Stuart J. Ferguson

The ATP synthase

Publisher Summary ATP synthase is highly conserved; it is present in mitochondria, chloroplasts, both aerobic and photosynthetic bacteria, and even those bacteria that lack a functional respiratory chain as well as where the enzyme generates Δp (protonmotive force) at the expense of hydrolysing ATP produced in glycolysis. The function of the ATP synthase is to utilize Δp to maintain the mass-action ratio for the ATPase reaction 7–10 orders of magnitude away from equilibrium, or in the case of fermentative bacteriato utilize ATP to maintain Δp for the purpose of transport. The ATP synthase can be visualized under the electron microscope in preparations of submitochondrial particles (SMPs) that have been negatively stained with phosphotungstate. The complexes appear as roughly spherical knobs projecting from the original matrix side of the membrane.

Mitochondria in the cell

This chapter focuses on four major areas of current interest—the monitoring of mitochondrial membrane bioenergetics and ATP synthesis in intact cells, mitochondria and cellular Ca 2+ homeostasis, mitochondria and oxidative stress, and mitochondria as well as the control of apoptotic and necrotic cell death. The bioenergetic behavior of the mitochondrion in the intact cell is governed by the supply of substrate from the cytoplasm, the turnover of ATP by cytoplasmic and plasma membrane energy-requiring processes, the ionic environment of the cytoplasm, particularly in relation to Ca 2+ , and the redox state of the cell. Alterations in these parameters occur in response to changed energy demand, particularly in excitable cells, such as those in muscle and brain. The bioenergetics of the in situ mitochondria can be influenced by a wide variety of cellular stimuli, including plasma ion channel activation, hormonal signalling, and oxidative stress.

The chemiosmotic proton circuit

This chapter introduces the central concept of bioenergetics, the circuit of protons linking the primary generators of protonmotive force with the ATP synthase. Its purpose is to discuss experimental approaches to monitoring the functioning of the proton circuit under a wide range of conditions. The close analogy between the proton circuit and the equivalent electrical circuit are emphasized, not only as a simple model but also because the same laws govern the flow of energy around both circuits. In an electrical circuit the two fundamental parameters are potential difference (in volts) and current (in amps). From measurements of these functions, other factors may be derived, such as the rate of energy transmission (in watts) or the resistance of components in the circuit (in ohms). The chapter presents a simple electrical circuit, together with the analogous proton circuit across mitochondrial inner membrane.

Metabolite and ion transport

Mitochondria and bacteria continuously exchange metabolites and end products with the cell cytoplasm or external environment. At the same time the membranes maintain a high ° p for ATP synthesis. Since most metabolites are charged and/or weak acids, it follows that their distribution will be affected by δψ or δpH (Chapter 3). In practice, transport mechanisms are not only designed to operate under the constraints of a high δψ and/or δpH gradient, but may also exploit these gradients to drive the accumulation of substrates or the expulsion of products across the membrane. Some of the more common strategies (Fig. 8.1) are: (a) Proton symport with a neutral species leading to an accumulation driven by the full δ p , e.g. the lac permease of E. coli .

Quantitative Bioenergetics: The Measurement of Driving Forces

This chapter provides an introduction to the thermodynamics of quantitative bioenergetics. Thermodynamics include three types of system—isolated, closed, and open systems. The complexity of the thermodynamic treatment of these systems increases as their isolation decreases. Open systems strictly require a non-equilibrium thermodynamic treatment; classical equilibrium thermodynamics cannot be applied precisely to open systems because the flow of matter across their boundaries precludes the establishment of a true equilibrium. The most significant contribution of equilibrium thermodynamics to bioenergetics comes from considering individual reactions or groups of reactions as closed systems. Equilibrium thermodynamics is immensely powerful in bioenergetics as it can be applied to many problems. It helps in calculating the conditions required for equilibrium in an energy transduction, such as the utilization of the protonmotive force to produce ATP, and by extension determining how far such a reaction is displaced from equilibrium under actual experimental conditions.

The Chemiosmotic Proton Circuit in Isolated Organelles: Theory and Practice

The purpose of this chapter is to focus on the primary role of the proton circuit linking electron transport to ATP synthesis, discussing experimental approaches to monitoring the functioning of the proton circuit under a wide range of conditions. Although the analysis is focused on isolated mitochondria, it is equally applicable to bacteria and chloroplasts.

Ion transport across energy-conserving membranes

This chapter describes the basic permeability properties of membranes and the abilities of ionophores to induce the additional pathways of ion permeation. For an ion to be transported across a membrane, both a driving force and a pathway are required. These driving forces can be metabolic, concentration gradients, or electrical potentials, or combinations of all these. To reduce the complexity of membrane transport events, it is useful to classify any transport process. A consequence of the fluid-mosaic model of membrane structure is that transport can occur either through lipid bilayer regions of the membrane or be catalyzed by integral, membrane-spanning proteins. The distinction between protein-catalyzed transport and transport across the bilayer regions of the membrane is fundamental. Ions can be accumulated without direct metabolic coupling if there is a membrane potential or if transport is coupled to the “downhill” movement of a second ion.

ATP Synthases and Bacterial Flagella Rotary Motors

The proton-translocating ATP synthase is a marvellous example of biological nanotechnology, combining a proton-driven turbine (Fo) and an FI component that is massaged by an eccentric spindle driven by the turbine to generate ATP from ADP and Pi. High-resolution structures are available for nearly all the components, and the molecular mechanism is known in considerable, but not complete, detail. Archaea and a few eubacteria possess distinct but related ATP synthases, while the first molecular rotator motor to be described was that which drives the bacterial flagellum.

Photosynthetic generators of protonmotive force

A central feature of photosynthesis is the conversion of light energy into redox energy; photon capture causes a component to change its redox potential from being relatively electropositive to being highly electronegative. The production of ATP by photosynthetic energy-transducing membranes involves a proton circuit, which is closely analogous for mitochondria and respiratory bacteria. The two features that are unique to photosynthetic systems are the antennae that are responsible for the trapping of photons and the reaction centers to which the energy from light is directed. A component in the reaction center becomes electronically excited as a result of the absorption of a photon. Use of antennae to speed up the rate of photochemistry in the reaction centers is clearly more effective in biosynthetic terms than inserting very many copies of the reaction center into the membrane to achieve a high rate of overall photochemistry.

Transporters: Structure and Mechanism

Structures are now beginning to become available for some of the key proteins catalysing ion and metabolite transport across the mitochondrial and bacterial membranes. For the first time this allows informed hypotheses to be made about the molecular mechanisms of these proteins.