title: Methemoglobin reduction by NADH-cytochrome b(5) reductase in Propsilocerus akamusi larvae.
abstract: For oxygen respiration, a methemoglobin (metHb) reduction system is needed in the cell because metHb cannot bind oxygen. We examined the insect Propsilocerus akamusi larvae to elucidate the metHb reduction system in an organism that inhabits an oxygen-deficient environment. NADH-dependent reduction of metHb in coelomic fluid suggested the coexistence of cytochrome b5 reductase (b5R) and cytochrome b5 with hemoglobin in the fluid and that these proteins were involved in physiological metHb reduction in the larvae. The presence of b5R was revealed by purifying b5R to homogeneity from the midge larvae. Using purified components, we showed that larval metHb was reduced via the NADH-b5R (FAD)-cytochrome b5-metHb pathway, a finding consistent with that in aerobic vertebrates, specifically humans and rabbits, and b5R function between mammal and insect was conserved. b5R was identified as a monomeric FAD-containing enzyme; it had a molecular mass of 33.2 kDa in gel-filtration chromatography and approximately 37 kDa in SDS-PAGE analysis. The enzyme's optimal pH and temperature were 6.4 and 25 °C, respectively. The apparent Km and Vmax values were 345 μM and 160 μmol min(-1) mg(-1), respectively, for ferricyanide and 328 μM and 500 μmol min(-1) mg(-1), respectively, for 2,6-dichlorophenolindophenol. The enzyme reaction was inhibited by benzoate, p-hydroxymercuribenzoate, iodoacetamide, and iodoacetate, and was not inhibited by metal ions or EDTA.
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo 101-8308, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Maeda
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo 101-8308, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kobori
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo 101-8308, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Tanigawa
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo 101-8308, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Sato
authors.affiliation: Department of Biochemistry, Faculty of Science, Okayama University of Science, Okayama 700-0005, Japan.
authors.forename: Toshitsugu
authors.initials: T
authors.lastname: Yubisui
authors.affiliation: Division of Bioengineering, Graduate School of Engineering Science, Osaka University, Osaka 560-8531, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hori
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo 101-8308, Japan. Electronic address: ocean_nagata@axel.ocn.ne.jp.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nagata
mesh_terms: D000818:Animals; D042966:Cytochrome-B(5) Reductase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004175:Diptera / Q000201:enzymology* / Q000378:metabolism; D007814:Larva / Q000201:enzymology / Q000378:metabolism; D008706:Methemoglobin / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D008706:Methemoglobin; D042966:Cytochrome-B(5) Reductase
keywords: Hemoglobin; Methemoglobin; NADH–cytochrome b(5) reductase; Propsilocerus akamusi