BioChem 330 - Course Outline ATP.Synthase

BioChem 330 - Course Outline • Metabolism and Bioenergetics (II) – ENZYME CATALYSIS: • kinetic constants kcat, Km • Catalytic strategies, the serine proteases – CATABOLISM (breakdown) • Carbohydrates – – – – Glycolysis Tricarboxylic Acid Cycle Electron Transport Chemiosmosis and ATPase • Fatty acids and amino acids Overview for Today, November 17, 2011 •Coupling proton pumping to ATP production through the electrochemical potential •Smallest little motor in the world, the F0F1ATPase ET coupled to ATP synthesis • ET chain induces electrochemical potential gradient by pumping protons across energy transducing inner mitochondrial membrane against proton and voltage gradient: ‒ ΔpH = 1.4 ΔV = 0.14 V ‒ ΔG = 2.303 RT Δ pH + Z F ΔV = • ATP synthesis takes those protons pumped by ET and allows them to flow back into the cell through a clever little motor known as ATP synthase, catalyzes: ET coupled to ATP synthesis • ET chain induces electrochemical potential gradient by pumping protons across energy transducing inner mitochondrial membrane against proton and voltage gradient: ‒ ΔpH = 1.4 ΔV = 0.14 V ‒ ΔG = 2.303 RT Δ pH + Z F ΔV = 5.2 kcal/mole H • ATP synthesis takes those protons pumped by ET and allows them to flow back into the cell through a clever little motor known as ATP synthase, catalyzes: ΔGo’ = + 7.3 kcal/mole Chemiosmosis: the picture •Understanding  the   detailed  book-­‐keeping   of  H+  stoichiometry   has  changed  over  ;me,   your  text’s  accoun;ng   at  odds  with  newer   texts  which  cite  3H+   through  Complex  I   •The  intermembrane  space  is  essen;ally  in  equilibrium  with  the   cytoplasm,  which  is  buffered  at    pH  higher  than  the  mito,  and  so,   added  protons  don’t  change  the  pH  of  the  cytoplasm  but  have   profound  effect  on  the  pH  of  the  matrix  (like  adding  a  few  drops   of  acid  to  the  ocean  would  have  no  effect,  but  adding  it  to  a   thimble  would  have  a  large  effect).   Chemiosmosis: the picture  Electron  flow  is   accompanied  by  proton   transfer  across  the   mitochondrial   membrane,  producing   both  a  chemical  (ΔpH)   and  an  electrical  (ΔV)   gradient    3H +  The  proton-­‐mo;ve   force  that  drives  protons   back  into  the  matrix   provides  the  energy  for   ATP  synthesis,  catalyzed   by  the  F1  complex   associated  with  Fo.    – ΔG  =  2.3RT  ΔpH    +  F  ΔV   – F  =  23.06  kcal/mole/V   • Mitochondria:     – ΔpH  =  1  (alkaline  inside)   – ΔV    =  0.1-­‐0.2  V  (inside  nega;ve)     Basic  evidence   • Electron  transport  is  coupled  to  ATP   synthesis  (more  detail  in  a  moment)   • Coupling  requires  an  intact  membrane   • Electron  flow  through  individual   complexes  results  in  proton  pumping   • Proton  carriers  uncouple  electron   transport  and  ATP  synthesis   What experiments look like… Coupling: An Electron Source An inhibitor Of electron transport (Binds to cytochrome oxidase) Or, more subtly… “Uncoupler” ATP synthesis (“ATPase”) Inhibitors What they do… “Uncouplers” What they look like… A natural version of these things in brown fat… Crea;on  of  an   electrochemical   gradient  is   sufficient…   Structure of FoF1 ATPase: the littlest motor in the world 1. Fo unit: proton conducting unit, integral membrane proteins, proton channel formed by a ring of 10-14 c subunits and a single a subunit which binds to outside of c ring. 2. F1 catalytic unit (α3β3γδε) which consists of a hexamer of alternating α and β subunts (only β is active), a central stalk of γ and ε long α-helical proteins, which secure F1 to Fo. Assymetry of γ is key to activity 3. b subunit of Fo interacts with F1 through d. Looking down through the top of F1 * γ subunit breaks the symmetry, interacting differently with each of the β subunits (color), •ATP binds differently to each conformer: •L is loose (ADP and Pi bound), •T is tight (ATP made), •O is open, (bound ATP released). •Intact assembly, no proton gradient, ATP is formed, but not released. Binding Change Model for coupling Perspective from matrix above F1 : rotation of γ counterclockwise ATP synthesis 1. Each counterclockwise step of the red wedge (γ subunit) by 120o changes β conformation: O -> L or L->T, or T -> O 2. Each full cycle of γ (3x120o steps) makes 3ATP(1 from each β) 3. Catalysis only happens in T 4. Motor can go backwards for hydrolysis (ATPase) in bacteria, but only ATP synthesis in mitochondria and chloroplasts. F1 the first single molecule experiments ATP Hydrolysis: Energy Wasted H.Noji*, R.Yasuda*, M. Yoshida, and K. Kinosita, Jr. (1997). Nature 386:299-302 Equally contributed F1 the first single molecule experiments 1. Experimental witness to rotation of γ subunit with single molecule spectroscopy. 2. NO Fo subunit, DRIVING FORCE IS ATP HYDROLYSIS 3. When ATP is hydrolyzed, shaft goes in the opposite direction as before L → O; T → L: O → T releasing Rotation is in 120o steps ADP product Rotor speed is 100 Hz, 3 ATP/cycle Rate is 100 ATP/s/subunit, Why does γ rotate in membrane? γ runs through F0, where protons are pumped Coupling of Fo toUnlike F1what seems to be illustrated in this image, it Intermembane Side S u b u n i t c Matrix Side is not the a subunit that rotates, but the c subunits. The c subunits each contain an aspartic acid group centrally located within the helix within the membrane. When subunit a is in association with this subunit c, this asp is connected through a half channel to either the matrix space or the intermembrane space. When the channel connects to the matrix space, the asp deprotonates because of the H+ gradient. This deprotonation is followed by a conf change which ratchets the newly deprotonated c into alignment with the second half channel, in which it reprotonates from the intermembrane space. Meanwhile, the next c has just deprotonated to matrix, and the assembly ratchets again, moving the newly protonated asp out of alignment with any channel. it will undergo a full rotation around the assembly, driven by the successive protonation/ratchet events, until it comes full circle to the a subunit and the matrix channel, where it once again, deprotonates, ratchets and protonates. Coupling of Fo to F1 ………...Meanwhile, the rotation of the c subunits within Fo transfer this rotation to the γ subunit which acts as a shaft connecting Fo to F1. Rotation of γ about the hexamer of α3β3 converts b subunits successively from O to T to L. Matrix Side γ subunit shaft communicates c rotation to F1 Nature Struct. & Mol. Bio. 11 Feb 2004 110-112 At low dilution, only one ATP synthase containing liposome will migrate into irradiation volume at a time Green sphere is fluorescence donor on γ (which rotates) and red sphere is fluorescence acceptor on stator, which doesn’t move. In a FRET experiment, whenever the donor (green) comes close to the acceptor (red) energy will be transferred to acceptor. Figure  2.  Photon  bursts  from  single  F0F1-­‐ATP  synthases  in  liposomes.   (a,b)  Photon  bursts  during  ATP  hydrolysis.  (c,d)  Photon  bursts  during  ATP  synthesis.  FRET  donor   is  TMR  in  a  and  c,  Rh110  in  b  and  d;  FRET  acceptor  is  Cy5bis  in  all  traces.  Fluorescence  intensity   of  the  donor,  FD,  and  acceptor,  FA,  are  green  and  red,  respec;vely,  in  a  and  c  (;me  window  1   ms).  Corrected  intensity  ra;os  FD  /  FA  are  orange  in  all  panels  (;me  window  1  ms),  fluorescence   life;mes  of  the  donor  Rh110  are  green  in  b  and  d  (total  ;me  window  5  ms,  shiaed  by  2.5  ms  per   data  point).  Three  dis;nct  FRET  levels  are  abributed  (1,  2  or  3)  at  top  of  panels.