Respiratory complex I in mitochondrial membrane catalyzes oversized ubiquinones

NADH-ubiquinone (UQ) oxidoreductase (complex I) couples electron transfer from NADH to UQ with proton translocation in its membrane part. The UQ reduction step is key to triggering proton translocation. Structural studies have identified a long, narrow, tunnel-like cavity within complex I, through which UQ may access a deep reaction site. To elucidate the physiological relevance of this UQ-accessing tunnel, we previously investigated whether a series of oversized UQs (OS-UQs), whose tail moiety is too large to enter and transit the narrow tunnel, can be catalytically reduced by complex I using the native enzyme in bovine heart submitochondrial particles (SMPs) and the isolated enzyme reconstituted into liposomes. Nevertheless, the physiological relevance remained unclear because some amphiphilic OS-UQs were reduced in SMPs but not in proteoliposomes, and investigation of extremely hydrophobic OS-UQs was not possible in SMPs. To uniformly assess the electron transfer activities of all OS-UQs with the native complex I, here we present a new assay system using SMPs, which were fused with liposomes incorporating OS-UQ and supplemented with a parasitic quinol oxidase to recycle reduced OS-UQ. In this system, all OS-UQs tested were reduced by the native enzyme, and the reduction was coupled with proton translocation. This finding does not support the canonical tunnel model. We propose that the UQ reaction cavity is flexibly open in the native enzyme to allow OS-UQs to access the reaction site, but their access is obstructed in the isolated enzyme as the cavity is altered by detergent-solubilizing from the mitochondrial membrane

Diverse reaction behaviors of artificial ubiquinones in mitochondrial respiratory complex I

The ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQ(m-1)/pUQ(p-1) and pUQ(m-2)/pUQ(p-2)), with each pair having the same chemical properties except for a similar to 1.0 angstrom difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [I-125]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [I-125]pUQs used, indicating that [I-125]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model.Peer reviewe

Inhibitors of a Na⁺-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function

The Na⁺-pumping NADH-ubiquinone (UQ) oxidoreductase (Na⁺-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na⁺-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na⁺-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na⁺-NQR

22449 Skeleton curve and increase of strength due to strain hardening of buckling restrained brace using steel mortar planks

研究報告書ページ(143)-(144

03 【3.1.1 鋼製デバイスの実験および開発】成果 鋼モルタル板を用いた座屈拘束ブレース －履歴型ダンパーとしての性能評価－

３．研究成果の概要　３.１〈研究テーマ①〉制振・免震デバイスの性能確認実験および開

Buckling-restrained braces using high performance hard polyurethane foam

The authors developed a buckling‐restrained brace for using steel framed houses．The buckling‐restrained brace is small‐sized and light‐weighted with a restraining part of hard polyurethane or mortar．Conducting static and cyclic axial loading tests，elastoplastic and stiffening properties are difined．Specimens with the restraining parts of various restraining forces were fabricated．The authors considere the test data on cumulative ductility factors and cumulative plastic strain energy，and evaluate its performance