No abstract

The NDI1 gene encoding rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria was cotransfected into the complex I-deficient Chinese hamster CCL16-B2 cells. Stable NDI1-transfected cells were obtained by screening with antibiotic G418. The NDI1 gene was shown to be expressed in the transfected cells. The expressed Ndi1 enzyme was recognized to be localized to mitochondria by immunoblotting and confocal immunof luorescence microscopic analyses. Using digitonin-permeabilized cells, it was shown that the transfected cells, but not nontransfected control cells, exhibited the electron transfer activities with glutamate͞malate as the respiratory substrate. The activities were inhibited by f lavone, antimycin A, and KCN but not by rotenone. Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the inner mitochondrial membranes. Furthermore, although nontransfected cells could not survive in a medium low in glucose (0.6 mM), which is a substrate of glycolysis, the NDI1-transfected cells were able to grow in the absence of added glucose. When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration is required for cells to survive. The mutant cells do not survive at low glucose or in galactose, but they can be rescued by Ndi1. These results indicated that the S. cerevisiae Ndi1 was expressed functionally in CCL16-B2 cells and catalyzed electron transfer from NADH in the matrix to ubiquinone-10 in the inner mitochondrial membranes. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.Mammalian NADH-quinone (Q) oxidoreductase (complex I) is composed of at least 43 distinct subunits and has the most intricate structure of the membrane-bound mitochondrial enzyme complexes (1). Of these subunits, seven are encoded by mitochondrial DNA and others are encoded by nuclear DNA (2, 3). Complex I contains noncovalently bound FMN and at least five EPR-detectable iron-sulfur clusters as prosthetic groups (4-7). It has been shown in recent years that structural and functional defects of complex I are involved in many human mitochondrial diseases (8-10). At present, mutations and deletions of the seven mtDNA-encoded subunits are not correctable and mutations of multiple subunits encoded by nuclear DNA are difficult to repair. Various chemotherapies have been reported to be ineffective at the present time (11). Dysfunction of complex I presents three problems (12): (i) impairment of the ability of the respiratory chain to oxidize NADH to NAD; (ii) impairment of the ability of this enzyme to pump protons, which results in a decrease in the rate of ATP synthesis; and (iii) production of superoxide radicals, causing mitochondrial DNA mutations, lipid peroxidation, and protein denaturation. Of the three problems, the impairment of proton pumping by one of the three proton translocation sites d...

Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI (ϳ40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having alltrans retinal as a chromophore. UV-Vis, low-temperature UVVis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.

Photosynthetic water oxidation takes place at the Mn 4 CaO 5 cluster in photosystem II. Although the atomic structures of its intermediates called S states have recently been reported, the catalytic mechanism of water oxidation has not been well understood. Here, to investigate the involvement of the O4 site of the Mn 4 CaO 5 cluster and a water channel from O4 in the water oxidation reaction, we examined the effects of D1-S169A mutation, which perturbs the interaction of a water molecule hydrogen-bonded with O4, by thermoluminescence (TL), Fourier transform infrared (FTIR) difference, and time-resolved infrared (TRIR) measurements. The observed upshifts of TL peaks and some changes in FTIR spectra upon S169A mutation revealed the perturbations of the redox potential of the Mn 4 CaO 5 cluster and the interactions of the surrounding hydrogen bond network. In contrast, FTIR oscillation patterns and TRIR traces showed only minor effects of the mutation on the efficiencies and kinetics of individual S-state transitions. It was thus concluded that the O4 site plays a role in retaining the redox potential and the structure of the hydrogen bond network, whereas it is unlikely to be directly involved in the catalytic reaction of substrate water except for proton transfer through the O4 water chain.

No abstract

In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and 'the rest' of the present family Halobacteriaceae.

The NDI1 gene encodes the internal rotenoneinsensitive NADH-quinone oxidoreductase localized in the inner mitochondrial membranes of Saccharomyces cerevisiae. The T7 tag-fused mature NDI1 was overexpressed in Escherichia coli. The overexpressed NDI1 was exclusively found in the membrane fraction. The NDI1-overexpressed membranes showed significantly increased activities of NADH oxidase and NADHubiquinone-1 (UQ I ) reductase when compared with the control membranes. Flavone, which is a specific inhibitor of the S. cerevisiae NDI1, inhibited almost completely NADH oxidase and NADH-UQ I reductase activities of NDI1-overexpressed membranes but scarcely inhibited these activities of the control membranes. In addition, the NADH oxidase activity of the NDI1-overexpressed membranes was also inhibited by KCN as well as the control membranes. These results indicate that the overexpressed NDI1 worked as a member of the respiratory chain in the host cells, even though E. coli membranes are different from S. cerevisiae inner mitochondrial membranes in terms of quinones and lipid composition.z 1998 Federation of European Biochemical Societies.

Photosynthetic oxygen evolution is performed at the Mn cluster in photosystem II (PSII). The advent of this reaction on ancient Earth changed its environment by generating an oxygenic atmosphere. However, how oxygen evolution originated during the PSII evolution remains unknown. Here, we characterize the site-directed mutants at the carboxylate ligands to the Mn cluster in cyanobacterial PSII. A His residue replaced for D1-D170 is found to be post-translationally converted to the original Asp to recover oxygen evolution. Gln/Asn residues in the mutants at D1-E189/D1-D342 are also converted to Glu/Asp, suggesting that amino-acid conversion is a common phenomenon at the ligand sites of the Mn cluster. We hypothesize that post-translational generation of carboxylate ligands in ancestral PSII could have led to the formation of a primitive form of the Mn cluster capable of partial water oxidation, which could have played a crucial role in the evolutionary process of photosynthetic oxygen evolution.