Specific Modification of a Na+ Binding Site in NADH:Quinone Oxidoreductase from Klebsiella pneumoniae with Dicyclohexylcarbodiimide

ABSTRACT The respiratory NADH:quinone oxidoreductase (complex I) (NDH-1) is a multisubunit enzyme that translocates protons (or in some cases Na+) across energy-conserving membranes from bacteria or mitochondria. We studied the reaction of the Na+-translocating complex I from the enterobacterium Klebsiella pneumoniae with N,N′-dicyclohexylcarbodiimide (DCCD), with the aim of identifying a subunit critical for Na+ binding. At low Na+ concentrations (0.6 mM), DCCD inhibited both quinone reduction and Na+ transport by NDH-1 concurrent with the covalent modification of a 30-kDa polypeptide. In the presence of 50 mM Na+, NDH-1 was protected from inhibition by DCCD, and the modification of the 30-kDa polypeptide with [14C]DCCD was prevented, indicating that Na+ and DCCD competed for the binding to a critical carboxyl group in NDH-1. The 30-kDa polypeptide was assigned to NuoH, the homologue of the ND1 subunit from mitochondrial complex I. It is proposed that Na+ binds to the NuoH subunit during NADH-driven Na+ transport by NDH-1.

[1]  B. Böttcher,et al.  A Novel, Enzymatically Active Conformation of theEscherichia coli NADH:Ubiquinone Oxidoreductase (Complex I)* , 2002, The Journal of Biological Chemistry.

[2]  Patrick Polzer,et al.  Structure of the Rotor Ring of F-Type Na+-ATPase from Ilyobacter tartaricus , 2005, Science.

[3]  P. Dimroth,et al.  The Respiratory Complex I (NDH I) from Klebsiella pneumoniae, a Sodium Pump* , 2002, The Journal of Biological Chemistry.

[4]  K. Altendorf,et al.  Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae , 1987, Archives of Microbiology.

[5]  G. Krishnamoorthy,et al.  Studies on the electron transfer pathway, topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase. , 1988, The Journal of biological chemistry.

[6]  Y. Bertsova,et al.  The origin of the sodium‐dependent NADH oxidation by the respiratory chain of Klebsiella pneumoniae , 2004, FEBS letters.

[7]  T. Friedrich,et al.  The Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is a Primary Proton Pump but May Be Capable of Secondary Sodium Antiport* , 2004, Journal of Biological Chemistry.

[8]  C. Schmid,et al.  Na+ translocation by complex I (NADH:quinone oxidoreductase) of Escherichia coli , 2000, Molecular microbiology.

[9]  P. Dimroth,et al.  Characterization of the Na+-stimulated ATPase of Propionigenium modestum as an enzyme of the F1F0 type. , 1987, European journal of biochemistry.

[10]  M. Hayashi,et al.  Covalently bound flavin in the NqrB and NqrC subunits of Na+‐translocating NADH‐quinone reductase from Vibrio alginolyticus , 2000, FEBS letters.

[11]  T. Yagi,et al.  Functional Roles of Four Conserved Charged Residues in the Membrane Domain Subunit NuoA of the Proton-translocating NADH-Quinone Oxidoreductase from Escherichia coli* , 2004, Journal of Biological Chemistry.

[12]  M. Hayashi,et al.  Modifications by Na+ and K+, and the site of Ag+ inhibition in the Na+-translocating NADH-quinone reductase from a marine Vibrio alginolyticus , 1993 .

[13]  P. Dimroth.  Chapter 2 Sodium ion coupled F1F0 ATPases , 1996 .

[14]  R. Hedderich.  Energy-Converting [NiFe] Hydrogenases from Archaea and Extremophiles: Ancestors of Complex I , 2004, Journal of bioenergetics and biomembranes.

[15]  E. Bakker.  Alkali Cation Transport Systems in Prokaryotes , 1992 .

[16]  P. Dimroth,et al.  The Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus--redox states of the FAD prosthetic group and mechanism of Ag+ inhibition. , 1997, European journal of biochemistry.

[17]  H. W. Veen,et al.  Handbook of Biological Physics , 1996 .

[18]  C. Wahlestedt,et al.  A two‐dimensional protein map of Caenorhabditis elegans , 2001, Electrophoresis.

[19]  P. Dimroth,et al.  Formation of a functionally active sodium-translocating hybrid F1F0 ATPase in Escherichia coli by homologous recombination. , 1993, European journal of biochemistry.

[20]  J. Walker,et al.  Structural analysis of NADH: ubiquinone oxidoreductase from bovine heart mitochondria. , 1995, Methods in enzymology.

[21]  H. Beinert,et al.  Micro methods for the quantitative determination of iron and copper in biological material. , 1967, Methods in enzymology.

