subunit induced a partial effect. We here conclude that the ε subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the β subunit.

The γ subunit in chloroplast F1-ATPase can rotate in a unidirectional and counter-clockwise manner

Rotation of the γ subunit in chloroplast F1‐ATPase (CF1) was investigated by using a single molecule observation technique, which is developed by Noji et al. to observe the rotation of a central γ subunit portion in the α3β3γ sub‐complex of F1‐ATPase from thermophilic Bacillus PS3 (TF1) during ATP hydrolysis [Noji, H. et al. (1997) Nature 386, 299–302]. We used two cysteines of the γ subunit (Cys‐199 and Cys‐205) of CF1‐ATPase, which are involved in the regulation of this enzyme, to fix the fluorochrome‐labeled actin filament. Then we successfully observed a unidirectional, counter‐clockwise rotation of the actin filament with the fluorescent microscope indicating the rotation of the γ subunit in CF1‐ATPase. We conclude that the rotation of the γ subunit in the F1‐motor is a ubiquitous phenomenon in all F1‐ATPases in prokaryotes as well as in eukaryotes.

THERMOPHILIC F1-ATPASE IS ACTIVATED WITHOUT DISSOCIATION OF AN ENDOGENOUS INHIBITOR, EPSILON SUBUNIT

Subunit complexes (α3β3γ, α3β3γδ, α3β3γε, and α3β3γδε) of thermophilic F1-ATPase were prepared, and their catalytic properties were compared to know the role of δ and ε subunits in catalysis. The presence of δ subunit in the complexes had slight inhibitory effect on the ATPase activity. The effect of ε subunit was more profound. The (−ε) complexes, α3β3γ and α3β3γδ, initiated ATP hydrolysis without a lag. In contrast, the (+ε) complexes, α3β3γε and α3β3γδε, started hydrolysis of ATP (<700 μm) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (+ε) complexes became shorter, and it was not observed above 1 mm ATP. Analysis of binding and hydrolysis of the ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP, suggested that the (+ε) complexes bound substrate only slowly. Differing fromEscherichia coli F1-ATPase, the activation of the (+ε) complexes from the lag phase was not due to dissociation of ε subunit since the re-isolated activated complex retained ε subunit. This indicates that there are two alternative forms of the (+ε) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

Structural Asymmetry of F1-ATPase Caused by the γ Subunit Generates a High Affinity Nucleotide Binding Site

The α3β3γ and α3β3 complexes of F1-ATPase from a thermophilic Bacillus PS3 were compared in terms of interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) that differed from ATP and ADP and did not destabilize the α3β3 complex. The results of equilibrium dialysis show that the α3β3γ complex has a high affinity nucleotide binding site and several low affinity sites, whereas the α3β3 complex has only low affinity sites. This is also supported from analysis of spectral change induced by TNP-ADP, which in addition indicates that this high affinity site is located on the β subunit. Single-site hydrolysis of substoichiometric amounts of TNP-ATP by the α3β3γ complex is accelerated by the chase addition of excess ATP, whereas that by the α3β3 complex is not. We further examined the complexes containing mutant β subunits (Y341L, Y341A, and Y341C). Surprisingly, in spite of very weak affinity of the isolated mutant β subunits to nucleotides (Odaka, M., Kaibara, C., Amano, T., Matsui, T., Muneyuki, E., Ogasawara, K., Yutani, K., and Yoshida, M.(1994) J. Biochem. (Tokyo) 115, 789-796), a high affinity TNP-ADP binding site is generated on the β subunit in the mutant α3β3γ complexes where single-site TNP-ATP hydrolysis can occur. ATP concentrations required for the chase acceleration of the mutant complexes are higher than that of the wild-type complex. The mutant α3β3 complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP rather slowly, and there is no chase acceleration. Thus, the γ subunit is responsible for the generation of a high affinity nucleotide binding site on the β subunit in F1-ATPase where cooperative catalysis can proceed.

A facilitated electron transfer of copper--zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode.

The direct electrochemical redox reaction of bovine erythrocyte copper--zinc superoxide dismutase (Cu(2)Zn(2)SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65 +/- 3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant k(s) is 1.2 +/- 0.2 s(-1) and the anodic (alpha(a)) and cathodic (alpha(c)) transfer coefficients are 0.39 +/- 0.02 and 0.61 +/- 0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu(2)Zn(2)SOD), its Cu- or Zn-free derivatives (E(2)Zn(2)SOD and Cu(2)E(2)SOD, E designates an empty site) and the SOD reconstituted from E(2)Zn(2)SOD and Cu(2+). The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion.