Chapter 19

Transmembrane Movement of Reducing Equivalents Under aerobic conditions, extramitochondrial NADH must undergo oxidation by the mitochondrial respiratory chain. Consider a preparation of rat hepatocytes containing mitochondria and all the cytosolic enzymes. After the introduction of $[4-{}^3\mathrm{H}]\mathrm{NADH}$, radioactivity soon appears in the mitochondrial matrix. Conversely, no radioactivity appears in the matrix after the introduction of $\left[7^{-14}\mathrm{C}\right]$ NADH. What do these observations reveal about the oxidation of extramitochondrial NADH by the respiratory chain?

Time Scales of Regulatory Events in Mitochondria Compare the likely time scales for the adjustments in respiratory rate caused by a. increased [ADP] and b. reduced ${\mathrm{pO}}_2$. What accounts for the difference?

The Pasteur Effect When investigators add ${\mathrm{O}}_2$ to an anaerobic suspension of cells consuming glucose at a high rate, the rate of glucose consumption declines greatly as the cells consume the ${\mathrm{O}}_2$, and accumulation of lactate ceases. This effect, first observed by Louis Pasteur in the 1860 s, is characteristic of most cells capable of both aerobic and anaerobic glucose catabolism. a. Why does the accumulation of lactate cease after the addition of ${\mathrm{O}}_2$ ? b. Why does the presence of ${\mathrm{O}}_2$ decrease the rate of glucose consumption? c. How does the onset of ${\mathrm{O}}_2$ consumption slow down the rate of glucose consumption? Explain in terms of specific enzymes.

Mitochondrial Disease and Cancer Mutations in the genes that encode certain mitochondrial proteins are associated with a high incidence of some types of cancer. How might defective mitochondria lead to cancer?

Dariable Severity of a Mitochondrial Disease Different individuals with a disease caused by the same specific defect in the mitochondrial genome may have symptoms ranging from mild to severe. Explain why.

Diabetes as a Consequence of Mitochondrial Defects Glucokinase is essential in the metabolism of glucose in pancreatic $\beta$ cells. Humans with two defective copies of the glucokinase gene exhibit a severe, neonatal diabetes, whereas those with only one defective copy of the gene have a much milder form of the disease (maturity onset diabetes of the young, MODY2). Explain this difference in terms of the biology of the $\beta$ cell.

Effects of Mutations in Mitochondrial Complex II Single nucleotide changes in the gene for succinate dehydrogenase (Complex II) are associated with midgut carcinoid tumors. Suggest a mechanism to explain this observation.

Membrane Fluidity and Respiration Rate The mitochondrial electron transfer complexes and the ${\mathrm{F}}_0{\mathrm{F}}_1$ ATP synthase are embedded in the inner mitochondrial membrane in eukaryotes and in the inner membrane of bacteria. Electrons are shuttled between complexes in part by coenzyme Q, or ubiquinone, a factor that migrates within the membrane. Jay Keasling and coworkers explored the effect of membrane fluidity on rates of respiration in $E$. coli. E. coli naturally adjusts its membrane lipid content to maintain membrane fluidity at different temperatures. Workers in the Keasling lab bioengineered an $E$. coli strain to allow them to control expression of the enzyme FabB, which catalyzes the limiting step in the synthesis of unsaturated fatty acids in $E$. coli. a. How does the content of unsaturated fatty acids affect membrane fluidity? b. The researchers were able to modulate the content of unsaturated fatty acids in the membrane lipid from $15\mathrm{\%}$ to $80\mathrm{\%}$. They did not try to completely block synthesis of unsaturated fatty acids to extend the experimental range in the membrane to $0\mathrm{\%}$. Why not? c. When the cells were grown under aerobic conditions, the researchers found that bacterial growth rate increased as the concentration of unsaturated fatty acids in the membrane increased. However, when oxygen was very limited, the unsaturated fatty acid content of the membrane had no effect on growth rate. How might you explain this observation? d. The researchers measured rates of respiration, finding a strong correlation between those rates and the fraction of membrane fatty acids that was unsaturated. When the unsaturated fatty acid content of the membranes was kept low, the cells accumulated pyruvate and lactate. Explain these observations. e. Next, they measured rates of diffusion of membrane phospholipids and ubiquinone in vesicles derived from $E$. coli membranes. The diffusion rates increased as a function of the content of unsaturated fatty acids. These measured rates were consistent with simulations carried out to model the effects of ubiquinone diffusion on respiration. What overall conclusion can be drawn from this work?