NOTE WE WILL NOT DISCUSS GLYCOLYSIS OR KREB'S CYCLE IN DETAIL: THIS IS COVERED IN BIOCHEMISTRY; BUT ELECTRON TRANSPORT AND CHEMIOSMOSIS IS OFTEN NOT SO WE WILL EMPHASIZE THESE LATTER SUBJECTS. YOU DON'T HAVE TO MEMORIZE MEMBRANE PROTEINS OF ELECTRON TRANSPORT CHAIN, OR THE STEPS OF GLYCOLYSIS, THE INTERMEDIATE STEP OR THE KREB'S CYCLE. NOTE THAT THERE IS A LOT OF EXTRA MATERIAL IN THESE 2 TEXT CHAPTERS THAT WE WILL NOT COVER.

THIS SECTION ON MITOCHRONDRIA PROVIDES AN OPPORTUNITY TO APPLY THE EQUATION FOR THE D G FOR REDOX REACTIONS (I.E., THE ELECTRON TRANSPORT CHAIN), D GINWARD, AND NERNST EQUATIONS ALONG WITH APPLICATION OF ACTIVE TRANSPORT AND FACILITATED DIFFUSION IN ATP SYNTHESIS.

IN 1948 MITOCHONDRIA ISOLATED BY DIFFERENTIAL CENTRIFUGATION FOR FIRST TIME.

MITOCHONDRIAL STRUCTURE FIG. 14-4A:

OUTER MEMBRANE READILY PERMEABLE TO MOLECULES<5000 DALTONS; HAS PORINS.

INNER MEMBRANE STRONG BARRIER

INTERMEMBRANE SPACE

MATRIX (WHAT IS PH HERE RELATIVE TO CYTOPLASM OR INTERMEMBRANE SPACE?)

FOLDING OF INNER MEMBRANE ARE CRISTAE (PLURAL)--WHY ARE THEIR INFOLDINGS?

AEROBIC RESPIRATION;

MAKING ATP WITH OXYGEN.

IDEA FOR STUDYING AT HOME: MAKE AN ENERGY DIAGRAM OF RESPIRATION (WHAT ARE ON THE AXES?)

STEPS:

1. GLYCOLYSIS (CHAPTER 13). GLUCOSE IS BROKEN DOWN TO STRIP OFF THE HIGHLY ENERGETIC ELECTRONS. GIVE THEM TO NAD+. THIS IS OXIDATION (MNEMONIC: OILRIG) OF GLUCOSE, REDUCTION OF NAD+ TO FORM NADH.

EVENTUALLY, ALL SIX CARBON ATOMS OF GLUCOSE ARE OXIDIZED COMPLETELY TO WHAT?

(ANSWER: C0₂).

2. INTERMEDIATE STEP: MOVEMENT OF WHAT WAS GLUCOSE (NOW CALLED PYRUVATE; IT HAS THREE CARBONS). MORE ELECTRONS ARE STRIPPED OFF AS PYRUVATE IS BROKEN DOWN TO ACETYL CoA AND THE ELECTRONS ARE GIVEN TO NADH.

3. KREB'S CYCLE (OR TCA CYCLE); ACETYL CoA IS BROKEN DOWN TO CO₂, AND MORE EXCITED ELECTRONS ARE GIVEN TO FADH₂ AND NADH.

4. ALL ELECTRON CARRIERS GO TO THE ELECTRON TRANSPORT CHAIN. THE MEMBRANE PROTEINS OF THE ELECTRON TRANSPORT CHAIN ACCEPT THE ELECTRONS, STRIP OFF THEIR ENERGY AND USE THE ENERGY TO PUMP PROTONS (H+) FROM THE MATRIX TO THE INTERMEMBRANE SPACE. THIS IS ACTIVE TRANSPORT.

5. FINALLY, WE MAKE ATP. THE PROTONS MOVE DOWN THEIR ELECTROCHEMICAL GRADIENT THROUGH A MEMBRANE PROTEIN CALLED AN ATPase. THE MOVEMENT IS FROM THE INTERMEMBRANE SPACE TO THE MATRIX. THIS IS FACILITATED DIFFUSION.

AS THE PROTONS MOVE THROUGH THE ATPase, ADP AND P ARE FORCED TOGETHER TO MAKE ATP.

ELITCH MODEL: AS YOU LINE UP AT THE TURNSTILE AT ELITCHE'S, AND A BUSLOAD OF KIDS COME UP BEHIND YOU. YOU AND THE KIDS ARE PROTONS AND THE TURNSTILE IS THE ATPase. AS YOU ARE FORCED THROUGH THE TURNSTILE BY THE KIDS (YOUR ELECTROCHEMICAL GRADIENT), THE ENERGY OF YOUR MOVEMENT IS USED TO ATP.

FIG. 14-1 REVIEW OF AEROBIC RESPIRATION

WHY DO WE NEED OXYGEN TO LIVE? WE SUFFOCATE BUT WHAT IS THIS?

ANSWER: BECAUSE OXYGEN IS THE FINAL ELECTRON ACCEPTOR IN THE ELECTRON TRANSPORT CHAIN. FOR ANIMALS AND MOST PLANTS. NO OXYGEN, NO ELECTRON TRANSPORT AND NO ATP MADE.

USE OF THE NERNST EQUATION AND THE D G_INWARD EQUATION WITH MITOCHONDRIA:

USE THE NERNST EQUATION: DIRECTION AND MAGNITUDE OF DIFFUSION

EQUATION ONE:. WE NOTED BEFORE THAT WE CAN CALCULATE THE DIRECTION AND MAGNITUDE OF DIFFUSION (DUE TO ELECTROCHEMICAL GRADIENT):

EMF = BOOK CALLS IT "PMF" ( QUESTION: WHAT DOES P STAND FOR IN PMF?)

= ACTUAL MEMBRANE POTENTIAL - EQUILIBRIUM POTENTIAL.

= E_M - E_H+

= E_M - (RT/F) Ln ([H+]_OUTS/[H+]_INSIDE)

WHERE R = 1.987 CAL/MOL-DEG, T IS DEG. KELVIN (310 K), F IS 23062 CAL/MOL-V) AND EM IS MEMBRANE POTENTIAL IN VOLTS (TAKEN WITH REFERENCE TO THE CYTOPLASM).

NOTE THAT THE "INSIDE" IS ALWAYS THE CYTOPLASM,

THE OUTSIDE IN THIS CASE IS THE MATRIX OF THE MITOCHONDRION.

SINCE THE OUTER MITOCHONDRIAL MEMBRANE IS LIKE A SIEVE, WE IGNORE IT. THE INNER MEMBRANE IS VERY "TIGHT" SO IT IS THE MEMBRANE THAT PREVENTS PROTON MOVEMENT (PROTONS PASS THROUGH THE OUTER MEMBRANE).

ELECTRICAL GRADIENT:

OUR TYPICAL MITOCHONDRION (IE. 37 DEG. C) HAS AN E_M OF 0.16 VOLTS; THIS MEANS THAT THERE IS A BUILD UP OF POSITIVE IONS IN THE INTERMEMBRANE SPACE/CYTOPLASM BECAUSE H+ IS PUMPED OUT OF THE MATRIX AND THERE IS A SLIGHT NEGATIVE BUILD UP IN THE MATRIX.

JUST AS WITH THE PLASMA MEMBRANE, THE MEMBRANE POTENTIAL E_M TAKES THE CHARGE OF THE CYTOPLASM.

CHEMICAL GRADIENT:

ALSO, THE MATRIX pH IS 1-2 PH UNITS HIGHER THAN THE CYTOPLASM (E.G., CYTOPLASMIC pH_IN IS 7, pH_OUT IN MATRIX IS 8).

DRAW A MITOCHONDRION AND SHOW CHEMICAL AND ELECTRICAL GRADIENTS:

SO, USING OUR EQUATION FOR THE DIRECTION AND MAGNITUDE OF PASSIVE DIFFUSION:

EMF OR PMF= E_M - [(1.987*310)/23062]* Ln([H+]_MATRIX/[H+]_{INTERME SPACE OR CYTOPLASM})

= 0.16 - (-0.0615)

= 0.222 V

SO THE FORCE IS A POSITIVE NUMBER FOR THE POSITIVELY CHARGED H+; SIGNIFICANCE????

ANSWER: NET FORCE IS INWARD TO THE CYTOPLASM OR OUTWARD INTO THE MATRIX OF THE MITOCHONDRIA?

SECOND EQUATION; THE D G_INWARD EQUATION:

D G_INWARD= RTLn ([H]_IN)/([H]_OUT) +ZF E_M

REMEMBER THAT THIS EQUATION HAS THE RATIO OF CONC. IN OVER OUT!! [H]_IN IS PROTON CONCENTRATION IN THE CYTOPLASM (OR 10^-7 M OR PH 7). THE OUTSIDE CONCENTRATION (IN THE MATRIX) IS 10^-8 M OR pH 8.

D G_INWARD = (1.987*310) Ln(10) + ((1)23062* 0.16)

= 1419 + 3690 CAL/MOL

= 5109 CAL/MOL

SO THE POSITIVE D G MEANS THAT PROTONS WILL NOT SPONTANEOUSLY MOVE INWARD (OR INTO THE CYTOPLASM) BUT WILL MOVE SPONTANEOUSLY INTO THE MATRIX (OR OUT OF THE CYTOPLASM). AS THE PROTONS MOVE INTO THE MATRIX, THEY WILL MAKE ATP. THAT IS, AS THEY MOVE, THEY CAN DO WORK.

THUS, ONE MOLE OF PROTONS MOVE FROM THE CYTOPLASM TO THE MATRIX CAN DO THIS MUCH WORK. SOME RESEARCHERS ESTIMATE THAT TWO PROTONS MOVE TO MAKE ONE ATP (THAT IS, ONE MOLE OF ATP IS WORTH ABOUT 10 KCAL).

ASK YOURSELF THESE TWO QUESTIONS --------------------------------

A. IF YOU MAKE THE CYTOPLASM BASIC, THIS WILL MAKE THE INTERMEMBRANE SPACE BASIC ALSO BECAUSE THE OUTER MEMBRANE IS A POOR BARRIER. BUT THE INNER MEMBRANE PREVENTS IONS FROM MOVING INTO THE MATRIX.

