title: Investigation of NADH binding, hydride transfer, and NAD(+) dissociation during NADH oxidation by mitochondrial complex I using modified nicotinamide nucleotides.
abstract: NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. During catalysis, NADH oxidation by a flavin mononucleotide is followed by electron transfer to a chain of iron-sulfur clusters. Alternatively, the flavin may be reoxidized by hydrophilic electron acceptors, by artificial electron acceptors in kinetic studies, or by oxygen and redox-cycling molecules to produce reactive oxygen species. Here, we study two steps in the mechanism of NADH oxidation by complex I. First, molecular fragments of NAD(H), tested as flavin-site inhibitors or substrates, reveal that the adenosine moiety is crucial for binding. Nicotinamide-containing fragments that lack the adenosine do not bind, and ADP-ribose binds more strongly than NAD(+), suggesting that the nicotinamide is detrimental to binding. Second, the primary kinetic isotope effects from deuterated nicotinamide nucleotides confirm that hydride transfer is from the pro-S position and reveal that hydride transfer, along with NAD(+) dissociation, is partially rate-limiting. Thus, the transition state energies are balanced so that no single step in NADH oxidation is completely rate-limiting. Only at very low NADH concentrations does weak NADH binding limit NADH:ubiquinone oxidoreduction, and at the high nucleotide concentrations of the mitochondrial matrix, weak nucleotide binding constants assist product dissociation. Using fast nucleotide reactions and a balance between the nucleotide binding constants and concentrations, complex I combines fast and energy-conserving NADH oxidation with minimal superoxide production from the nucleotide-free site.
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: James A
authors.initials: JA
authors.lastname: Birrell
authors.forename: Judy
authors.initials: J
authors.lastname: Hirst
mesh_terms: D000241:Adenosine / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D003067:Coenzymes / Q000737:chemistry; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D008958:Models, Molecular; D009243:NAD / Q000031:analogs & derivatives* / Q000737:chemistry; D009537:Nicotinamide Mononucleotide / Q000737:chemistry; D010084:Oxidation-Reduction; D011485:Protein Binding
chemical_list: D003067:Coenzymes; D005415:Flavins; D009243:NAD; D009537:Nicotinamide Mononucleotide; D000246:Adenosine Diphosphate Ribose; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D006859:Hydrogen; C005941:3-acetylpyridine adenine dinucleotide; D042967:Electron Transport Complex I; D000241:Adenosine