Second Half 
         Chapter 10 of Becker et al. 6th edition 
                 Chemotrophic Energy Metabolism:
                      Aerobic Respiration

back to First Half of Aerobic Respiration

  I. Electron Carriers in ETC
     1. relate to previous diagram
     1. flavoproteins
        a. flavoproteins = proteins which use a flavin 
            containing coenzyme (usually FMN or FAD)
           (1)   FMN = flavin mononucleotide
           (2)   FAD = flavin adenine dinucleotide
        b. eg: NADH dehydrogenase
           (1)   uses FMN to remove e- from NADH & to
                   transfer e- to coenzyme Q
        c. eg: succinate-coenzyme Q reductase
           (1)   uses FAD to remove 2e- from succinate & 
                    transfer e- onto coenzyme Q
     2. coenzyme Q = ubiquinone
        a. ubiquinone + 2H+ +2e-  --> dihydroubiquinone
        b. see Fig 14-15
        c. as ubiquinone is reduced, H+s and e-s are picked
            up at the inner surface inner mitochondrial
            membrane
        d. when ubiquinone is oxidized, H+s and e-s are given up
             at the outer surface of the inner mitochondrial 
             membrane 
        e. Net Effect: dihydroubiquinone carries H+s and 
            e-s across hydrophobic interior of membrane by 
            diffusion
           (1)  this shuttling redox reaction serves as a 
                  proton (H+) pump
           (2) see cartoon of a quinone shuttle
    3. iron-sulfur proteins
        a. iron and sulfur atoms complexed with four "R"
           groups of cysteine residues
           (1)   Fe and S in equimolar amts
              (a)   Fe2S2 & Fe4S4 are common
              (b)   Fe can be reversibly oxidized and reduced
                 i) Fe+3 + e-  <--> Fe+2
      b. eg: NADH dehydrogenase is also a iron-sulfur protein
           (1)   e- first go from NADH to FMN and then from 
                   FMNH2 to the Fe-S and then to ubiquinone
                   binding site and on to ubiquinone
       c.  eg: eg: succinate-coenzyme Q reductase is also 
            a iron-sulfur protein 
                 -image of SDH  
                 -chime
     4. cytochromes (cyt)
           -eg: cytochrome C
        a. cytochromes are proteins
        b. cytochromes always contain heme groups spacefilling
                -heme groups give cytochromes their red color 
        c. heme groups always have a central Fe
           (1)   Fe of heme group can be reversibly oxidized 
                   or reduced
                  (a)   Fe+3 + e-   <--> Fe+2  
        d. electron transport from coenzyme Q to O2 requires 
            several cytochromes 
        d. eg: five different cytochromes in mitochondrial ETC
           (1)   cytochrome b
              (a)   integral membrane protein
              (b)   part of the bc1 complex
           (2)   cytochrome c1
              (a)   integral membrane protein
              (b)   part of the bc1 complex
           (3)   cytochrome c
              (a)   first protein crystal structure
              (a)   small peripheral protein (~10,000 daltons)
              (b)   one of the most studied proteins in all of science
              (c)    related cytochrome in almost all organisms
                    i) amino acid sequence of cytochrome c 
                        from 100's of different species are known
                    i)  differences in the amino acid sequences 
                         used to determine evolutionary "relatedness"
           (4)   cytochrome a1 + a3 = cytochrome oxidase
                    -chime 
             (a)   very large, integral, transmembrane 
                      protein complex
              (b)   two hemes (a1 and a3) combined in 
                       the cytochrome oxidase complex
 J. organization of mitochondrial ETC
     1. see Fig 14-18
             -possible test question
     2. four transmembrane protein complexes 
        a. NADH dehydrogenase (26 polypeptides)
        b. coenzyme Q-cytochrome c reductase 
            (10 polypeptides)
        c. cytochrome c oxidase (9 polypeptides)
        d. succinate coenzyme Q reductase 
            (5 polypeptides)
     3. NADH, succinate, coenzyme Q and cytochrome c are 
         connecting links
         a. NADH links TCA cycle to ETC via 
            NADH dehydrogenase
         b. coenzyme Q links both NADH dehydrogenase 
            and succinate coenzyme Q reductase to the
            cytochrome complex
         c.  cytochrome c  links the cytochrome complex 
              to cytochrome oxidase
         d. succinate also links the TCA cycle to ETC 
              via succinate dehydrogenase
     4.  NADH dehydrogenase is one entry point
            into the ETC
         a. electrons pass through the protein complex 
             and on to coenzyme Q
         b. sequence of components carrying the electrons is:
              NADH --> FMN --> Fe-S --> coenzyme Q
              (1) enzyme binds NADH at a hydrophilic site 
              (2) enzyme has a bound FMN which receives 
                    the  2e- from NADH
              (3) FMN passes electrons on to Fe-S
              (4) Fe-S passes electrons on coenzyme Q
                    (a) enzyme binds coenzyme Q at a hydrophobic site
                    (b) coenzyme Q is reduced to 
                          dihydroubiquinone
                    (c) protons for the reduction of 
                          coenzyme Q come from 
                          mitochondrial matrix
     5. succinate-coenzyme Q reductase is a second entry 
           point into the ETC 
         a. electrons go from succinate to coenzyme Q
         b. sequence of components carrying the electrons is: 
              succinate  --> FAD  --> Fe-S  --> coenzyme Q
              (1) enzyme binds succinate at a hydrophilic site
                     (a) succinate is oxidized to fumerate
              (2) enzyme has a bound FAD (FAD / FADH2) 
              (3) enzyme has bound nonheme iron (Fe-S) 
                    which transfers e- from FADH2 to the 
                    ubiquinone binding site
              (4) enzyme binds coenzyme Q at a hydrophobic site
                     (a) coenzyme Q is reduced to 
                           dihydroubiquinone
                     (b) protons for the reduction come
                           from mitochondrial matrix
     6. only the cyt a1+a3 complex can transfer e- to O2
        a. cyt a1+a3 is only link between cell's 
            chemistry and O2
     7. see animation of mitochondrial electron transport
        a. Note movement of the electrons
        b. Note the movement of the protons from matrix to the 
            space between the mitochondrial membranes
     8. see ETC for NADH to O2
VII. Oxidative Phosphorylation = ATP synthesis 
       associated with mitochondrial electron transport
  A. called oxidative phosphorylation since linked to 
       oxidatively driven electron transport
     1. distinguish from substrate level phosphorylation
        a. see glycolysis for several
            examples of substrate level phosphorylation
        b. another example of substrate level phosphorylation 
            is pyruvate kinase 
             (1) has two substrates
                   (a) ADP is one of the substrates
                   (b) phosphoenolpyruvate (PEP) is one of 
                         the substrates 
             (2) phosphate transferred from PEP to ADP
                   making ATP
             (3) this is substrate level phosphorylation
        c.  eg: phosphoglycerokinase
             (1) ADP is one of the substrates 
             (2) 1,3-bisphosphoglycerate is the other substrate
             (3) phosphate transferred from 
                  1,3-bisphosphoglycerate
                   to ADP making ATP
        d. substrate level phosphorylation occurs as part 
            of an enzyme catalyzed reaction which is part 
            of a metabolic pathway 
  B. the link between ET and ATP synthesis
     1. once very controversial
     2. now complete agreement
     3. linkage is via electrochemical proton gradient
        a. gradient established by directional H+ pumping 
            concomitant with ET
              -H+s pumped from matrix to intermembrane space
        b. see animation of electron transport and H+ pumping
  C. ET is coupled to ATP synthesis
     1. mito ATP generation requires ET
     2. mito ET requires ATP generation
     3. normally, the two events tightly (but not directly) coupled
     4. tight coupling is part of important regulatory process
        a. ATP/ADP ratio regulates rate ET
        b. thus ATP/ADP ratio controls rate of 
            mitochondrial TCA cycle
     5. ATP generation can be "uncoupled" from ET
        a. uncoupled ET goes very fast
        b. no ATP is synthesized
        c. no work done
        d. heat is liberated instead
        e. eg:  DNP = dinitrophenol, a man-made uncoupler 
            of ATP synthesis
           (1)   uncouples mitochondrial  ATP synthesis
                   -high rate ET
                   -only heat is produced
         f. certain proteins are uncouplers
                  - eg:  thermogenin = uncoupling protein 1 = UCP1
                  - special proteins are expressed
                     brown adipose tissue
                  -found only in mammels
                           -including human infants
                           -make mito membrane highly permeable to H+
                                   -can't make H+ gradient
                                   -can't develop membrane potential
                           -can be inhibited (regulated) with GDP
                            -see comparative image of
                              adipose tissues
                                     Note: white adipose has little cytoplasm
                                                 white adipose has huge oil droplet
                                                  brown adipose has more capillaries
                                                  brown adipose has much more cytoplasm
                              -higher magnification of brown adipose
                                       Note: brown adipose has many more mitochondria
                                       Note: mito in brown fat express a specific 
                                                   uncoupling protein
                                                   -TCA cycle runs, ETC runs, 
                                                     BUT, NO ATP synthesized!
                                                    -only heat is produced
  D. # of "coupling sites" = # of sites where ET is 
        connected to ATP synthesis
     1. Ochoa measured (early 1940s) the moles of 
         ATP generated and moles of oxygen atoms 
         consumed during the ATP synthesis
        a. calculated a ratio of mol ATP/ mol O2 = ATP/O ratio
        b.  ATP/O ratio depended on the source of electrons
        c. if NADH supplied e- to O2, then   ATP/O = 3
              (1) 3 ATP/2e-
                    - 2e- since each O accepts 2e-
              (2) 2 e- since NADH provides 2 e- for each O atom
        d. if succinate supplied e- (via FADH2)  to O2, then   ATP/O = 2
              (1) 2 ATP/2e-
        e. data suggested there must be at least 3 coupling 
            sites during ET from NADH to oxygen and only 2 
            coupling sites between FADH2 and O2
         f.  understood now in the energy available
     2. Racker showed by reconstitution experiments
          (isolated and purified mitochondrial protein complexes
           were reconstituted into artificial PL vesicles containing 
          CoQ) that 3 ATP could be synthesized per 2 e- passed 
          through the ETC from NADH to oxygen
         a. this experiment provided data supporting 
             the model presented in this class

  E. mechanism of coupling of ET with ATP synthesis
     1. chemiosmotic coupling model
        a. proposed 1961
        b. Peter Mitchell
           (1)   English gentleman
              (a)   independently wealthy
              (b)   did experiments in his own private lab
                       at a country estate
              (c)   did not use vast government resources
              (d)   did experiments that led him to propose
                      the chemiosmotic model
           (2)   got Nobel prize (1978) for the model and his work
        c. the chemiosmotic model (know it)
           (1) the 3 coupling sites are sites where ET is
                 accompanied by unidirectional pumping of
                 H+s across the imm from matrix to
                 intermembrane space
           (2) electrochemical H+ gradient is 
                 created as protons are transported
           (3) electrochemical gradient contains energy
           (4) electrochemical H+ gradient drives ATP synthesis
                 elsewhere on the membrane
           (5) be able to describe this model of energy coupling
  F. electrochemical proton gradient
     1. pmf = proton motive force (expressed in V)
          a. pmf is the force tending to drive the H+s 
               back inside the mitochondrion
          b.  units for pmf are Volts (force) 
          c.  units for energy are calories
          d. pmf describes the force produced by
              the energy contained in the 
              electrochemical gradient
     2. H+ gradient (+ outside) tends to drive H+s back 
         inside the mitochondrion
        a. hence, pmf
     3. basic concepts of chemiosmosis
        a. electron transport chain has a concomitant H+ pump 
             (1) eg: see the animation of mitochondrial electron transport
        b. H+ pump produces an electrochemical H+ gradient
        c.  the electrochemical H+ gradient produces the pmf 
        b. energy in the electrochemical gradient 
             (expressed as pmf) is used as energy source for  
             enzymes that  synthesize ATP
            (1)  see animation of H+ driven ATP synthesis
     4. see Fig 
     5. ATP is synthesized by reversing an 
         ATP driven H+ pump
        a. in evolutionary terms:
           (1)   start with enzyme using ATP to pump 
                   H+s out of cell
           (2)   modification of enzyme allows ATP to 
                   be synthesized when H+s come back in 
                   (recall: all chemical reactions are reversible)
                   (recall: the ATP driven Ca+2 pump of the 
                     sarcoplasmic reticulum)
                    (recall: the ATP driven H+ pump in the lysosome)
     6. what produces the required H+ gradient?
