Salvage: by PHOSPHORYLATION... adds a phosphate to ADP                 ADP   +   P    <------>    ATP     (recycle*)   cell ratio of ATP/ADP is about 10:1 (a non-equilibrium state)

How do cell's phosphorylate ADP to make ATP?                    a)  Substrate Level Phosphorylation...                  where a substrate molecule ( X-P ) donates*  its P to ADP making ATP     fig 9.6*              b) Oxidative Phosphorylation... involves capturing e-'s for use in Electron Transfer Chain...                  1. food molecules donate e- & protons to acceptor molecules via oxidation NADH*                  2. NADH passes on electrons & protons are pumped out of mitochondria...                                                   (or the chloroplast membranes in photosynthesis);                  3. protons diffuse back into mitochondria thru an enzyme = ATP synthase,                      the ATP synthase enzyme makes ADP + P  -->  ATP                                figure*                                                                                                                                          c) Photophosphorylation...                 e- of light energy, instead of food covalent bonds, are captured by chlorophylls                     to make a proton gradient across the chloroplast membranes...                figure*              protons move through a chloroplast ATP synthase enzyme to make ATP

   
1st - Oxidative Metabolism...     (Cell Respiration in animal cells)
             occurs in heterotrophic organisms that consume foods
                      ... we say organisms oxidize (consume) foods (often glucose) to make energy
                                                        because it removes & captures electrons...
                      ... where is energy in foods?     it's in the covalent bonds (e-)
             thus =  oxidative metabolism is...   cells capturing e^- via REDOX reactions.
   
        what is a REDOX  REACTION*...       methane example - fig 9.2*
                      in step 6 of glycolysis  e- passed  from   PGAL --> to  diPGA*
                      energy is transferred via a redox couple with NAD⁺ by e-
  
        OXIDATION     =    removal of electron &/or protons from food covalent bonds
  
        REDUCTION   =    gaining electron &/or protons; adds an electron to an acceptor molecule
   

       Redox state* of molecules     -->     the more reduced  =  the more energy it holds 

  
a model redox reaction... 
   
      A-H   +  B-O        <--->           A     +        B-O-H
         donor         acceptor          (:H)              acceptor                  donor

Let's look at a redox reaction in the Krebs cycle pathway using NAD⁺.
         isocitrate + NAD⁺  < --- >   alpha-ketoglutarate*      +       NADH
  
              reducing    oxidizing                       becomes                 becomes
                agent         agent                          oxidized                  reduced

              a 2nd redox coenzyme (couple) of living cells is   FAD⁺  <-->  FADH₂*  
                                                                                                      acceptor       reduced
 
        Transition metals [metallic elements with unfilled valances that accept e's...
        Cu, Fe, Zn, Mn] and may have been involved in origin biological redox reactions
        and evolved into today's oxidoreductase (dehydrogenase) enzymes?
           Structural similarity suggests an early origins of biological electron transfer* 
 
Thus : heterotrophic metabolism is the stepwise OXIDATION of molecules (foods)...
  
                    if AEROBIC - directly requires oxygen directly as the electron acceptor
     
                    if ANAEROBIC - requires no oxygen directly (uses other e- acceptors) 

          

 
  CELL RESPIRATION...  is the oxidation of food stuffs that produces ATP.

                                      Overview of Cellular Respiration*   

                                                                          

      1.  oxidation of GLUCOSE  (6C)    -->    PYRUVATE   2 (3C)   -->   GLYCOLYSIS
  
      2.  oxidation of pyruvate --> CO₂ + H₂O                                    --> KREBS CYCLE                           

      3.  reduction O₂  to  H₂O                              --> ELECTRON TRANSPORT CHAIN
     
                     C₆H₁₂O₆ + 6O₂   <---->   6 CO₂ + 6 H₂0    + e- --->    36-38 ATP
                      ΔG  =  -686 Kc/mole                                              -270Kc  = 39%

                     called oxidation...  because e- are removed from glucose
                 
                     called reduction...  because e- passed to O₂ making water
  
      4.  phosphorylation of ADP  via  ATP synthase
                     thus the terminology... oxidative phosphorylation  [chemiosmosis]
           
                    Cell Respiration is a multi-pathway process*...     

 a more complete description of Cell Respiration :
 
    -  series of enzyme rx's (in biochemical pathways) in the cytoplasm & mitochondria
                   (glycolysis, fermentations, pyruvate oxidation, Krebs cycle, electron transport),
  
    -  removes e- (oxidation) from covalent bonds of substrates (as glucose), and
  
    -  pass e- to acceptor molecules [redox coenzymes] such as NAD⁺ & FAD*
                   which become reduced to  NADH  & FADH₂
  
    -  the reduces these coenzymes [ NADH & FADH₂ ]  passing e- to other e- acceptors...
                   a series of metalloprotein Electron Transport carriers, including cytochromes,
  
