Kohei Suruga

Structure of cytochrome c6 from the red alga Porphyra yezoensis at 1.57 Å resolution

The crystal structure of cytochrome c(6) from the red alga Porphyra yezoensis has been determined at 1.57 A resolution. The crystal is tetragonal and belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 49.26 (3), c = 83.45 (4) A and one molecule per asymmetric unit. The structure was solved by the molecular-replacement method and refined with X-PLOR to an R factor of 19.9% and a free R factor of 25.4%. The overall structure of cytochrome c(6) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys14 and Cys17, and the iron has an octahedral coordination with His18 and Met58 as the axial ligands. The sequence and the structure of the eukaryotic red algal cytochrome c(6) are very similar to those of a prokaryotic cyanobacterial cytochrome c(6) rather than those of eukaryotic green algal c(6) cytochromes.

Increasing the conformational stability by replacement of heme axial ligand in c‐type cytochrome

To investigate the role of the heme axial ligand in the conformational stability of c‐type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c 6 in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (ΔG°unf=1.48 kcal/mol) was lower than that of the wild‐type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a ΔG°unf of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild‐type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild‐type according to the heme‐bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c‐type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.

Crystal structure of oxidized cytochrome c6A from Arabidopsis thaliana

Compared with algal and cyanobacterial cytochrome c 6, cytochrome c 6A from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c 6A from Arabidopsis thaliana at 1.5 Å resolution in order to help elucidate its function. The overall structure of cytochrome c 6A follows the topology of class I c‐type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c 6A. Our model provides a chemical basis for the known low redox potential of cytochrome c 6A which makes it an unsuitable electron carrier between cytochrome b 6 f and PSI.

Appearance of Nitrite Reducing Activity of Cytochrome c upon Heat Denaturation

The appearance of NO2 − reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100°C for 30 min, had NO2 − reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, exposure of the heme, dissociation of the sixth ligand methionine sulfur, and appearance of autoxidizability. The dCyt c catalyzed NO2 − reduction to NH4 + via ferrous-NO complexes, and this reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized dCyt c had activity similar strength to that in solution. The resin retained the activity after five uses and even after storage for 1 year. On the basis of these results, we concluded that Cyt c acquired a new catalytic activity upon heat treatment, unlike to other familiar biological molecules.

Antitumour and Apoptotic Effects of a Plant Extract Mixture Containing Rhus verniciflua and Other Herbs in Human Leukaemia Cells.

The apoptotic effects of a novel antitumour agent (Rv-PEM01) prepared from 6 kinds of herbs, including Rhus verniciflua were investigated using flow cytometry and western blot analysis. Rv-PEM01 induced apoptosis but not necrosis in MOLT-3, KG-1, and K562 human leukaemia cell lines. Further, Rv-PEM01-treated cells showed significantly upregulated expression of caspase-3 and 9 and cleaved caspase-3 and 9 compared to the control cells. Taken together, the results suggest that Rv-PEM01 induced apoptosis via the mitochondrial-mediated pathway, and is a potential natural anticancer agent and/or a functional food material.

“Okara” a New Preparation of Food Material with Antioxidant Activity and Dietary Fiber from Soybean

Okara (OC) is a byproduct of the production of soybean foods such as tofu and soy milk. It is a nutrient-rich product, containing about 25% protein, 20% fat and 33% dietary fiber on a dry basis. Approximately 700,000 tons of okara are produced in Japan each year, some of which has been utilized as a feed for domestic animals and as a fertilizer (O’Toole, 1999). Most of it, however, is discarded as industrial waste because is perishable and other uses for it have not been identified (Ohno & Shoda, 1993), which has created social and environmental problems. Although many papers have reported methods for fermentation (Matsuo, 1997; Jiang et al., 2005; Mizumoto et al., 2006), extraction (Quitain, 2006) and digestion (Kasai et al., 2004), development of effective methods for OC utilization remain an important and difficult challenge. So, as part of a study increase utilization of OC, we have developed a new food product by combining fermented OC with fruits, especially banana. We investigated two properties of this food, reactive oxygen scavenging activity, the effect of including it as part of diet therapy for obesity using dogs as a model.

Changes in Plasma Metabolites Concentrations in Obese Dogs Supplemented With Anti-oxidant Compound

The aim of this study is to discuss the effect of anti-oxidant supplement (Rv-PEM01-99, Kibun Foods, Inc., Tokyo, Japan) on changes in energy metabolism in obese dogs. 200 mg/kg/day of Rv-PEM01-99 (equivalent to 5 mg kg/day of quercetin derivative) were applied for 6 weeks to the Beagle dogs fed high fat diet (HFD) or control diet (CD). In the present study, body weight (BW) decreasing effect of Rv-PEM 01-99 in obese dogs was not clear. However, plasma alkaline phosphatase (ALP) activities at the end of experiment were significantly decreased compared to those at the start of experiment in obese dogs supplemented with Rv-PEM 01-99 (paired-t test, p < 0.05). In control dogs supplemented with Rv-PEM 01-99, Plasma malondialdehyde (MDA), and triglycerides (TG) levels and lactate dehydrogenase (LDH) activities were significantly decreased compared to those at the start of experiment (paired-t test, p < 0.05). From these findings, Rv-PEM 01-99 seems to be not harmful for dogs. Anti-lipid peroxide effect and liver function improvement are expected in the dogs supplemented with Rv-PEM 01-99.