Mitochondrial Electron Transfer

Mitochondrial Membrane Transport & Electron Transfer
   membranes = impermeant to most everything, esp to H⁺       [mito* & ecb 14.3 pg457]
    outer membrane - porin* - channel protein; diffuses 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. α-glycerol-P & malate shuttles
e. lipid metabolism enzymes (β-oxidation)
Mitochondrial aerobic respiration 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 & replication.
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 mitochondrial eve genetic variation among peoples mitochondrial diseases forensic uses of Mito-DNA

How Electron Transfer Works

REDOX POTENTIAL                     ^{*a
text description}

is an empirical measure of tendency of molecular couple (donor/acceptor) to gain e's
       - strong reducing agent (electron donor - NADH) has negative    - D Eo'
       - strong oxidizing agent (electron acceptor - O₂) has positive     +   D Eo'
                   (how its measured – Reference half-cell* & table )        ecb2/e panel 14.1 pg471

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

         NADH <---> NAD⁺ + H⁺ + 2e^--0.32V   (-320 millivolts)
               H₂O <---> ½ O₂+ 2H⁺ + 2e^-   +0.82V   (+820 millivolts)
                  DGo' =   - (1) (0.023) (1.14)   =   - 26.2 Kcal

                                    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...    via increasing Redox Potential... table*
         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

--> membranes themselves have no electrical charge, but instead separate electrical
                         charges making it an insulator
                         an insulator that separates electric charges until used is a battery
                                                                          system not unlike a battery (ecb 14.11 pg 463)

Major Components of the ETC * 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⁺² <--> ferric⁺³) 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^{+3 ox} vs. ferrous^{+2 red}] via iron porphyrin (heme) bound protein carriers

How Oxidative Phosphorylation Works

Mitochondrial Respiratory Assemblies
   I. NADH-Q reductase
      II. Succinate dehydrogenase    *
    III. Cytochrome-C-Reductase
    IV. Cytochrome Oxidase
  overview: locations & Karp 5.16pg199 & Karp fig 5.30

   ETC* - passes e thru ETC carrier proteins
   PMF* - 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)

Chemiosmosis [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
     F₁ (extrinsic) & F₀ (intrinsic)
     Humbeto Fernandez (60's) sees lollipops on inner mito membranes
      Efraim Racker (1966) isolates lollipop - Coupling Factor 1 - F1
             EM's*


ATP synthase of liver mitochondria number about 15,000

   F₁ 5 polypeptides (nuclear DNA) 3α , 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
1. 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 (γ)
           α & β subunits of F1 form hexagonal ring that rotates around central axis
           γ stalk extends from F_o & interacts with 3 β's differently as it rotates thru 360^o

     step animation*

*
makes 100 ATP per 300 H+ per sec

     Boyer Figure*

   experimental
         rotation of F1*

Proton Pathway thru Fo - a rotational model of C-ring & γ stalk
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

*
                      click

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

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

                    NDSU- animation of ATP synthase*

Biovisions animation of ATP synthase*

Chemiosmosis *[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 F₁ (extrinsic) & F₀ (intrinsic) Humbeto Fernandez (60's) sees lollipops on inner mito membranes Efraim Racker (1966) isolates lollipop - Coupling Factor 1 - F1	EM's*
ATP synthase of liver mitochondria number about 15,000 F₁ 5 polypeptides (nuclear DNA) 3α , 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 1. 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 (γ) α & β subunits of F1 form hexagonal ring that rotates around central axis γ stalk extends from F_o & interacts with 3 β's differently as it rotates thru 360^o
step animation*	* makes 100 ATP per 300 H+ per sec
Boyer Figure*
experimental rotation of F1*

Proton Pathway thru Fo - a rotational model of C-ring & γ stalk		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		* click
each C protein has a half-channel space with a charged aspartate^- C's bind H⁺ on pms side & via shape changes each C-rotates 30^o CCW next C in ring picks up H⁺ - thus 12 C's rotates ring cycles thru 360^o release of H⁺ into matrix happens at end of cycle Karp 5.29 4 H⁺moves ring 120^o (γ stalk) shifts 120^o --> β's change 4 H⁺ result in one ATP being made
rotation of C-ring drives γ stalk through 360^o & µ 3 conformations of F1 (L-T-O) to make ATP		ATP synthase animated movie (Wang & Oster)
NDSU- animation of ATP synthase*