        a. e- transport and the concomitant H+ pumps
  G.  Mechanism of H+ driven  ATP synthesis, local
  H.  movie

  H. energetics of proton gradients
     1. for the inward movement of H+s across
         inner mitochondrial membrane 
         H+out --> H+in
     2. the energy in the gradient is related to the 
         H+ concentration gradient  ([H+]in/[H+]out) across 
         membrane and the membrane potential
     3. deltaG'inward = RTln([H+]in / [H+]out) + nFVm
         a. see eqn in Becker et al.
         b. BUT recall: lnx = 2.303 logx 
     4. so:  deltaG'inward = 2.303RTlog([H+]in/[H+]out) + nFVm
         a. ALSO recall: pH = -log[H+]   
         b. and [H+] concentrations are usually expressed as pH
     5. so:  deltaG'inward = -2.303RT(pHin-pHout) + nFVm
     6. OR: deltaG'inward = 2.303RT(pHout-pHin) + nFVm
     7. OR: deltaG'inward = 2.303RTdeltapH + nFVm
        a. T in K
        b. F is 23,062 cal/mol V
        c. R is 1.987 cal/mol K
        d. Vm is membrane potential in V
        e. [H+] is the H+ concentration in mol/L
   I. calculating the pmf
     1. pmfinward = deltaG'inward / nF = 2.303RTdeltapH/nF + Vm
     2. eg: typical mitochondrial pmf at 37oC
        a. Vm = 0.16 V
        b. matrix pH 1.0 units higher than cytoplasmic pH
        c. pmf = (2.303)(1.987)(310)(1.0) / 23,062 + 0.16
        d. pmf = 0.06 V + 0.16 V = 0.22 V
        e. recall:  deltaGo' = -nFdeltaEo'
               -introduced under reduction potentials (E)
               -works here as well because we are dealing
                 with total energy associated with both the membrane
                 potential and the proton concentration gradient      
        f. for pmf we write deltaGo= -nF(pmf)  = -(1)(23,062)(0.22)
        f.  Go = -5.07 kcal/mol
           (1)   if standard conditions (except pH)
              (a)   reactants and products at 1 M
              (b)   T = 298oK
              (c)   P = 1 atm
           (2)   then, 1 mol of H+s moving across the membrane
                 will release 5.07 kcal of energy
        g. BUT: under standard conditions ATP synthesis
             requires more than 7 kcal/mol!!!  
        h. so, recalculate deltaGo' allowing the movement of 
            2 H+s to be coupled to ATP synthesis
           (1)    Go' = -nF(pmf) = -(2)(23062)(0.22)
           (2)    Go' = -10.14 kcal/mol
           (3)   NOW (if two H+s cross) there is enough energy 
                 (a) so, with a small pmf, more H+s must move through
                        membrane to make ATP
                 (b) big pmf, fewer H+s need to pass through membrane
                        to make same amount of ATP
           (4)   how test the chemiosmotic hypothesis?
