Chapter 3 – Cellular Energy Metabolism
- Mitochondrial oxidation of diet-derived carbon-based fuels
Metabolic fuel is imported into cells in one of three main forms: lipid, carbohydrate and amino acids. In all cases, the carbon backbone of these molecules is eventually imported into mitochondria for oxidation if required to maintain ATP synthesis. The exception to this rule occurs in the complete absence of oxygen, where metabolic fuels are broken down to lactate (in mammals; other endproducts are possible in non-mammals) via anaerobic glycolysis. In most animal cells, this is not sustainable over the long term, and upon return to normoxia any accumulated lactate is oxidized. Oxidation of metabolic fuel in mitochondria is directly coupled to the creation of a proton motive force (PMF) across the mitochondrial inner membrane, and indirectly coupled to the synthesis of ATP from ADP by the ATP synthase (also called the FoF1ATPase). The term ‘oxidative phosphorylation’ (OxPhos) refers to the process in its entirety. It is important to understand how OxPhos is organized.
The fundamental unit of the bioenergetic organelle is the mitochondrion (plural = mitochondria). However, it should be noted that most recent data demonstrate that mitochondria normally exist as a reticulum, with tubule-like branches that extend throughout the cell. Furthermore, this reticulum is dynamic. It appears to be continually altering its shape, with individual tubules ‘pinching off’ from the network and then rejoining elsewhere. Nonetheless, it remains common for people to refer to a ‘mitochondrion’, implying a discrete ovoid entity (the first good electron microscopy images implied this kind of structure), and I will use this terminology here.
Mitochondria are enveloped by two distinct membranes: the inner membrane and the outer membrane. For the purpose of the following discussion of bioenergetics, we will basically ignore the outer membrane, which is quite freely permeable to molecules with molecular weights <1500 Daltons and therefore does not contribute directly to OxPhos The inner membrane is actually organized into two regions, though the regions are fully continuous. The first region is referred to as the ‘inner boundary membrane’, which encapsulates the interior of the organelles. The second region, ‘cristae’, refers to tubular processes which penetrate into the interior of the mitochondrial matrix and emerge out the other side. It is within the cristae that the machinery of OxPhos is localized.
The interior of the mitochondrion defined by the inner boundary membrane is the ‘matrix’. It is in the mitochondrial matrix that the enzymes of the tricarboxylic acid (TCA) cycle reside. Molecules to be oxidized within mitochondria are transformed into one of the TCA cycle intermediates so that they can enter the cycle. (It’s called a cycle because it continually regenerate its intermediates) The enzymes of the TCA cycle catalyze the reduction of the so-called ‘reducing equivalents’, NADH and FADH2 from NAD+ and FAD+, respectively. It is these reducing equivalents that will donate the high energy electrons to the ‘electron transfer chain’, thus driving the process of OxPhos.
There are five respiratory complexes: Complex I accepts electrons from NADH and passes them, via Coenzyme Q, to complex III; Complex II accepts electrons from FADH2 and passes them, via Coenzyme Q, to complex III; Complex III accepts electrons from Coenzyme Q, transferring them via cytochrome C to Complex IV. This latter enzyme transfers the electrons to oxygen, in the process reducing oxygen to water (thus oxygen is the ‘electron sink’). This is illustrated in movie form at this website:
In complexes I, III and IV the redox reactions undergone during electron transfer are linked to the extrusion (pumping) of protons out of the mitochondrial matrix. This thus establishes the PMF. The PMF provides the energy to drive ATP synthesis from ADP and Pi. The ATP synthase (complex V) is a huge, multi-protein-subunit rotary motor that spans the crystal membrane (there are many of them per unit mitochondrion). As protons flow down their concentration gradient and back into the matrix, they propel the ATP synthase, thus driving the synthesis of ATP. Peter Mitchell received the Nobel Prize in Chemistry in 1978 for outlining the above in his ‘chemiosmotic hypothesis, first presented in 1961.
