Mitochondrial respiratory enzymes play a central role in energy production in aerobic organisms. They differentiated from the ␣-proteobacteria-derived ancestors by adding noncatalytic subunits. An exception is Complex II (succinate: ubiquinone reductase), which is composed of four ␣-proteobacteria-derived catalytic subunits (SDH1-SDH4). Complex II often plays a pivotal role in adaptation of parasites in host organisms and would be a potential target for new drugs. We purified Complex II from the parasitic protist Trypanosoma cruzi and obtained the unexpected result that it consists of six hydrophilic (SDH1, SDH2 N , SDH2 C , and SDH5-SDH7) and six hydrophobic (SDH3, SDH4, and SDH8 -SDH11) nucleus-encoded subunits. Orthologous genes for each subunit were identified in Trypanosoma brucei and Leishmania major. Notably, the iron-sulfur subunit was heterodimeric; SDH2 N and SDH2 C contain the plant-type ferredoxin domain in the N-terminal half and the bacterial ferredoxin domain in the C-terminal half, respectively. Catalytic subunits (SDH1, SDH2 N plus SDH2 C , SDH3, and SDH4) contain all key residues for binding of dicarboxylates and quinones, but the enzyme showed the lower affinity for both substrates and inhibitors than mammalian enzymes. In addition, the enzyme binds protoheme IX, but SDH3 lacks a ligand histidine. These unusual features are unique in the Trypanosomatida and make their Complex II a target for new chemotherapeutic agents.The parasitic protist Trypanosoma cruzi is the etiological agent of Chagas disease, a public health threat in Central and South America. These parasites are normally transmitted by reduviid bugs via the vector feces after a bug bite and also via transfusion of infected blood. About 16 -18 million people are infected, and 100 million are at risk, but there are no definitive chemotherapeutic treatments available (1). Despite having potential pathways for oxidative phosphorylation (2), all trypanosomatids (Trypanosoma and Leishmania species) analyzed so far are characterized by incomplete oxidation of glucose with secretion of end products, such as succinate, alanine, ethanol, acetate, pyruvate, and glycerol (3, 4) (Fig. 1). Major routes for formation of succinate in Trypanosoma brucei are via NADH-dependent fumarate reductase in glycosomes and mitochondria (5, 6). In trypanosomatid mitochondria, the Krebs cycle is inefficient, and pyruvate is principally converted to acetate via acetate:succinate CoA transferase (7). A part of the Krebs cycle operates the utilization of histidine in the insect stage of T. cruzi (8).Mitochondrial Complex II (succinate:quinone reductase (SQR) 5 and succinate dehydrogenase (SDH)) serves as a membrane-bound Krebs cycle enzyme and often plays a pivotal role in adaptation of parasites to environments in their host (9, 10). In general, Complex II consists of four subunits (11). A flavoprotein subunit (SDH1, Fp) and an iron-sulfur subunit (SDH2, Ip) form a soluble heterodimer, which then binds to a membrane anchor heterodimer, SDH3 (CybL) and SDH4 (CybS)....

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Two forms of NAD-dependent d-mandelate dehydrogenase (d-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different Km and V max values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.

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The gene for the NAD-dependent formate dehydrogenase (FDH) of Paracoccus sp. 12-A, a formate-assimilating bacterium, was cloned through screening of the genomic library with activity staining. The FDH gene included an open reading frame of 1,200 base pairs, and encoded a protein of 43,757 Da, which had high amino acid sequence identity with known FDHs, in particular, with bacterial enzymes such as those of Moraxella sp. (86.5%) and Pseudomonas sp. 101 (83.5%). The gene was highly expressed in Escherichia coli cells using an expression plasmid with the pUC ori and tac promoter. The recombinant enzyme was somewhat inactive in the stage of the cell-free extract, but its activity markedly increased with purification, in particular, with the step of heat-treatment at 50 degrees C. The purified enzyme showed essentially the same properties as the enzyme from the original Paracoccus cells.

Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 μm × 1.2 μm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 μm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect.