✓ 4 Identify the ultimate determinant of the rate of cellular respiration.

Mitochondria contain an evolutionarily conserved protein, inhibitory factor 1 (IF1), that specifically inhibits the potential hydrolytic activity of the F₀F₁ ATP synthase. What is the function of IF1? Consider a circumstance where tissues may be deprived of oxygen (ischemia). Without oxygen as the electron acceptor, the electron transport chain will be unable to generate the proton-motive force. The ATP in the mitochondria would be hydrolyzed by the synthase, working in reverse (problem 22). The role of IF1 is to prevent the wasteful hydrolysis of ATP by inhibiting the hydrolytic activity of the synthase.

IF1 is over-expressed in many types of cancer. This over-expression plays a role in the induction of the Warburg effect, the switch from oxidative phosphorylation to aerobic glycolysis as the principle means for ATP synthesis.

Some organisms possess the ability to uncouple oxidative phosphorylation from ATP synthesis to generate heat. Such uncoupling is a means to maintain body temperature in hibernating animals, in some newborn animals (including human beings), and in mammals adapted to cold. In animals, brown fat or brown adipose tissue (BAT) is specialized tissue for this process of nonshivering thermogenesis. In contrast, white adipose tissue (WAT), which constitutes the bulk of adipose tissue, plays no role in thermogenesis but serves as an energy source.

Brown adipose tissue is very rich in mitochondria, often called brown-fat mitochondria. The inner mitochondrial membrane of these mitochondria contains a large amount of uncoupling protein 1 (UCP-1), also called thermogenin. UCP-1 transports protons from the intermembrane space to the matrix with the assistance of fatty acids. In essence, UCP-1 generates heat by short-circuiting the mitochondrial proton battery. The energy of the proton gradient, normally captured as ATP, is released as heat as the protons flow through UCP-1 to the mitochondrial matrix (Figure 21.18). This dissipative proton pathway is activated when the core body temperature begins to fall. In response to a temperature drop, a adrenergic hormones stimulate the release of free fatty acids from triacylglycerols stored in cytoplasmic lipid droplets. Long chain fatty acids bind to the cytoplasmic face of UCP-1, and the carboxyl group binds a proton. This causes a structural change in UCP-1 so that the protonated carboxyl now faces the proton-poor environment of the matrix, and the proton is released. Proton release resets UCP-1 to the initial state.

We can witness the effects of a lack of nonshivering thermogenesis by examining pig behavior. Pigs are unusual mammals in that they have large litters and are the only ungulates (hoofed animals) that build nests for birth (Figure 21.19). These behavioral characteristics appear to be the result of a biochemical deficiency. Pigs lack UCP-1 and, hence, brown fat. Piglets must rely on other means of thermogenesis. Nesting, large litter size, and shivering are means by which pigs compensate for a lack of brown fat.

Until recently, adult humans were believed to lack brown fat tissue. However, new studies have established that adults, women especially, have brown adipose tissue on the neck and upper chest regions that is activated by cold (Figure 21.20). Obesity leads to a decrease in brown adipose tissue.

Many potent and lethal poisons exert their effects by inhibiting oxidative phosphorylation at one of a number of different locations.

Inhibition of the electron-transport chain The four complexes of the electron-transport chain can be inhibited by different compounds, blocking electron transfer downstream and thereby shutting down oxidative phosphorylation. NADH-Q oxidoreductase (Complex I) is inhibited by rotenone, which is used as a fish and insect poison, and amytal, a barbiturate sedative. Inhibitors of Complex I prevent the utilization of NADH as a substrate (Figure 21.21). Rotenone exposure, along with a genetic predisposition, has been implicated in the development of Parkinson disease, a condition characterized by resting tremor, slowness of movement, inability to initiate movement, rigidity, and postural instability. Inhibition of Complex I does not impair electron flow from FADH₂, because these electrons enter through QH₂, beyond the block. Q-cytochrome c oxidoreductase (Complex III) is inhibited by antimycin A, an antibiotic isolated from Streptomyces that is used as a fish poison. Furthermore, electron flow in cytochrome c oxidase(Complex IV) can be blocked by cyanide (CN⁻), azide (N₃⁻), and carbon monoxide (CO). Cyanide and azide react with the ferric form (Fe³⁺) of heme a₃, whereas carbon monoxide inhibits the ferrous form (Fe²⁺). Inhibition of the electron-transport chain also inhibits ATP synthesis because the proton-motive force can no longer be generated.

Inhibition of ATP synthase Oligomycin, an antibiotic used as an antifungal agent, and dicyclohexylcarbodiimide (DCC), used in peptide synthesis in the laboratory, prevent the influx of protons through ATP synthase by binding to the carboxylate group of the c subunits required for proton binding. Modification of only one c subunit by DCC is sufficient to inhibit the rotation of the entire c ring and hence ATP synthesis. If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron-transport chain ceases to operate. This observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled.

Uncoupling electron transport from ATP synthesis The tight coupling of electron transport and phosphorylation in mitochondria can be uncoupled by 2,4-dinitrophenol (DNP) and certain other acidic aromatic compounds. These substances carry protons across the inner mitochondrial membrane, down their concentration gradients. In the presence of these uncouplers, electron transport from NADH to O₂ proceeds in a normal fashion, but ATP is not formed by mitochondrial ATP synthase, because the proton-motive force across the inner mitochondrial membrane is continuously dissipated. This loss of respiratory control leads to increased oxygen consumption and the oxidation of NADH. Indeed, in the accidental ingestion of uncouplers, large amounts of metabolic fuels are consumed, but no energy is captured as ATP. Rather, energy is released as heat. DNP is the active ingredient in some herbicides and fungicides. Remarkably, some people consume DNP as a weight-loss drug, despite the fact that the U.S. Food and Drug Administration (FDA) banned its use in 1938. There are also reports that Soviet soldiers were given DNP to keep them warm during the long Russian winters. Chemical uncouplers are nonphysiological, unregulated counterparts of uncoupling proteins.

Drugs are being sought that would function as mild uncouplers, uncouplers not as potentially-lethal as DNP, for use in treatment of obesity and related pathologies. Xanthohumol, a prenylated chalcone found in hops and beer, shows promise in this regard. A chalcone is an aromatic ketone. Xanthohumol also scavenges free radicals and is used for treatment of certain types of cancers.

Inhibition of ATP export ATP-ADP translocase is specifically inhibited by very low concentrations of atractyloside (a plant glycoside) or bongkrekic acid (an antibiotic found in fermented coconut contaminated by the bacterium Burkholderia gladioli). Atractyloside binds to the translocase when its nucleotide site faces the cytoplasm, whereas bongkrekic acid binds when this site faces the mitochondrial matrix. Oxidative phosphorylation stops soon after either inhibitor is added, showing that ATP-ADP translocase is essential for maintaining adequate amounts of ADP to accept the energy associated with the proton-motive force.

The number of diseases that can be attributed to mitochondrial mutations is steadily growing in step with our growing understanding of the biochemistry and genetics of mitochondria. Mitochondrial diseases are estimated to affect from 10 to 15 per 100,000 people, roughly equivalent to the prevalence of the muscular dystrophies. The first mitochondrial disease to be understood was Leber hereditary optic neuropathy (LHON), a form of blindness that strikes in midlife as a result of mutations in Complex I. Some of these mutations impair NADH utilization, whereas others block electron transfer to Q. Mutations in Complex I are the most frequent cause of mitochondrial diseases. The accumulation of mutations in mitochondrial genes in a span of several decades may contribute to aging, degenerative disorders, and cancer.

A human egg harbors several hundred thousand molecules of mitochondrial DNA, whereas a sperm contributes only a few hundred and thus has little effect on the mitochondrial genotype. Because the maternally-inherited mitochondria are present in large numbers and not all of the mitochondria may be affected, the pathologies of mitochondrial mutants can be quite complex. Even within a single family carrying an identical mutation, chance fluctuations in the percentage of mitochondria with the mutation lead to large variations in the nature and severity of the symptoms of the pathological condition as well as the time of onset. As the percentage of defective mitochondria increases, energy-generating capacity diminishes until, at some threshold, the cell can no longer function properly. Defects in cellular respiration are doubly dangerous. Not only does energy transduction decrease, but also the likelihood that reactive oxygen species will be generated increases. Organs that are highly dependent on oxidative phosphorylation, such as the nervous system, the retina and the heart, are most vulnerable to mutations in mitochondrial DNA.