[22]  P. Dimroth,et al.  Specific protection by Na+ or Li+ of the F1F0-ATPase of Propionigenium modestum from the reaction with dicyclohexylcarbodiimide. , 1993, The Journal of biological chemistry.

[23]  D. Wink,et al.  Nitric Oxide Reactions Important to Biological Systems: A Survey of Some Kinetics Investigations , 1995 .

[24]  R. Roth,et al.  Transmembrane orientation and topology of the NADH:quinone oxidoreductase putative quinone binding subunit NuoH. , 2001, Biochimica et biophysica acta.

[25]  M. Huynen,et al.  Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (Complex I). , 2005, Journal of molecular biology.

[26]  C. Ragan,et al.  Photolabelling of a mitochondrially encoded subunit of NADH dehydrogenase with [3H]dihydrorotenone , 1987, FEBS letters.

[27]  S. Dunn,et al.  Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. , 1986, Analytical biochemistry.

[28]  F. Neese,et al.  NADH Oxidation by the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae , 2004, Journal of Biological Chemistry.

[29]  P. Dimroth,et al.  Na+ translocation by the NADH:ubiquinone oxidoreductase (complex I) from Klebsiella pneumoniae , 1999, Molecular microbiology.

[30]  J. Steuber.  The C-terminally Truncated NuoL Subunit (ND5 Homologue) of the Na+-dependent Complex I from Escherichia coli Transports Na+* , 2003, Journal of Biological Chemistry.

[31]  P. Dimroth,et al.  Aspartate 203 of the oxaloacetate decarboxylase beta‐subunit catalyses both the chemical and vectorial reaction of the Na+ pump. , 1996, The EMBO journal.

[32]  P. Dimroth.  Preparation, characterization, and reconstitution of oxaloacetate decarboxylase from Klebsiella aerogenes, a sodium pump. , 1986, Methods in enzymology.

[33]  P. Dimroth,et al.  Sodium ion cycling mediates energy coupling between complex I and ATP synthase , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  H. Reichenbach,et al.  Two binding sites of inhibitors in NADH: ubiquinone oxidoreductase (complex I). Relationship of one site with the ubiquinone-binding site of bacterial glucose:ubiquinone oxidoreductase. , 1994, European journal of biochemistry.

[35]  S. Yoshikawa,et al.  Steady-State Kinetics of NADH:coenzyme Q Oxidoreductase Isolated from Bovine Heart Mitochondria , 2002, Journal of bioenergetics and biomembranes.

[36]  I. Hassinen,et al.  Reaction of dicyclohexylcarbodiimide with mitochondrial proteins. , 1993, Biochimica et biophysica acta.

[37]  P. Dimroth,et al.  Kinetics of inactivation of the F1Fo ATPase of Propionigenium modestum by dicyclohexylcarbodiimide in relationship to H+ and Na+ concentration: probing the binding site for the coupling ions. , 1993, Biochemistry.

[38]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[39]  P. Dimroth,et al.  Molecular mechanism of the ATP synthase's F(o) motor probed by mutational analyses of subunit a. , 2002, Journal of molecular biology.

[40]  Ichiro Yamato,et al.  Structure of the Rotor of the V-Type Na+-ATPase from Enterococcus hirae , 2005, Science.

[41]  T. Yagi.  Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms. , 1987, Biochemistry.

[42]  P. Rich.  The molecular machinery of Keilin's respiratory chain. , 2003, Biochemical Society transactions.

[43]  Robert R. Ishmukhametov,et al.  Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase. , 2005, Biochimica et biophysica acta.

[44]  J. Walker,et al.  Cryo-electron crystallography of two sub-complexes of bovine complex I reveals the relationship between the membrane and peripheral arms. , 2000, Journal of molecular biology.

[45]  K. Kogure,et al.  Generalized distribution and common properties of Na+-dependent NADH:quinone oxidoreductases in Gram-negative marine bacteria , 1989 .

[46]  T. Friedrich,et al.  Isolation and characterization of the proton-translocating NADH: ubiquinone oxidoreductase from Escherichia coli. , 1995, European journal of biochemistry.

[47]  J. Casida,et al.  Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling. , 2001, Biochimica et biophysica acta.

[48]  M. Finel,et al.  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. , 2000, Biochemistry.

[49]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[50]  T. Yagi,et al.  Identification of the dicyclohexylcarbodiimide-binding subunit of NADH-ubiquinone oxidoreductase (Complex I). , 1988, Journal of Biological Chemistry.

[51]  H. Beinert.  Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. , 1983, Analytical biochemistry.

[52]  G. Unden,et al.  Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications. , 1997, European journal of biochemistry.

[53]  J. Weber,et al.  ATP synthesis driven by proton transport in F1F0‐ATP synthase , 2003, FEBS letters.