Mitochondrial Membrane Transport & Electron Transfer
   membranes =
impermeant to most everything,  esp to H+       [mito*  &  ecb 14.3 pg457]
    outer membrane
- porin* - channel protein; passes molecules up to 5,000+ daltons
    inner membrane
- 70% protein & 30% lipid... contains:

           a redox proteins of Electron Transfer Chain
           b.  ATP synthase                             -->
           c.  many carrier proteins:     phosphate translocases,
                ADP/ATP translocases
*,   pyruvate/H+ symporter
           d glycerol-P   &   malate shuttles
           e lipid metabolism enzymes   (β-oxidation)

   Mitochondrial membrane transport driven by chemiosmosis*








  mito-DNA...   16,569+ np's...                                 mcb 6.21
                                     holds some
37 genes that
                                     codes  for 
20%  of mitochondrial proteins
Mitochondrial DNA functions
  5 subunits of NADH dehydrogenase (complex I),
cytochrome oxidase subunits I, II, III (complex IV),
  ATP synthase : subunits 6
& 8 (complex V),
  RNA polymerase,  
22 tRNA's  &  2 rRNA's

Nuclear encoded components include:

 lipid metabolism, nucleotide metabolism, aa
 metabolism, carbo metabolism, heme synthesis,
 Fe-S synthesis, ubiquinone synthesis, proteases,
 chaperones, signal pathways, & DNA repair &
                                   proteins encoded by nuclear & mitochondrial DNA* 
       1,000's copies per cell; maternally inherited; lots of short tandem repeat sequences;
       frequent point mutations; thus, sequence analysis can indicate phylogeny:  

                            mtDNA & Human Evolution            
genetic variation among peoples   
forensic uses of Mito-DNA            mitochondrial diseases




How Electron Transfer Works

REDOX POTENTIAL                     Text description - REDOX REACTION*a text description 

    is an empirical measure of tendency of molecular couple to gain e's
     - strong reducing agent (electron donor) has negative        -   D Eo'
          - strong oxidizing agent (electron acceptor) has positive    +   D Eo'
                               (how its measured – Reference half-cell*  &   ecb2/e panel 14.1 pg471)

Free Energy   &   Redox Potential
:      D Go' =  -nf D Eo’

         NADH <---> NAD+ + H+ + 2e-   
   (-320 millivolts)

           H2O <--->  ½ O2 + 2H+ + 2e-    +0.82V   (+820 millivolts)

                      DGo'  =   - (1)  (0.023)  (1.14)   =   - 26.2 Kcal          table
                                             P to O ratio is  1 NADH = 3 ATP = 21.9 Kcal








Electron Transfer Chain and the order of its elements...
        ETC is a series of electron CARRIER MOLECULES that that transfer e-'s from
        a more negative redox potential to a more positive redox potential,
        while "pumping" protons out of the mitoplasm into perimitochondrial space. 

--> carriers are aligned linearly
increasing Redox Potential
from more electronegative [
- ]  toward more electropositive to [ + ]
                and therefore by their energy differentials:

     sequence of components*        mcb 12.18 pg500            Karp fig 5.13 p197

                                              system not unlike a battery (ecb 14.11 pg 463)









 Major Components of the ETC  text description - Components of ELECTRON TRANSFER CHAIN* text description         carrier structures Karp 5.11
  Pyridine nucleotides NAD+       2.33a p60*                           ecb-3.25 
enzyme bound hydrogen carriers                                       karp-3.26
     accept  2e's and/or protons                                        ecb-14.4
     show spectral shifts @ 340nm NADH vs. NAD
  Flavoproteins FMN & FAD      2.33b p60*                                
protein bound hydrogen carriers                                         ecb-13.12
     spectral shift @ 340, 370, & 460 nm
  Iron sulfur proteins FeS        12.14b p495*                       ecb-14.22
     non-heme iron electron carriers  (ferrous <--> ferric        karp-5.12
  Ubiquinone CoQ - semiquinone & hydroquinone  8.16a-1e*     ecb-14.20
       mobile, membrane bound, non-protein hydrogen carriers
Cytochromes ( a, a3, b562, b566, c1, c)   12.14a.heme*        ecb-14.23 
   "colored proteins" with bound Fe atoms [ ferric vs. ferrous]
        via iron porphyrin (
heme) bound protein carriers