    -  the electron carriers (cytochromes) pass e- to O2  --- reducing it to --->   H₂O
  
    -  while pumping protons [H⁺]  out of mitochondria into the peri-mitochondrial space,
                    creating a proton gradient as an diffusive energy source
  
    -  the protons move back into mito thru a special enzyme (ATP synthase)  &  make ATP

          
                         let's look at the details of each of these processes...                     

all the details:   PATHWAYS of CELL RESPIRATION which make ATP...             what should we know - what goes in,  what comes out, and where,            also the types of chemical reactions that occur in the pathways.
Glyco-lysis : 10 step pathway converts 1 glucose (C6) to 2 pyruvate (C3)         products :  2 molecules of pyruvate,   2 NADH,   &   2 ATP (net)         occurs in :  cytoplasm  [and is anaerobic - does not require O2]            may lead to:   alcoholic fermentation     =   glucose --> alcohol                                   lactic acid fermentation   =   glucose --> lactic acid
KREBS Cycle  :   oxidizes  2 pyruvates  from glycolysis  to   CO2  +  H2O                               produces :         8 NADH,   2 GTP (ATP equivalent),    2 FADH2                               releases :          6 CO2   (thus called...  Cell Respiration)                               occurs in the mitochondria  [and is aerobic = directly requires O2]
ETC - Electron Transport Chain  :   uses e- carrier proteins (including cytochromes, etc...)                     to pass   e- & H+  from  NADH & FADH2  to   O2   making H2O   &                    generates a proton gradient (chemiosmosis) across the inner mitochondria membrane
ATP synthase :  a carrier enzyme in the inner mitochondrial membrane that                             passively transports  H+  back into mitoplasm & while making ATP directly

  
1.  GLYCOLYSIS...                          
  
 KEY ENERGY REACTIONS of GLYCOLYSIS...     Animation of Glycolysis*                     
                                            don't memorize the pathway, but learn the major reaction types...
          1.  substrate level phosphorylation* [occurs twice* in glycolysis]
          2.  redox reaction   step 6*  involving   NAD⁺    
          3.  reaction paths -->    investment phase*  &  payoff phase*  -  Summary of glycolysis* 

FATES of the products:              1. PYRUVATE*...              if  anaerobic - involves alcoholic fermentation  &   lactic acid respiration                   alcoholic fermentation*          "history of wines"  animation                      lactic acid fermentation*         also called anaerobic respiration                 if  aerobic - involves pyruvate dehydrogenase  +  Krebs Cycle  +  ETC                                                                          2. cytosolic NADH... - it holds captured e- energy, but how some cells can use it?          mito membrane is impermeable to NADH -> cytoplasmic & mitoplasmic pools         Shuttles*  malate shuttle        (liver, kidney, heart) =   NADHc --> NADHm                          glycerol-P shuttle   (muscle/brain)          =   NADHc --> FADH2m         Purpose:  to move electrons captured in cytosolic NADHc into mitochondria

    
  
        SUMMARY of GLYCOLYSIS

              -  2 ATP to initiate
              -  2 substrate level phosphorylation steps  =  4 ATP gross (2 ATP net) and
              - 1 redox step making NADH

                thus Glycolysis makes:   (figure*)  
                          2   ATP (net),
                          2   NADH, and
                          2   PYRUVATES 
                                    remember the role of the ...  Fermentations & Shuttles

               END OF MATERIAL FOR EXAM #3 (electron microscopy to here) 
 
                                                          the Glucose song^{listen at home}

   
 
Heterotrophic Metabolism (cell respiration) in Aerobic Organisms...   
               if aerobic then the Fate of Pyruvate*  is oxidation in mitochondria

1.  PYRUVATE DEHYDROGENASE Reaction... [before Krebs cycle itself]             in mitoplasm (Fig 9.10)*  oxidizes  PYR --> acetyl-CoA             a multienzyme complex of 60 proteins and 5 coenzymes             involves CoASH*    --ADP*-->  acetyl coenzyme-A [Fritz Lippman]             reactions:  1.   decarboxylation (-CO2),                               2.   reduction of 2 NAD+  -->  2 NADH                                         3.   acylation with synthesis of 2 Acetyl-coA  .
2. KREBS CYCLE [Sir Hans Krebs]   animation of cycle*  &   cycle summary*             Key Reactions                full cycle details       &   enzymes involved                  1.   NAD+ is reduced  (6 NADH)   and   FAD is also reduced (2 FADH2)             2.   substrate level phosphorylation occurs  (2 GTP <--> 2 ATP)             3.   decarboxylation occurs -4x [-COOH]             4.* 2 acylation reactions via coenzyme-A  (forms 2 Succinyl-coA)
-->   how many actual ATPs per glucose are made so far?  only 4 (net) by SLP

 
   
