Oxidative PhosphorylationLecture 30Key Concepts• Structure and function of the ATP synthase complex• Transport systems in mitochondria• Regulation of oxidative phosphorylationHow does proton flow through the F1F0ATP synthase complex drive ATP synthesis? Why does glucose oxidation in muscle cells produce two less ATP than in liver cells?Biochemical Application of the Oxidative PhosphorylationThe F1 component of the ATP synthase complex can be used as a "nanomotor" to drive ATP synthesis by attaching a magnetic bead to the gamma subunit and forcing clockwise rotation using electromagnets. Nano-biotechnology is a rapidly growing field that utilizes biomolecules to build molecular machines.The ATP Synthase Comples uses the proton-motive force generated via the electron transport system to synthesize ATP through conformational changes in a process called oxidative phosphorylation.ATP Currency Exchange Ratios of NADH and FADH2Experimental measurements demonstrate 3 H+are required to synthesize 1 ATPwhen they flow back down the electrochemical proton gradient through the ATP synthase complex, and 1 H+is needed to transport each negatively-charged Pi molecule into the matrix. ATP Currency Exchange Ratios of NADH and FADH2Taking into account the requirement of 3 H+/ATP synthesized, and the use of 1 H+ to translocate ADP, We can now see where the ATP currency exchange ratios of ~2.5 ATP/NADH and ~1.5 ATP/FADH2come from: oxidation of NADH by complex I leads to10 H+/4 H+= 2.5 ATPoxidation of FADH2by complex II yields6 H+/4 H+= 1.5 ATP for FADH2Structure and Function ofthe ATP Synthase Complex• ATP synthase complex represents one of the quintessential protein machines found in living cells.• Mitochondrial ATP synthase complex consists of two large structural components called F1which encodes the catalytic activity, and F0which functions as the proton channel crossing the inner mitochondrial membraneThree functional unitsof ATP Synthase1. the rotor rotates as protons enter and exit the ring2. the catalytic head piececontains the enzyme active site in each of the three beta subunits3. the stator consists of the α subunit imbedded in the membrane which contains two half channels for protons to enter and exit the F0component, and a stabilizing arm.3D view movieA 3D view of alpha3-beta3-gammaClick hereto go to UC Berkeley sitefor ATP synthase movies.Proton flow through F0alters the conformation of F1subunitsThe realization that the catalytic activity of the three beta subunits was regulated by conformational changes induced by the rotating g subunit provided the key to understanding the enzyme mechanism of the F1F0ATP synthase complex. Nucleotide binding studies revealed that it was the affinity of the beta subunit for ATP, not the rate of ATP synthesis (or ATP hydrolysis in isolated F1fragments), that was altered by proton flow through the F0component. This conclusion came from studies showing that in the presence of proton-motive force the dissociation constant (KD) decreased by a million-fold. Based on these results, and on what was known about the subunit composition of the F1component, Paul Boyer at UCLA proposed the binding change mechanism of ATP synthesis to explain how conformational changes in β subunits control ATP productionTop view movie:A top view of alpha3-beta3-gammaClick hereto go to UC Berkeley sitefor ATP synthase movies.The binding change mechanism incorporatesthree basic principles1. The gamma subunit directly contacts all three beta subunits, however, each of these interactions are distinct giving rise to three different βsubunit conformations.2. The ATP binding affinities of the three beta subunit conformations are defined as: T, tight; L, loose; and O, open; in which ADP and Pi bind to the O and L conformations, and ATP binds tightly to the T conformation but is released from the enzyme when the β subunit is in the O conformation. 3. As protons flow through F0the gamma subunit rotates in a counter-clockwise circle (looking at F1from the matrix side) such that with each 120º rotation the b subunits sequentially undergo a conformational change from L --> T --> O --> L.The binding change mechanism model predicts that one full rotation of the gamma subunit should generate 3 ATP since each beta subunit will have cycled once through the T state.Cross section view movie:A cross-section view of alpha-beta-gammaClick hereto go to UC Berkeley sitefor ATP synthase movies.Note that the ratio of 3 H+/ATP is not yet certain because there are unanswered questions regarding the molecular mechanism of the proton-driven rotor (see below). Nevertheless, we will use 3 H+/ATP here because it is consistent with the ATP currency exchange ratio of ~2.5 ATP/NADH, as well as the proton pumping ratio of 10 H+/NADH by the electron transport system (see lecture 29), both of which have been empirically determined. Boyer's model predicts that ATP hydrolysis by the F1headpiece should reverse the direction of the γ subunit rotor. To test this idea, Masamitsu Yoshida and Kasuhiko Kinosita of Tokyo Institute of Technology used recombinant DNA methods to modify the alpha, beta and gamma subunits of the E. coli F1component in order to build a synthetic molecular motor. Movie: atp.movHow does proton movement through the c subunit ring cause rotation of the γ subunit? A proposed model for the F0"rotary engine" is shown below based on structural analysis of the yeast mitochondrial c subunit ring that was found to contain 10 identical subunits. Since the concentration of H+on the P side (positive side; inter-membrane space) is higher than it is on the N side (negative side; matrix), a H+(P) will readily enter the half channel in the a subunit where it then comes in contact with a negatively charged aspartateresidue (D61) in the nearby c subunit. A Carousel Ride at a Carnivalthe carousel is the c subunit ringthe entrance and exit lines for the carousel are the two different proton channels in the a subunit.Transport Systems In The MitochondriaKey element of the Chemiosmotic Theory:The inner mitochondrial membrane must be impermeable to ions in order to establish the proton gradientBiomolecules required for the electron transport system and oxidative phosphorylation must be transported, or "shuttled," back and forth across the inner mitochondrial membrane by specialized proteins Accomplished by two translocase proteins located in the inner mitochondrial membrane Two Translocase Proteins1. ATP/ADP
View Full Document
Unlocking...