Novel FMN-containing rotenone-insensitive NADH dehydrogenase from Trypanosoma brucei mitochondria: isolation and characterization.
A rotenone-insensitive NADH dehydrogenase has been isolated from the mitochondria of the procyclic form of African parasite, Trypanosoma brucei. The active form of the purified enzyme appears to be a dimer consisting of two 33-kDa subunits with noncovalently bound FMN as a cofactor. Hypotonic treatment of intact mitochondria revealed that the NADH dehydrogenase is located in the inner membrane/matrix fraction facing the matrix. The treatment of mitochondria with increasing concentrations of digitonin suggested that the NADH dehydrogenase is loosely bound to the inner mitochondrial membrane. The NADH:ubiquinone reductase activity is insensitive to rotenone, flavone, or dicumarol; however, it was inhibited by diphenyl iodonium in a time- and concentration-dependent manner. Maximum inhibition by diphenyl iodonium required preincubation with NADH to reduce the flavin. More complete inhibition was obtained with the more hydrophobic electron acceptors, such as Q(1) or Q(2), as compared to the more hydrophilic ones, such as Q(0) or dichloroindophenol. Kinetic analysis of the enzyme indicated that the enzyme followed a ping-pong mechanism. The enzyme conducts a one-electron transfer and can reduce molecular oxygen forming superoxide radical.
Fang
Jing
J
Department of Biochemistry and Molecular Pharmacology, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9142, USA.
Beattie
Diana S
DS
D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology; D002384:Catalysis; D019281:Dimerization; D005486:Flavin Mononucleotide / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D021122:Protein Subunits; D012402:Rotenone / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014451:Ubiquinone / Q000378:metabolism
D001713:Biphenyl Compounds; D009861:Onium Compounds; D021122:Protein Subunits; D012402:Rotenone; C031291:diphenyliodonium; D013481:Superoxides; D014451:Ubiquinone; D005486:Flavin Mononucleotide; D009245:NADH Dehydrogenase; D010100:Oxygen