ATP synthase

Introduction

Respiration is crucial to life and in all organisms this is performed by a series of protein complexes located in the mitochondrial inner membrane, cholorplast thylakoid membrane or plasma membrane of bacteria or archaea (1). These respiratory complexes make up the so-called electron transport chain (ETC) and transfer protons across a biological membrane, which is coupled to the shuttling of electrons. The simultaneous transfer of protons and electrons generates an electrochemical proton gradient (proton motive force) which fuels the synthesis of ATP from ADP and inorganic phosphate. In mitochondria, the ETC comprises four protein complexes, termed complex I, III, IV and V.  Of these, complex I is the entry point of electrons into the ETC and together with complex III and IV functions as proton translocating enzyme to generate the proton motive force (PMF). While complex V (ATP synthase) utilizes the energy of the PMF for the production of ATP. The basic architecture of the mitochondrial ETC is shown in figure 1 (adopted from 1). Interestingly, accumulating evidence suggests that respiratory complexes are organized into large super complexes, termed the respirasome (2). Despite the wealth of structural data on respiratory complexes and individual subunits thereof, the precise molecular composition of the respirasome remained elusive. However, recent studies, employing cryo-electron microscopy, assessed the structure of different mammalian respirasomes, including the human (3,4,5,6). These studies revealed different respirasome stoichiometries, namely CI₂CIII₂CIV₂  for the human super complex and CICIII₂CIV for the sheep, porcine and bovine respirasome. In contrast to the mitochondrial ETC, the (aerobic) respiratory system of E. coli is more complex as it is made up of three dehydrogenases, two NADH dehydrogenases, three ubiquinol oxidases and two bo₃-type cytochromes (7).  Different respiratory supercomplexes have been observed in bacteria. For example, a complex of both NADH dehydrogenases, a large assembly of formate dehydrogenase and cytochrome bo₃ as well as a large complex containing at least both NADH dehydrogenases and other respiratory enzymes have been reported for E. coli (8,9). ATP synthase (complex V) is a highly conserved enzyme and a crucial part of the ETC. In fact, it is the primary means of energy production in most organisms because it is responsible for synthesis of the vast majority of cellular ATP by coupling the transport of protons across a biological membrane to the formation of a high-energy phosphate bond in ATP (10). Moreover, ATP synthase represents an attractive target for anticancer and antimicrobial drugs (11). Here, I will discuss recent structural and mechanistic advances of ATP synthase from E. coli. 

Biochemical properties and structure of ATP synthase

ATP synthase is a membrane-embedded enzyme that is ubiquitously present in all organisms. As a primary source of cellular ATP, it is essential for the functioning of a cell. The enzyme functions as a transporter and couples the movement of protons across the membrane to the production of ATP. All ATP synthases share a similar structure and exhibit the same biochemical and mechanistic properties (12,13). The enzyme is monomeric in photosynthetic organisms, while a dimer has been observed in the mammalian mitochondrial inner membrane. A schematic representation of E. coli ATP synthase is shown in figure 2 (adapted from 14). Like all ATP synthases, it comprises two parts, namely the membrane-integrated F_o sector and the cytosolic F₁ sector. Consequently, this enzyme is termed F_oF₁ or F-ATP synthase. E. coli ATP synthase has a molecular mass of about 530 kDa and is made up eight different subunits. The F_o part is organized into a ring-shaped structure and comprises the proton-conducting channel. It is believed that the interior of the c ring is filled with lipids. The F_o sector contains a, b and c subunits in the stoichiometry 1, 2 and 10 (ab_2­cc₁₀). The number of c subunits in the F_o sector is variable e.g. it numbers 8 in the ATP synthase of bovine mitochondria, 10 in the enzyme from yeast mitochondria and 14 in chloroplasts. In bacteria, the number of c subunits varies from 10 to 11 (REF).  The c-subunit is highly hydrophobic, has a molecular mass of about 8 kDa and comprises a hairpin of two transmembrane domains. The a-subunit consists of 5 transmembrane domains and is crucial for the translocation of protons because it forms the actual pathway for the transfer of protons in single file across the membrane. The F₁ sector represents the business part of the enzyme because it is responsible for the synthesis of ATP (12,13). It is made up of α, ß, δ, ε and γ subunits in the stoichiometry 3,3,1,1 and 1 (α₃ß₃ γ δ ε). Despite the overall structural similarity, ATP synthases display some variation in subunit composition. For example, the E. coli enzyme contains the ε subunit which is not present in bovine ATP synthase. Interestingly, the ε subunit is able to inhibit bacterial and chloroplast ATP synthases (12). The membrane-embedded and cytosolic sector are connected through a peripheral stalk consisting of two b subunits. The α and ß-subunits are alternately arranged into hexagon and contain six nucleotide-binding sites. All α-subunits are able to bind ATP but do not play a role in catalysis unlike the ß-subunits, which contain three nucleotide-binding sites that are catalytically active. These sites are termed ßTP, ßDP and ßE and are able to bind ATP, ADP and P_i. During catalysis, ßTP, ßDP and ßE undergo conformational changes that enables the following sequence of events: binding of substrates (ADP and P_i), ATP synthesis and release of ATP (12,13). These conformational changes are linked to rotation of the γ-subunit. This subunit extends into the α₃ß₃ of the F₁ sector and its rotation is powered by the rotation of the c subunits of the F_o sector upon proton translocation. Thus, in the holo enzyme the F_o sector and F₁ sector operate against each other (opposite rotational movements).  The net function of the enzyme is PMF-dependent e.g. in case of a strong PMF ATP is produced and when the PMF is weak ATP is hydrolyzed and protons are translocated across the membrane to generate a PMF (12,13). 

The following video nicely shows the principles of rotational catalysis.  

Several high resolution structures of ATP synthases from different organisms have been solved, including bovine mitochondrial ATP synthase and the yeast mitochondrial enzyme. Recently, a cryo-EM reconstruction of E. coli ATP synthase was reported (14). A complete molecular model of this reconstruction is depicted in figure 3 as spheres, revealing an overall structure typical of ATP synthases with a proton-powered F_o sector attached to a catalytic F₁ sector with a central and peripheral stalk. The left panel shows both sectors of the enzyme. The membrane-embedded F_o part is in grey, while the cytosolic F₁ sector is in green. The right panel reveals the subunit composition of both sectors. The c-subunit of the F_o sector is in blue, the a-subunit in green and the b-subunit is in yellow. While the α-subunit (in the ATP-bound state) of the F₁ sector is in pink, the ß-subunit is in light blue, the γ-subunit in orange, the ε-subunit in red and the δ-subunit in grey. The central stalk is formed by the γ and ε-subunits and the peripheral stalk is made up of two b-subunits.