              (a) measure moles ATP formed per mole of H+s
                    crossing mitochodrial membrane 
                     -  get ATP/2H+ ratio = 1
                     -  1 ATP per 2 H+ crossing membrane
  J.  NOTE:  pH gradient  plus membrane potential drives ATP synthesis here
                       -pH gradient alone COULD drive ATP synthesis under certain conditions
                       -membrane potential COULD drive ATP synthesis under other conditions
  J. testing chemiosmosis hypothesis
     1. chemiosmotic hypothesis requires unidirectional 
          pumping of H+s
        a. how unidirectional pumping of 
            H+s demonstrated?
        b. Andre Jagendorf and his graduate student Goeff Hind
           (1)   1963
           (2)   showed that isolated thylakoid membranes
                   pumped H+s into lumen of thylakoid when illuminated
              (a)   put thylakoid membranes into unbuffered media
                      containing e- acceptor
              (b)   turn on light
                 i) suspending media goes alkaline 
                    (pH increases)
                    a) pH rises because H+s pumped from
                         the suspending media into the 
                         thylakoid lumen
              (c)   turn off light
                 i) media returns to original pH
                 ii) pH recovers when pump stops and 
                     H+s leak back out of lumen and
                      return to suspending media
        b. similar changes in pH occur with 
             mitochondria and bacteria
             when they pump H+s driven
             by oxidative electron tranport
     2. chemiosmotic hypothesis requires an 
         ATP synthase and proton translocator
        a. how demonstrated?
        b. Efraim Racker and graduate students
           (1)   recall that imm has "knobs", cartoon
           (2)   Racker removed "knobs" from vesicles 
                   made from imm
              (a)   Fo remains in membrane (only F1 removed)
              (b)   w/o "knobs" ET still occured
                 i)    ET was much faster without knobs than with knobs
                 ii)   BUT, no ATP synthesis
                 iii)  NO  pH change could be observed
                 iv)  why rate ET faster???
                 v)   concludes that Fo is the H+ translocator
              (c)  isolated knobs (F1) were a good ATPase 
                  i)   ATP readily hydrolyzed to ADP + Pi
                  ii)  but, never see any ATP synthesis by F1 
                       when F1 off of the membrane
              (c)   put knobs back on memb 
                i)     ET slows down
                 ii)    pH change returns
                 iii)   ATP synthesis returns
                 iv)   why ET slower???
           c.  Racker concludes that the knobs are 
                the ATP synthase
                 -now accepted as fact
            d. Racker concludes that Fo and F1 are 
                 both needed for ATP synthesis
     3.  chemiosmotic hypothesis requires that a
          pH gradient is necessary and sufficient to drive 
          ATP synthesis
        a. how was this first demonstrated?
        b. Andre Jagendorf
           (1)   1966
           (2)   thylakoid membranes incubated in dark with
                   low pH buffer (pH = 4.0)