In 1997, John Walker and Paul Boyer shared a Nobel prize for their work in elucidating the structure of the ATP synthase using X-ray crystallography. With this information, we now understand the structure of this large mult-protein reverse pump to look something like this:
More recently, Masasuke Yoshida has demonstrated that the ATP synthase functions as a rotary motor. As H+ pass through the core of the enzyme from intermembrane space to matrix, the membrane-bound Fo portion rotates and this is linked to the phosphorylation of ADP. Yoshida and colleagues created a movie showing their experiment directly showing the rotary behaviour of the synthase. View a movie of their experiment in action at:
ATP is used up at approximately the same rate as its synthesis, so that there is no ‘storage’ of ATP within the cell; nor does the intracellular [ATP] change markedly in most animal cells, even when huge changes in ATP turnover may occur, as during the rest to work transition in skeletal muscle. Thus, this is a dynamical system. Also, in most cells, there is no way to ‘store’ oxygen (but see chapter 4). It must therefore be continually supplied at the same rate that it is used up. The rate at which it must be supplied to a cell is entirely dependent upon the rate at which that cell is cleaving ATP to ADP. This rate of ATP usage is a reflection of the work the cell is performing. This is referred to as ‘ATP turnover’. ATP turnover is sustained by oxidative phosphorylation, and therefore the rate of oxygen consumption during this process is an indication of the rate of cellular work.
The efficiency of OxPhos
An interesting observation that has been made in all eukaryotic cells in which it has been studied is that PMF formation (via respiration) is not perfectly coupled to ATP synthesis. One might expect that all of the protons pumped out of the mitochondrial matrix would re-enter via the ATP synthase; however, a substantial proportion of these protons is able to re-enter the matrix via other pathway(s).
In mitochondria isolated from any eukaryotic cell, the PMF established by respiration-linked proton translocation from the matrix is gradually dissipated unless there is an ongoing oxidation of metabolic fuel. Thus, even when the ATP synthase is not working, either in the absence of ADP, or in the presence of its inhibitor oligomycin (neither of these represents a physiologicallynormal situation in a live cell), mitochondria continue to consume oxygen and pump protons. This observation was made long ago in isolated mitochondria, but was assumed to be due to damage that occurred during isolation of the organelle. However, subsequent measurements in intact cells, and even in whole tissues, have indicated that it is a real property of mitochondria in their normal working environment.
Measurements made in cells and working tissues indicate that the proportion of oxygen consumption dedicated to simply maintaining the PMF in the face of this proton leak is about 20% of SMR, a substantial amount! The original observations were made in mammals, and so it was assumed that this was related to the strategy of endothermy, allowing heat production that could be harnessed to maintain body temperature above ambient. However, a similar 20% of cellular MR is consumed by proton leak in the cells of ectotherms, including reptiles. Therefore, it must have some other purpose.
The realization that cellular oxidative metabolism has this seemingly wasteful design has actually occurred fairly recently (in the past 10-15 years), and it remains unknown why this would be the case. 20% of SMR is a lot of energy to be expended on a process unless it is either an unavoidable design feature or has a specific function. Interestingly, mitochondrial membranes are more permeable to protons than other cellular membranes that are not involved in OxPhos, suggesting that it could be avoided. Perhaps then, there is a very good reason for the existence of the proton leak in mitochondria from apparently all animal cells. A number of theories have been put forth. Two leading theories are: (1) perhaps the uncoupling works similarly to a clutch in a car, allowing an idling speed where there is some energy consumption but the motor is not turned off completely. It may be that with this design, it is possible to get up and running again quickly; (2) at high PMF, mitochondria tend to produce free radicals (reactive oxygen species; see chapter 4) and the proton leak might be a mechanism to prevent PMF from becoming too high if [ADP] or Pi becomes temporarily diminished.
Research into the physiological significance of proton leak has benefited from the identification of specific proteins that catalyze OxPhos uncoupling. The first uncoupling protein (originally thermogenin; now UCP1; see Chapter 5) was discovered and described in brown adipose tissue, a specialized form of adipose found in small mammals. It is not normally present in any other cell types. In 1997, several homologues to UCP1 (UCP2, UCP3, and others) were discovered and found to be expressed in a variety of cell types in mammals. It was initially thought that these might provide the explanation for proton leak; however, although they are widely expressed they are not ubiquitous, and even mitochondria that have no UCPs nonetheless have proton leak. Thus, both the mechanism and physiological function of the mitochondrial proton leak remain incompletely understood, though it has now been established as one of the major contributors to cellular MR.
