OXIDATION PHOSPHORYLATION-2 BIOC 460 - DR. TISCHLER LECTURE 29 OXIDATION PHOSPHORYLATION-2

OBJECTIVES How pH and charge gradients form across mitochondrial inner membrane Electroneutral vs electrogenic transport; significance of electrogenic transport for adenine nucleotide transporter; role of this transporter. Chemiosmotic model. Components of the ATP synthase complex; describe their roles. How the malate-aspartate and -glycerol phosphate electron shuttles generate energy from NADH produced by glycolysis. Define respiratory control and uncoupling; physiological importance of these processes.

+ + + + + + + Outer Membrane - Inner Membrane - - - - - - High [H+] High [H+] + + + + + + H+ Outer Membrane - - Inner Membrane - - - - - - - _ - _ Low [H+] Intermembrane Space + Matrix H+ + + H+ + + + Cytoplasm + + + + + H+ H+ Figure 1. Generation of a pH gradient ([H+]) and charge difference (negative in the matrix) across the inner membrane constitute the protonmotive force that can be used to drive ATP synthesis and transport processes

– MITOCHONDRIAL TRANSPORT SYSTEMS Examples of Electroneutral Transport: Pyruvate1- moves into matrix and OH1- moves out Phosphate1- moves into matrix and OH1- moves out Citrate3- + H+ exchanges with malate2- Inner Membrane Matrix Side Intermembrane Space ATP4- Adenine nucleotide transporter ADP3- Net negative charge moves out – Electrogenic Transport Figure 2. Electrogenic transport system in mitochondria.

OH- ADP3- ATP4- INTERMEMBRANE SPACE ADP3- 4H+ ATP4- OH- ADP3- ADP3- NADH + H+ complex I FMNH2 NAD+ ATP4- 4H+ e- OH- ADP3- e- ADP3- e- inner membrane e- 3H+ ATP4- Pi- ADP3- ADP3- CoQ complex III 4H+ 2H+ e- cyt b e- 2H+ F1 C1 ½O2 H2O e- cyt MATRIX e- C e- a-a3 stalk e- 4H+ Fo complex IV Proton gradient/ Charge gradient 3H+ Figure 3. Oxidative Phosphorylation

FEATURES OF THE CHEMIOSMOTIC MODEL: mitochondrial inner membrane is impermeable to protons by simple diffusion measurable proton (pH) gradient exists across the inner membrane collapse of the proton gradient (uncoupling) abolishes ATP synthesis but accelerates O2 consumption inhibition of the respiratory chain prevents ATP synthesis because pumping of protons ceases

Figure 4. The malate-aspartate shuttle. e = electrons INNER MEMBRANE NAD+ Glucose Pyruvate GLYCOLYSIS NADH OUTER MEMBRANE e- Complex I e- OAA NADH NAD+ (3) e- Glu0 Asp-1 (4) OAA Malate (1) e- NAD+ Glu0 (6) (5) Asp-1 KG Malate (2) e- CYTOPLASM MATRIX Figure 4. The malate-aspartate shuttle.

e = electrons CoQ e O2 INNER MEMBRANE OUTER MEMBRANE CYTOPLASM NADH Glucose Pyruvate GLYCOLYSIS NAD+ MATRIX FADH2 e Dihydroxyacetone phosphate (DHAP) DHAP NAD+ 3-phosphate Glycerol e (1) (2) Glycerol-3-phosphate dehydrogenase G3P FAD Figure 5. Glycerol phosphate shuttle. Cytoplasmic glycerol 3-phosphate dehydrogenase (1) oxidizes NADH. Glycerol 3-phosphate dehydrogenase in the inner membrane (2) reduces FAD to FADH2.

RESPIRATORY CONTROL depends on the availability of ADP increased ADP in matrix opens proton channel protons move through channel down pH gradient respiration increases to compensate for decline in pH gradient; oxygen consumption (respiration) controlled at low ADP, ATP synthesis ceases, pH gradient builds up, oxygen use diminishes as ATP needs rise (i.e., ADP increases) respiration is again accelerated inhibition of respiratory chain causes loss of control

UNCOUPLER EFFECTS hydrophobic molecules that bind protons take protons into matrix to collapse pH gradient without pH gradient, synthesis of ATP ceases electron transport chain operates at high rate; protons are pumped out rapidly in attempt to restore pH gradient energy is released as heat and the body temperature rises respiratory control is lost uncoupled brown fat mitochondria generates body heat in infants until shivering reflex develops