Mitochondrial disease occurs about 1 in 5000 births. The enzyme most commonly affected is
mitochondrial Complex I (NADH:ubiquinone oxidoreductase), the entry point of the electron
transport chain. It oxidizes NADH, reduces ubiquinone, and couples the free energy from those
reactions to the translocation of protons across the membrane, which are used to drive the
synthesis of ATP. In humans, 7 of the 45 subunits of Complex I are encoded by mitochondrial
genes. Advances in sequencing of genomes and exosomes has greatly increased the number of
gene variants found in mitochondrial disease, but it has been difficult to determine if these alleles
are causative for disease. In the case of mitochondrial genes, it is complicated both by
heteroplasmy, in which both wild type and mutant alleles can co-exist in the same cell, and by the
various haplotypes of mitochondrial DNA that can influence the effects of a mutation. A bacterial
model system allows individual mutations to be examined in a consistent background. Recent
structural studies have demonstrated that the 14 core subunits of Complex I from mitochondria
are nearly identical to the 14-subunit bacterial enzyme. The goal of this proposal is to
characterize about 24 clinical mutations in a bacterial version of Complex I, to provide insight into
their effects on assembly and function, and to improve disease diagnosis. These mutations are
associated with disease, but have not been shown to be causative. In the first aim, mutations that
map to interfaces of ND2, ND4, and ND5 subunits at the distal end the membrane arm will be
analyzed. It will be determined whether the mutations impair enzyme activity, or prevent the
binding of one of the partner subunits, and how that impacts the assembly process. In the second
aim, mutations that map to the integrated pair of ND6-ND4L will be analyzed. It will be determined
whether these mutations disrupt mutual interactions, or interactions with neighboring subunits,
such as ND2 and ND5, and how that affects assembly of Complex I. In the third aim, mutations
will be modeled that map to ND1-ND3, at the interface between the membrane subunits and the
peripheral arm. Effects on enzyme activity and assembly will be examined. Assembly will be
verified by native gel electrophoresis and western blotting. The results will provide insights into
the assembly pathways of Complex I, and the nature of disruptions by clinically-identified
mutations in the core subunits. Results will provide guidance in the diagnosis of patients with
currently-known, and newly-identified mutations.