Cellular Energy Utilization and the Molecular Origin of Standard Metabolic Rate in Mammals
Animals may be composed of enormous numbers of cells. For example, it takes approximately 300 trillion cells to make an adult human. These cells may bear virtually no physical resemblance to each other: a neuron and a liver cell appear to have very little in common. However, they do have the same basic metabolic design in that many of the energy-consuming reactions are driven by ATP, and oxidative phosphorylation continually synthesizes ATP to replenish it as it is used up. Oxidative phosphorylation can be considered as two distinct but coupled processes: (1) oxidation of molecules derived from the diet (or mitochondrial respiration); (2) ATP synthesis using the PMF.
While ATP can also be made anaerobically (anaerobic glycolysis), this pathway can sustain the ATP demand for only a short time. Over the long haul, mammalian metabolism is aerobic. That is, all of the reactions of energy metabolism eventually end up supplying electrons to oxidative phosphorylation, with the concomitant conversion of oxygen to water. Because of this, we can measure metabolic rate as the rate at which the body consumes oxygen.
Thus, whole body metabolic rate is the culmination of all individual cellular metabolic rates. We can examine whole body metabolic rate as the sum of its constituent parts. An initial observation is that different tissues and organs make different contributions to the metabolic rate of the body under standard conditions (see glossary for definition of standard metabolic rate; SMR). For example, in humans the liver, heart, kidney and brain are responsible for 55% of oxygen consumption (i.e. metabolic rate) under standard conditions, despite combining for only 4.9% of overall body mass. Clearly, the remaining 95% of the human body by mass must be considerably less active under standard conditions. This remaining proportion includes the skeleton, adipose tissue and inactive skeletal muscle, all of which have very low metabolic rates under SMR conditions. However, skeletal muscle can become the major oxygen-consuming tissue under working conditions.
As the human SMR was partitioned into contributions made by individual tissues and organs, so can the metabolic rate of a cell be partitioned. When removed from the body (isolated) and placed into a bathing medium, an individual cell continues to have a measurable metabolic rate. This can be measured readily using an oxygen electrode. The oxygen electrode, contained within a sealed chamber, records the rate at which isolated animal cells (also in the chamber) use up oxygen. Now, if individual activities that the cell engages in are inhibited, one can estimate the contribution they make to overall cellular metabolism. Before examining these contributions, we must remind ourselves of the way cellular energy metabolism works.
The cell is organized into discrete compartments separated by membranes. With respect to cellular energy metabolism, the important compartments are the cytosol (contained within the cell membrane but not including the interior of organelles) and the mitochondria (enclosed within a double membrane structure). The individual metabolic reactions can then be considered with respect to their location:
Cytosol: glycolysis – one molecule of glucose split into to molecules of pyruvate
Mitochondria: pyruvate conversion to acetyl-CoA
lipid conversion to acetyl-CoA
amino acid conversion to acetyl-CoA or other TCA cycle intermediates
TCA cycle - acetyl-CoA derived from above substrates enters it; NADH
and FADH2 (reducing equivalents) are produced from NAD+ and FAD+
respiratory chain – NADH & FADH2 from TCA cycle provide electrons to
complexes I or II, respectively. As electrons passed from one respiratory
complex to anotherrespiratory complexes pump protons from matrix
ATP synthase – proton motive force drives synthesis of ATP from ADP
As you can see, the majority of energy metabolism relies on the mitochondria. For this reason, mitochondria are often referred to as the ‘powerhouses’ of the cell. This is true, but keep in mind that these organelles do not produce energy. Rather, they ‘transduce’ energy, i.e. convert it from one form to another. To do this, they require oxygen, as the energy transduction is dependent on the presence of an electron sink. Actually, there are other possibilities: some deep sea hydrothermal vent animals utilize hydrogen sulphide as an electron sink instead of oxygen.
