Oxidative Phosphorylation CH 19 (pp ) March 31, 2015 BC368Biochemistry of the Cell II

"Anyone who is not confused about oxidative phosphorylation just doesn't understand the situation." -Efraim Racker Oxidative phosphorylation is the coupling of energy release during electron transport to ATP synthesis.

Chemiosmotic Theory Fig 19-19

Proton Motive Force Fig = -

Case Study In 1933, Stanford biochemists Cutting and Tainter published a report in the Journal of the American Medical Association on the use of dinitrophenol (DNP) to treat obesity. After its first year on the market, an estimated 100,000 people had been treated with DNP in the United States, in addition to many others abroad. Unfortunately, in some cases the treatment eliminated not only the fat, but also the patient. How does DNP work as a diet pill, and what side effects would you expect?

Fig 19-21

Uncouplers Fig 19-20

Uncouplers Fig  Thermogenin dissipates the proton gradient…no work is done. Huffington Post

Uncouplers Fig 19-34

Other ways to waste energy  Bypassing the proton pumps leads to production of heat instead of ATP

Chemiosmotic Theory Fig 19-19

Mechanism of ATP Synthesis

ATP Synthase: F o and F 1  In the 1960’s, “lollipop” structures were evident through electron microscopy in samples of everted inner membranes from bovine mitochondria.

ATP Synthase: F o and F 1 Matrix side

ATP Synthase: Kinetics Fig 19-24

ATP Synthase: The Binding Change Mechanism Each β subunit has a different conformation:  β-ADP  β-ATP  β-empty Fig 19-26

1.ADP and Pi bind Fig ATP Synthase: The Binding Change Mechanism

2.Conformation changes, catalyzing ATP formation; energy provided by H + movement Fig ATP Synthase: The Binding Change Mechanism

3.Conformation changes; ATP dissociates; energy provided by H + movement Fig ATP Synthase: The Binding Change Mechanism

4. Conformation changes back to initial state so that cycle continues Fig ATP Synthase: The Binding Change Mechanism AnimationAnimation: Binding Change Mechanism

ATP Synthase ATP Synthase: The Binding Change Mechanism

AnimationAnimation: start at :23 ATP Synthase: Rotation of F o via the c Ring Each c subunit has two half- channels, open to either the intermembrane space or to the matrix, that allow protons to access a key Asp residue. Protonation of the Asp relieves the negative charge and allows rotation into the membrane. Rotation of negative Asp out of the membrane results in deprotonation.

Energy balance sheet

Mitochondrial “shuttles”  Functionally, transport of OH - out is the same as transport of H + in.

Pmf-driven transport Fig 19-30

Malate-Asp shuttle  Liver, kidney, and heart  Results in NADH in the matrix Fig  Complicated, but free!

Malate-Asp shuttle  Complicated, but free!  Liver, kidney, and heart  Results in NADH in the matrix Fig 19-31

Glycerol 3-P shuttle  Electrons enter at Q.  Skeletal muscle and brain  Easier, but costly!

Regulation Acceptor Control Fig 19-20

Regulation Coordinated Control Fig 19-35

In-Class Problem The mitochondria of a patient oxidize NADH irrespective of whether ADP is present. The P:O ratio (ATP synthesized per oxygen atom [or pair of electrons] consumed) for oxidative phosphorylation by these mitochondria is less than normal. Predict the likely symptoms of this disorder.

Hypoxia  Normally, the ATP synthase makes ATP, using the proton gradient  Sometimes, the ATP synthase uses ATP to generate a proton gradient (acts as a ATPase). makes (bacteria or hypoxia)

IF 1 inhibitor (a dimer at low pH) Hypoxia Inhibition of ATPase by IF 1 Fig  The protein IF 1 protects the cell from hypoxia-induced ATP hydrolysis.

Hypoxia  When O 2 is limiting, electrons may fall out of the electron transport chain, often at  Q −.

Hypoxia  When O 2 is limiting, electrons may fall out of the electron transport chain, often at  Q −.  Superoxide dismutase converts  O 2 − to H 2 O 2.  Glutathione peroxidase breaks down the H 2 O 2.

Hypoxia  Other protective effects are mediated by HIF-1: Decreased activity of PDH (via the kinase). Swapping out of a complex IV subunit.

Assign each inhibitor to one of the oxygen traces on the right (the y- axis is [O 2 ]; isolated mitochondria; succinate is the electron source)