 How Oxidative Phosphorylation Works

Mitochondrial Respiratory Assemblies
     I. NADH-Q reductase 
II. Succinate dehydrogenase     text description - Components of ELECTRON TRANSFER CHAIN*
III. Cytochrome-C-Reductase  
IV. Cytochrome Oxidase 
overview: locations & Karp 5.16pg199 Karp fig 5.30
 -  passes e thru ETC carrier proteins
* -  Proton Motive Force gradient 
               animation of PMF*view@home
             ex: how Q-cycle moves protons mcb-12.20
          pH difference   
ΔpH = 1.0 to 1.4 pH units 
    pH 8.0 matrix vs. pH 7.0 peri-mito. space
                membrane potential difference
  Δcharge =   140mV   in(-) vs.  out(+)
      pmf uses* (drives transport mcb-12.27)

cytochrome oxidase





Chemiosmosis [Oxidative Phosphorylation] –  Text description - OXIDATIVE PHOSPHORYLATION*   text description: the Making of ATP 
 Synthesis of ATP- made via a proton motive force gradient
H+ gradient generated by transfer of e's in ETC
           e's through series of redox proteins           fig*
           e's & H+ finally reduce  O2  &  make H2O
     Mechanism - Chemiosmotic Coupling*   Mitchell 1961  
       a fundamental cell energy mechanism that arose
early in
         evolution & was retained -
works like a fuel cell
    Evidence:    fractionation*  &  reconstitution*
pH gradients*  &  bacterio-rhodopsin*
   Chemiosmosis in bacteria, mitochondria, & chloroplasts*


 ATP Synthase   condenses ADP + Pi  --->  ATP   
                        has a hydrophilic channel (
F0) for H+ flow
                        makes 100 ATP per 300
H+ per sec

            F0 – membrane piece & stalk 
            F1 – soluble piece; 5 proteins 
(it is also hydrolytic)              
















 ATP Synthase Structure...
  'mushroom' shaped complex* composed of 2 membrane subunits
(extrinsic) & F0 (intrinsic)
       Humbeto Fernandez (60's) sees lollipops on inner mito membranes
       Efraim Racker (1966) isolates lollipop - Coupling Factor 1 - F1
 ATP synthase of liver mitochondria number about 15,000

   F1  5 polypeptides (nuclear DNA) 3
α ,  3β ,  1γ ,  1δ,  &   1ε
               arranged like sections of grapefruit 
         3 catalytic sites for ATP synthesis - 1 on each
β  subunit
   F0   3 polypeptides in ratio of  1a, 2b, and 12c (C-ring)

















Binding Charge Mechanism of ATP Synthesis - A Rotary Motor   Paul Boyer 1979 Nobel
 H+ movement changes binding affinity of synthases's active site, thus
          when ADP & P bind to active site,   they readily condense into ATP
(removed from aqueous solution Keq = 1   and   ΔG close to zero,  thus ATP forms easily)
  2.  active site is on [β-subunits] & it changes conformation through 3 successive shapes (L-T-O)
           O - open - site has low affinity to bind ATP - thus releases it  [4]

           L - loose - ADP & P loosely bound to site  [1 & 2]
           T - tight - ADP & P tightly bound favoring condensation without water  [3]
  3.  conformational changes result in rotation of subunits relative to central stalk (
α & β subunits of F1 form hexagonal ring that rotates around central axis
γ stalk extends from
Fo & interacts with 3 β's differently as it rotates thru 360o


makes 100 ATP per 300 H+ per sec

     Boyer Figure*

         rotation of F1






 Pathway of the Protons through Fo
                  rotational model of  C-ring  &
 γ stalk

text description - ATP SYNTHASE  a description

    12 C-proteins reside in lipid bilayer (C-ring)
        C-ring is attached to
γ stalk of F1 subunit
    H+ diffuse through
Fo half-channel
 rotating the 12-C's of the Fo ring


  each C protein has a half-channel space with a charged ASP- 
   C's bind H+ on  pms side & via shape changes each C-rotates 30o CCW
    next C in ring picks up H+ - thus 12 C's rotates ring cycles thru 360o
       release of H+ into matrix happens at end of cycle     Karp 5.29
             4 H+ moves ring 120o (
γ stalk) shifts 120o --> β's change
             4 H+ result in one ATP being made

            rotation of C-ring drives γ stalk through 360o  &
µ  3 conformations of F1 (L-T-O) to make ATP

ATP synthase animated movie
(Wang & Oster)

                    Biovisions animation of ATP synthase*  










      end of material