Approximately 90% of cellular oxygen usage is mitochondrial. But, there are some other reactions that consume oxygen: egs. NADPH oxidases, cytochrome P450 oxidases, xanthine oxidase, monoamine oxidase (see Chapter4). Also, not all of the oxygen consumed by mitochondria to build the PMF (∆Ψm in the figure above) is coupled to the synthesis of ATP (approximately 20% of oxygen consumption associated with proton pumping is uncoupled from ATP synthesis because some of the proton leak; see above). Thus, only 80% of the 90%, or approximately 72% of total body oxygen consumption, is linked to ATP synthesis.
The major consumers of ATP in the cell (SMR)
Remember that the vast majority of work done by a cell depends upon proteins. If we consider the case of muscle (particularly skeletal or cardiac), the shortening of individual cells occurs via interactions between myosin and actin that consume ATP. Also, the continual cycling of Ca2+ out of and into the sarcoplasmic reticulum (SR) requires the activity of the Ca2+ ATPase to pump Ca2+ into the SR. And, the activity of the Na+/K+ ATPase is required to maintain Na+ and K+ gradients across the sarcopasmic membrane. There are many other ATP-consuming reactions as well, but studies in which individual activities are inhibited suggest that a few individual proteins or processes are responsible for the bulk of ATP consumption in many types of cells.
Na+/K+ ATPase: 5 to 70% of cellular metablic rate
All cells (not just excitable ones) maintain Na+ and K+ gradients between the cytosol and extracellular fluid. One purpose of this gradient is to drive the import and export of specific molecules across the membrane (facilitated transport), often against the concentration gradient of that molecule (secondary active transport). For example, glucose import into many cells relies on this. Thus, the plasma membrane contains many co-transporters that will move molecules across the membrane in concert with Na+ and/or K+ movements (symport or antiport). The gradient itself is maintained by the Na+/K+ ATPase at the expense of ATP hydrolysis. As the metabolic activity of a cell increases, so does the rate of material movement across the membrane (in either direction). Therefore, simultaneously, the rate of Na+/K+ ATPase activity increases. The gradient never collapses; rather, these increases and decreases in activity of membrane transporters and Na+/K+ ATPase necessarily occur in parallel.
The contribution this one molecular machine (the Na+/K+ ATPase) makes to the energy budget of a cell (and by extrapolation of the body) can be estimated by inhibiting it and measuring the change in metabolic rate. Alternatively, it can be measured using radiolabelled K+ or Rb+ (the latter is recognized as K+ by the Na+/K+ ATPase). Experiments where this has been done indicate that the pump makes a substantial contribution to cellular metabolic rate, though the magnitude differs between tissues. In kidney and brain, Na+/K+ ATPase activity accounts for 50-60% of total cellular oxygen consumption! In other tissues, it is less, however. By experimentally measuring the contribution of Na+/K+ ATPase to the metabolic rates of individual cell types and then multiplying that number by the amount of that cell type present in the body, one can calculate that approximately 20% of whole body SMR is used to drive the Na+/K+ ATPase. Therefore, fully one-fifth of the oxygen you consume at rest goes simply to drive this one type of molecular machine.
Ca2+ ATPase: 5 to 30% of cellular metabolic rate
Ca2+ is an important intracellular messenger in the cytosol of most cells, and therefore its concentration is strictly regulated. Ca2+ is stored within specific organelles, particularly the endoplasmic reticulum (or sarcoplasmic reticulum (SR) in muscle) and mitochondria. The role of the Ca2+ ATPase in muscle provides an excellent example of its function. The cycles of contraction/relaxation that occur in working skeletal muscle are dependant upon very rapid increases and decreases in [Ca2+] in the cytosol, and this is accomplished by storing lots of Ca2+ in the SR and using a gated Ca2+ channel to release it, and the Ca2+ ATPase to pump it rapidly back in. The contribution this one activity makes to skeletal muscle metabolism is estimated to be 6% for muscles at rest, but up to 60% for rapidly contracting muscle. Similarly, it is <1% of MR of resting heart muscle, but 18-36% of MR of contracting heart (of course only the latter condition is physiologically relevant). It is substantially lower in other cell types. It is estimated that the Ca2+ ATPase accounts for about 5% of SMR. During exercise or work, this percent will increase as skeletal muscle makes a progressively greater contribution to whole body metabolic rate.