The structure of Escherichia coli respiratory complex I

The ability to capture energy from the environment to drive cellular processes
is fundamental for life. Heterotrophic organisms, including humans, consume
nutrients to synthesize adenosine triphosphate (ATP), the main energy currency
of the cell. The most effective process to achieve this is respiration. There,
macromolecules such as fats, proteins, and sugars are broken down into simpler
molecules with the concomitant release of energy. This energy is conserved as
an electrochemical gradient that among other processes is used for ATP
synthesis. In aerobic organisms, including humans and many bacteria, most of
the ATP is synthesized by the electron transport chain that consists of a set of
enzymes residing in the inner mitochondrial membrane of eukaryotes, or the
cytoplasmic membrane of bacteria.

Respiratory complex I is the first member of the electron transport chain,
creating 40 % of the electrochemical gradient for ATP synthesis during
respiration. It is a multi-subunit membrane protein complex that reversibly
couples NADH oxidation and ubiquinone reduction with proton translocation
against transmembrane potential with nearly 100% efficiency. The molecular
mechanism of this large protein complex has been puzzling scientists for more
than 30 years.

Complex I from Escherichia coli is the simplest and among the best functionally
characterized known complex I homologs. Thus, it is an attractive target for
mechanistic studies. However, such research has been hindered by the lack of
the complete structure of E. coli complex I. Historically, structural analysis of
this protein has been prevented for two main reasons. First, the fragility of
E. coli complex I made its purification in the intact form difficult. Second, the
high flexibility of the complex has made the actual structural studies very
challenging.

In the scope of this PhD thesis, a novel method for efficient expression and
purification of intact E. coli complex I have been developed. It allows obtaining
a catalytically active protein in conditions compatible with structural studies by
single-particle electron cryogenic microscopy. The entire E. coli complex I
structure has been solved to high resolution. It represents the first complete
reconstruction of complex I from a mesophilic bacterium. It has revealed the
architecture of the complex in atomic detail and explained the reasons for its
high flexibility. The complex is stabilized by the unique terminal extensions and
an insertion loop in the peripheral arm. An unusual conformation of the
conserved interface between the peripheral and membrane domains suggests
that the purified complex adopts an uncoupled conformation. Based on the
structural data, a new, hypothetical coupling mechanism for the complex I has
been proposed.