Methemoglobin reduction by NADH-cytochrome b5 reductase in Propsilocerus akamusi larvae

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 b(5) reductase (b5R) and cytochrome b(5) with hemoglobin in the fluid and that these proteins were involved in physiological metHb reduction in the larvae. The presence of b(5)R was revealed by purifying b(5)R to homogeneity from the midge larvae. Using purified components, we showed that larval metHb was reduced via the NADH-b5R (FAD)-cytochrome b(5)-metHb pathway, a finding consistent with that in aerobic vertebrates, specifically humans and rabbits, and b(5)R function between mammal and insect was conserved. b(5)R 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 degrees C, respectively. The apparent K-m and V-max values were 345 mu M and 160 mu gmol min(-1) mg(-1), respectively, for ferricyanide and 328 mu M and 500 mu mol 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. (C) 2015 Elsevier Inc. All rights reserved.