Showing papers by "Jun Minagawa published in 1998"

Substitutions of conserved aromatic amino acid residues in subunit I perturb the metal centers of the Escherichia coli bo-type ubiquinol oxidase.

Abstract: Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, whose axial ligands have been identified to be six invariant histidines. This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp. Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII−VIII, and Phe348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity. Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center. On the basis of these findings and recent crystallographic studies on c...

Characterization of Photosystem II Donor-Side Mutants in Chlamydomonas Reinhardtii

Abstract: By utilizing the energy of light Photosystem II (PSII) oxidizes water to molecular oxygen and reduces plastoquinone [1]. The membrane proteins D1 and D2 constitute the PSII reaction center and form a heterodimer that binds the redox components necessary for the primary electron transfer reactions. These bound redox components include P680, the acceptor complex including the pheophytin, the first and second quinone acceptors, QA and QB and the Fe2+ ion. On the donor side the heterodimer contains two redox active tyrosine residues, the immediate electron donor to P680+, Tyrz, and an accessory electron donor, TyrD. Probably the Dl/D2 heterodimer also binds the Mn-cluster involved in the water oxidation reaction consisting of four Mn ions and a Ca2+ ion [2–5].

Cloning and Characterization of a cDNA for Algal Homologue of CP26 in the Light-Harvesting Complex II of Chlamydomonas Reinhardtii

Abstract: CP29, CP26, and CP24 have been identified as ‘minor’ Chl-alb binding proteins, since they were less abundant and copurified in the ‘LHC II-fraction’. Due to the low amount of bound pigments, it seems unlikely that the main function of these Chl-alb binding proteins is to harvest light. In fact, important observations have been reported in the past decade, suggesting that these proteins play crucial role(s) in energy distribution and photoprotection in green plants. These observations include, 1) non-photochemical quenching is closely related to the xanthophyll cycle turnover, 2) qE-quenching is inhibited by DCCD, 3) DCCD is preferentially bound to CP26 and CP29, 4) xanthophyll violaxanthins are preferentially bound in these minor Chl-alb binding proteins, and 5) the minor Chl-alb binding proteins are localized between the outer antenna and the reaction center core (for review, see ref. 1, 2). The unicellular green alga Chlamydomonas reinhardtii provides an excellent system to study the photosynthetic apparatus because of the ability to grow heterotrophically, the feasibility of performing biophysical measurements in vivo and availability of well-established techniques for transforming cells with foreign DNA. Genes encoding CP26 have been previously characterized from scots pine (3), tomato (4), barley (5), and maize (6). However, whereas homologues of CP26 and CP29 in this alga, p10 and p9, respectively, had been identified (7), there has been no report on their polypeptide sequences. In this study, p 10 protein was purified from C. reinhardtii and the partial amino acid sequences were determined, we further report the isolation and characterization of a cDNA encoding p10 protein with the aim of understanding to what extent they are comparable to those in higher plants and, in the long run, of investigating physiological roles of the polypeptides in vivo.

Rapid Isolation and Characterization of His-Tagged PS II Core Complex from Chlamydomonas Reinhardtii

Abstract: Photosystem II (PS II) core complex is the structural minimum required for evolution of molecular oxygen from water, and consists of more than 10 subunit proteins including the heterodimer of D1 and D2 proteins, CM-binding inner antenna proteins of CP43 and CP47, a hemoprotein of Cyt b-559 and several low-molecular-weight membrane proteins. Aiming at elucidation of the mechanism of oxygen evolution, several types of oxygen-evolving PS II core complexes have been purified from thylakoids of higher plants, green alga, and cyanobacteria, by density-gradient centrifugation of detergent-solubilized thylakoids, followed by anion exchange and size-exclusion column chromatographies. The oxygen-evolving activities retained in these preparations differed variously, due probably to denaturation during the time-consuming purification steps required for eliminating LHC and PS I complex, so that development of a rapid and simple protocol for isolating the oxygen-evolving PS II core complex has been desired.