  ELECTRON TRANSFER CHAIN    &   OXIDATIVE PHOSPHORYLATION    
    the coupling of oxidation of substrates (-e) to phosphorylation of ADP to make ATP
                                    .                                                       
    ► remember, most of the energy of glucose's bonds is now carried in NADH & FADH₂  
          e- are passed from NADH/FADH₂  to  O₂ (aerobic)  ►  ETC animation*       
         these series of electron carrier proteins occur in 4 membrane subunits  fig 9.14*   
               I)    NADH Reductase,                   II )    Succinate Dehydrogenase
               III)  Cytochrome Reductase,         IV)    Cytochrome Oxidase
  
    
 CHEMIOSMOSIS: hypothesis of e- transport createing a PROTON MOTIVE FORCE  -  figure* 
           e- carriers release protons into perimitochondrial space making a proton gradient.
           1978 Nobel Prize (Chemistry) to Peter Mitchell for Chemiosmotic Hypothesis.

           Can a hydrogen ion gradient (H⁺) created by  e- flow thru the ETC also make ATP ??? 
                -  experimental evidence of a   H⁺ gradient  making ATP came via  bacteriorhodopsin*
                -  H⁺ diffuse back into mitoplasm thru ATP synthase  --->   ATP via a molecular motor*
               -  Boyer hypothesis* =  chemical to mechanical chemical energy (like a water wheel)*
                -  animation of ATP synthase action* 

                                                                                                               

       
  
  Overview of Cell Respiration*... 
        ►  A rule of thumb for the amount of ATP made per e- pair...                   
                  the Phosphate/Oxygen Ratio, or (P/O ratio), refers to the amount of ATP produced
                  from the movement of 2 e's through the ETC to the reduction of an oxygen atom.    
                  Measured empirically it is  -->    NADH  ≈  3 ATP     &      FADH₂  ≈  2 ATP

        So How much ATP can be made per Glucose...
                  the ATP/O₂ ratio...      based on 3 ATP per NADH  &  2 ATP per FADH₂
                                                     idealized = 36 to 38 ATP      [ a yield table ]
        
        What can serve as substrates for aerobic metabolism?*
                carbohydrates, proteins, lipids... can serve as substrates         
        Aerobic metabolism intermediates are precursors for other molecules*
   
        How is heterotrophic metabolism regulated?  
                1.  stiochiometric [substrate concentration] levels is a main control mechanism
 
                2.  allosteric controls include:  +AMP/ADP stimulate enzymes  &  +ATP inhibits enzymes
                        Key mammalian allosteric enzyme :   PHOSPHOFRUCTOKINASE*
                                                                                       feedback inhibition
                                                                                       allosteric regulation   
    

    
SUMMARY - Heterotrophic Metabolism - CELL RESPIRATION

    1. Substrates  =  sugars,   amino acids acids,   fatty acids

    2. Pathways  =  Glycolysis,  Krebs Cycle,  ETC,  &  ATP synthase are Universal to cells

    3. Products  =   C0₂, H₂0, and energy as  H⁺ gradient,   NADH,   FADH₂,  &   ATP

    4. part of process is Anaerobic (doesn't require -0₂ ; Glycolysis)
                    & may include alcohol & lactate fermentations    (...anaerobic respiration)
                    & part is  Aerobic    (requires +0₂  ...electron transfer chain),

► 5. Reaction types include:
                  oxidations,    reductions,    substrate level phosphorylations,
                  decarboxylations,    acylation,   oxidative phosphorylation,
                  & hydrolysis  (or dephosphorylation)

    
   6. Energy capture is via   electron transfers,  proton pumps,  H⁺ gradients,  &  ATP synthase*

   7. Regulation is by:
                stiochiometry,  feedback inhibition  &  allosteric modulation 
                (key enzyme:  phosphofructokinase)

   8. Intracellular compartmentation:      
                glycolysis occurs in the cytosol
                Krebs Cycle is mostly in the mitoplasm of mitochondria
                ETC is in the cristae membranes of mitochondria
                ATP synthase within the inner mitochondrial cristae membrane

   9. Cell Respiration is KEY and central to all of a cell's metabolic pathways
                                                            IUBMB-Nicholson Metabolic Pathways Chart
  

    back      next lecture* - photosynthesis                    Key Concepts*

      copyright c2023     Last update -  March  2023
        Charles Mallery,    Biology 150, Department of Biology,   U. of Miami,  Coral Gables, FL 33124

    SKIP THE MATERIAL & LINKS BELOW...

 If you have comments or suggestions, email me at  cmallery@miami.edu

 oxidative phosphorylation animation

-   Individual reaction steps of glycolysis

    anim ATP synthase rotary mech - side  & top
                -  ATP synthase & H-Gradient make ATP*^view@home  &  BioVisions-Powering the cell*^view@home      

     animation of ATP synthase action

      know what goes in & come out*

                                  Cancer reprograms energy metabolism

     "what are these carrier molecules*" 

         (figure)