Title | Investigation of NADH binding, hydride transfer, and NAD(+) dissociation during NADH oxidation by mitochondrial complex I using modified nicotinamide nucleotides. |
Publication Type | Journal Article |
Year of Publication | 2013 |
Authors | Birrell, JA, Hirst, J |
Journal | Biochemistry |
Volume | 52 |
Issue | 23 |
Pagination | 4048-55 |
Date Published | 2013 Jun 11 |
ISSN | 1520-4995 |
Keywords | Adenosine, Adenosine Diphosphate, Adenosine Diphosphate Ribose, Adenosine Monophosphate, Animals, Binding, Competitive, Cattle, Coenzymes, Electron Transport Complex I, Flavins, Hydrogen, Kinetics, Mitochondria, Heart, Models, Molecular, NAD, Nicotinamide Mononucleotide, Oxidation-Reduction, Protein Binding |
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. |
DOI | 10.1021/bi3016873 |
Alternate Journal | Biochemistry |
Citation Key | 10.1021/bi3016873 |
PubMed ID | 23683271 |
PubMed Central ID | PMC3680915 |
Grant List | MC_U105663141 / / Medical Research Council / United Kingdom |