Investigation of NADH binding, hydride transfer, and NAD(+) dissociation during NADH oxidation by mitochondrial complex I using modified nicotinamide nucleotides.

TitleInvestigation of NADH binding, hydride transfer, and NAD(+) dissociation during NADH oxidation by mitochondrial complex I using modified nicotinamide nucleotides.
Publication TypeJournal Article
Year of Publication2013
AuthorsBirrell, JA, Hirst, J
JournalBiochemistry
Volume52
Issue23
Pagination4048-55
Date Published2013 Jun 11
ISSN1520-4995
KeywordsAdenosine, 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.

DOI10.1021/bi3016873
Alternate JournalBiochemistry
Citation Key10.1021/bi3016873
PubMed ID23683271
PubMed Central IDPMC3680915
Grant ListMC_U105663141 / / Medical Research Council / United Kingdom