Mutagenesis of three conserved Glu residues in a bacterial homologue of the ND1 subunit of complex I affects ubiquinone reduction kinetics but not inhibition by dicyclohexylcarbodiimide

被引:41
作者
Kurki, S
Zickermann, V
Kervinen, M
Hassinen, I
Finel, M
机构
[1] Univ Helsinki, Inst Biomed Sci, Dept Med Chem, Helsinki Bioenerget Grp, FIN-00014 Helsinki, Finland
[2] Univ Helsinki, Biocentrum, FIN-00014 Helsinki, Finland
[3] Univ Oulu, Dept Med Biochem, Oulu, Finland
关键词
D O I
10.1021/bi001134s
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Steady-state kinetics of the H+-translocating NADH:ubiquinone reductase (complex I) were analyzed in membrane samples from bovine mitochondria and the soil bacterium Paracoccus denitrificans. In both enzymes the calculated K-m, values, in the membrane lipid phase, for four different ubiquinone analogues were in the millimolar range. Both the structure and size of the hydrophobic side chain of the acceptor affected its affinity fur complex I. The ND1 subunit of bovine complex I is a mitochondrially encoded protein that binds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hatefi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. debitrificans complex I is a homologue of ND1, and within it three conserved Glu residues that could bind DCCD, E158, E212, and E247, were changed to either Asp or Gin and in the case of E212 also to Val, The DCCD sensitivity of the resulting mutants was, however, unaffected by the mutations. On the other hand, the ubiquinone reductase activity of the mutants was altered, and the mutations changed the interactions of complex I with short-chain ubiquinones, The implications of the results for the location of the ubiquinone reduction site in this enzyme are discussed.
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页码:13496 / 13502
页数:7
相关论文
共 29 条
[1]   A COMPARISON OF THE RESPIRATORY-CHAIN IN PARTICLES FROM PARACOCCUS-DENITRIFICANS AND BOVINE HEART-MITOCHONDRIA BY EPR SPECTROSCOPY [J].
ALBRACHT, SPJ ;
VANVERSEVELD, HW ;
HAGEN, WR ;
KALKMAN, ML .
BIOCHIMICA ET BIOPHYSICA ACTA, 1980, 593 (02) :173-186
[2]   MOLECULAR-BASIS OF MITOCHONDRIAL-DNA DISEASE [J].
BROWN, MD ;
WALLACE, DC .
JOURNAL OF BIOENERGETICS AND BIOMEMBRANES, 1994, 26 (03) :273-289
[3]   PHOTOLABELING OF A MITOCHONDRIALLY ENCODED SUBUNIT OF NADH DEHYDROGENASE WITH [H-3] DIHYDROROTENONE [J].
EARLEY, FGP ;
PATEL, SD ;
RAGAN, CI ;
ATTARDI, G .
FEBS LETTERS, 1987, 219 (01) :108-113
[4]   Steady-state kinetics of the reduction of coenzyme Q analogs by complex I (NADH:Ubiquinone oxidoreductase) in bovine heart mitochondria and submitochondrial particles [J].
Fato, R ;
Estornell, E ;
DiBernardo, S ;
Pallotti, F ;
Castelli, GP ;
Lenaz, G .
BIOCHEMISTRY, 1996, 35 (08) :2705-2716
[5]   DETERMINATION OF PARTITION AND LATERAL DIFFUSION-COEFFICIENTS OF UBIQUINONES BY FLUORESCENCE QUENCHING OF NORMAL-(9-ANTHROYLOXY)STEARIC ACIDS IN PHOSPHOLIPID-VESICLES AND MITOCHONDRIAL-MEMBRANES [J].
FATO, R ;
BATTINO, M ;
ESPOSTI, MD ;
CASTELLI, GP ;
LENAZ, G .
BIOCHEMISTRY, 1986, 25 (11) :3378-3390
[6]   DISRUPTION OF THE GENE ENCODING THE NADH-BINDING SUBUNIT OF NADH-UBIQUINONE OXIDOREDUCTASE IN NEUROSPORA-CRASSA FORMATION OF A PARTIALLY ASSEMBLED ENZYME WITHOUT FMN AND THE IRON-SULFUR CLUSTER N-3 [J].
FECKE, W ;
SLED, VD ;
OHNISHI, T ;
WEISS, H .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1994, 220 (02) :551-558
[7]   RESOLUTION OF NADH-UBIQUINONE OXIDOREDUCTASE FROM BOVINE HEART-MITOCHONDRIA INTO 2 SUBCOMPLEXES, ONE OF WHICH CONTAINS THE REDOX CENTERS OF THE ENZYME [J].
FINEL, M ;
SKEHEL, JM ;
ALBRACHT, SPJ ;
FEARNLEY, IM ;
WALKER, JE .
BIOCHEMISTRY, 1992, 31 (46) :11425-11434
[8]   Genetic inactivation of the H+-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans is facilitated by insertion of the ndh gene from Escherichia coli [J].
Finel, M .
FEBS LETTERS, 1996, 393 (01) :81-85
[9]   REACTION OF DICYCLOHEXYLCARBODIIMIDE WITH MITOCHONDRIAL PROTEINS [J].
HASSINEN, IE ;
VUOKILA, PT .
BIOCHIMICA ET BIOPHYSICA ACTA, 1993, 1144 (02) :107-124
[10]   SENSITIVITY TO NN'-DICYCLOHEXYLCARBODI-IMIDE OF PROTON TRANSLOCATION BY MITOCHONDRIAL NADH - UBIQUINONE OXIDOREDUCTASE [J].
HONKAKOSKI, PJ ;
HASSINEN, IE .
BIOCHEMICAL JOURNAL, 1986, 237 (03) :927-930