WE SEE THE GRADIENT IS NOW FROM THE MATRIX TO THE INTERMEMBRANE SPACE, AND PROTONS MAY FLOW THIS WAY IF THIS CONC. OR CHEMICAL GRADIENT IS LARGER THAN ANY ELECTRICAL GRADIENT. WHAT WILL HAPPEN IN RELATION TO ATP? (WE BREAKDOWN ATP- WHY?)

B. WHAT IF WE MAKE THE CYTOPLASM VERY ACIDIC, STOP ELECTRON FLOW, WHAT WILL HAPPEN IN RELATION TO ATP? (STILL MAKE ATP- WHY?)

EVEN IF IT IS NOT COVERED IN LECTURE, I ENCOURAGE YOU TO READ THROUGH ALL OF THIS MATERIAL THIS AS IT REVIEWS EARLIER MEMBRANE TRANSPORT EQUATIONS.

THIRD EQUATION FOR MITOCHONDRIAL STUDIES (LESS IMPORTANT THAN THE OTHER TWO):

THIS IS THE REACTION THAT OCCURS AT THE ELECTRON TRANSPORT CHAIN/CHEMIOSMOSIS SITE AT THE INNER MITOCHONDRIAL MEMBRANE:

NADH + 2H + 1/2O₂ ==> NAD+ + H₂0

THE CHANGE IN GIBB'S FREE ENERGY FOR THE REDOX REACTION: D G '_O = -52.4 KCAL/MOL (SEE CALCULATION BELOW). SO NADH IS A HIGH ENERGY MOLECULE, BREAKDOWN IS SPONTANEOUS AND IT CAN DO WORK AS IT RELEASES ELECTRONS AND H ATOM. WORK PERFORMED BY THIS CHEMICAL REACTION IS TO PUMP PROTONS AND THIS IS USED TO EVENTUALLY MAKE ATP. BUT IF THIS REACTION TOOK PLACE ALL AT ONCE, WE WOULD LOOSE A LOT OF THE ENERGY; IT WOULD BE LIKE AN EXPLOSION. SO, THE BODY DIVIDES THE REACTION INTO A SERIES OF STEPS. INSTEAD OF DIRECTLY GIVING THE H AND ELECTRONS TO OXYGEN, THEY ARE PASSED TO A SERIES OF MEMBRANE PROTEINS- THIS IS THE ELECTRON TRANSPORT CHAIN. THEN A LITTLE BIT OF ENERGY IS TRANSFERRED AT EACH STEP; MAXIMIZING THE WORK.

FOR A REDOX REACTION; D G'_O = Z NF D EO

WHERE D EO = (EO, ACCEPTOR - EO,DONOR), IN VOLTS;

N = ELECTRONS TRANSFERRED;

Z IS -1 FOR ELECTRONS;

F = FARADAY CONSTANT, IN THIS CASE 23,062 CAL/MOL-V

MEASURE OF HOW EASILY A COMPOUND CAN BE REDUCED

MEASURE OF HOW GOOD AN ELECTRON ACCEPTOR IT IS.

A GOOD ELECTRON ACCEPTOR HAS A POSITIVE REDUCTION POTENTIAL.

A GOOD ELECTRON DONOR HAS A NEGATIVE RED. POT.

HOW DO YOU CALCULATE THE D G FOR A REDOX REACTION SUCH AS THE ELECTRON TRANSPORT CHAIN? REMEMBER D G TELLS US WHETHER THE REACTION IS SPONTANEOUS? HOW MUCH WORK CAN IT DO IF IT IS SPONTANEOUS?

IDENTIFY THE DONOR OF ELECTRONS: IT IS NADH BECAUSE IT LOOSES HYDROGEN; OXYGEN IS THE ACCEPTOR. WE HAVE DISCUSSED THE MNEMONIC "OILRIG." LOOK UP IN TABLE 12-2 OF THE TEXTBOOK, GET THE EO VALUES AND N VALUE.

D G'_O = (-1) (2) (23062) (0.816 - (-0.32) )

= -2 * 23062 * 1.136 VOLTS

= -52396.8 CAL/MOL

SO IT IS SPONTANEOUS. NOTE THAT A REACTION THAT IS SPONTANEOUS UNDER STANDARD CONDITIONS HAS A NEGATIVE D G'_O BUT POSITIVE E_O

SIGNIFICANCE; TRANSFER ELECTRONS ONLY FROM MOLECULE A TO B NOT IN REVERSE; WHICH WAY DOES THE ELECTRON FLOW SPONTANEOUSLY??

DRAW A REACTION ENERGY DIAGRAM OF ELECTRONS FLOWING DOWN THE MEMBRANE PROTEINS IN ELECTRON TRANSPORT CHAIN (HINT: THE DIAGRAM WOULD GO DOWNHILL) ---

FOR OUR EXAMPLE:

D G_O = ZF (E_M-EEQ)

= (1) F (EMF OR PMF) = 23062 * .222

= 5119 CAL/MOL OF PROTONS