                   -over time, the low pH buffer crosses the
                     membrane and brings lumen to pH = 4
           (3)   suddenly expose thylakoids to pH = 8.0 buffer
                   containing ADP and Pi
                   -now have pH = 8 outside and pH = 4 
                     inside thylakoid membrane
           (4)   get short burst of ATP synthesis as the 
                   deltapH decayed
                   -conclude that deltapH is necessary and sufficient 
                     to drive ATP synthesis
        b. other experiments showed that electric 
            potential would work too
                   -conclude that a membrane potential is necessary
                     and sufficient to drive ATP synthesis 
review
VIII.   Uncouplers of phosphorylation
  A. uncouplers = molecules that disconnect 
       electron transport from ATP synthesis
  B. recall thermogenin = uncoupling protein 1 = UCP1
       -found brown adipose tissue
       -protein makes inner mito memb permeable to H+s
  C. recall DNP = dinitrophenol
     1. DNP is weak acid
          a.  DNP -> DNP-  +  H+
     2. DNP is hydrophobic and membrane permeable
          a.   DNP- is ALSO membrane permeable
          b.   the extra e- of DNP- gets delocalized over the whole ion
                 and so the ion does not interact strongly with H2O and 
                 can enter the hydrophobic phase of the membrane
                 and diffuse across to the other side
     3. mechanism of uncoupling
        a. DNP- ion dissolves in hydrophobic phase of membrane
        b. DNP-  picks up H+ on one side membrane
        c.  protonated form of DNP still soluble in membrane
        c. diffuses back across membrane
        d. releases H+ on other side
        e. H+'s move both ways
         f. net movement H+'s from where [H+] is hi 
            to where [H+] is low
         g. net effect to reduce  pH gradient and electric potential
              -NO ATP SYNTHESIS w/o deltapH and/or Vm
  D. other hydrophobic weak acids and hydrophobic 
       weak bases are also good uncouplers by 
       same mechanism
  E. ionophores as uncouplers
     1. some ionophores allow H+s to pass 
          through membrane
        a.  pH gradient is dissipated
     2. some ionophores allow other ions to pass
         through membrane
        a. electric potential dissipated
     3. together, either reduces the electrochemical gradient 
         and uncouples ATP synthesis from electron transport
IX.  ATP Yield from Respiratory Metabolism
  A. how much ATP per glucose? ((KNOW THIS FOR EXAM!!))
     1. follow yields of ATP, NADH, FADH & GTP during
        glycolysis and TCA cycle
        a. glycolysis
           (1)   2 ATP/glucose
           (2)   2 NADH/glucose (aerobic conditions only)
        b. TCA cycle
           (1)   8 NADH/glucose
           (2)   2 FADH/glucose
           (3)   2 GTP = 2 ATP/glucose
     2. recall: NADH yields 3 ATP 
         a.  also see coupling sites graphically
     3. recall: FADH yields 2 ATP
     4.   10 NADH   => 30 ATP
     5.   2 FADH  => 4 ATP
     6.   4 more ATP (2 from glycolysis and 2 from TCA cycle)
     7. ATPtotal = 30 + 4 + 4 = 38
        a. actual efficiency might be lower since 
            NADH transport into mitochondrion via 
            shuttle "costs" one ATP (see below)
        b. but, NADH can be transfered to NADPH in 
            cytoplasm and used for biosynthetic reactions
           (1)   thus, the energy can be conserved, although
                   not as ATP directly
X.   Efficiency of respiratory metabolism
  A. recall complete oxidation glucose
     1.  deltaGo'glucose oxidation = -686 kcal/mol
     2.  deltaGo'ATP hydrolysis= -7.3 kcal/mol
     3.   (38 mol ATP/ mol glucose)(-7.3 kcal/mol) = -277 kcal/mol glucose
     4.   efficiency of ATP synthesis = 277/686*100 = 40%
XI.  Transport across imm  A. requires specific transporters
  B. see Fig 12-27
     1. pyruvate/H+ symporter
        a. actively pumps pyruvate into mitochondria
           (1)   costs 1 H+/ pyruvate
        b. see an animation of a symporter
     2. dicarboxylic acid antiporter
        a. reciprocal exchange
           (1)   malate
           (2)   succinate
           (3)   fumerate
           (4)   Pi
     3. tricarboxylic acid antiporter
        a. reciprocal exchange
           (1)   citrate
           (2)   isocitrate
           (3)   dicarboxylic acids too
     4. ATP/ADP antiporter
        a. maintains mitochondrial concentration  ADP
     5. phosphate/OH- antiporter
        a. net effect is loss of one H+ outside
        b.   Pi is actively transported
     6. no transporter for NADH
        b. shuttle e-s from NADH via a shuttle
           (1) NADH used to reduce DHAP to glycerol-3-P 
                 in cytoplasm
           (2) glycerol-3-P oxidized back to DHAP with a imm enzyme 
                 which uses FAD as the e- acceptor
           (3)   enzyme binding site for g-3-P faces
                   intermembrane space
                   which is equivalent to the cytoplasm
           (4)   enzyme transfers 2e- to FAD --> FADH2
           (5)   FADH2 directly reduces coenzyme Q
                    to dihydroubiquinone
           (4)   get 2 ATP per FADH2 just as with the 
                   succinate coenzyme Q reductase
        d. DHAP converted back to G-3-P with NADH
           (1)   note: net loss of 1 ATP when exchanging
                   NADH for an FADH
XII  Mitochondrial Diseases
   A  most mitochondrial proteins are 
        coded on nuclear genes
   B  a few mitochondrial proteins are
        coded on mitochondrial DNA
            -defects in these genes are 
              inherited maternally
    C  some mitochondrial disorders

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