21b-Biochemistry-Lecture21b PDF

PROTON-MOTIVE FORCE Proton-motive force. The inner mitochondrial membrane separates two compartments of different [H+], resulting in a difference in chemical concentration (ΔpH) and in a difference charge distribution (Δψ) across the membrane. The electrochemical energy inherent in this difference i...

PROTON-MOTIVE FORCE Proton-motive force. The inner mitochondrial membrane separates two compartments of different [H+], resulting in a difference in chemical concentration (ΔpH) and in a difference charge distribution (Δψ) across the membrane. The electrochemical energy inherent in this difference in proton concentration (proton gradient) and in the separation of charge (electrical gradient) represents a temporary conservation of the energy of electron transfer. The free-energy stored in such a gradient, the proton-motive force, has two components: 1/ difference in chemical concentration (ΔpH) 2/ difference in charge distribution (Δψ) across the membrane and can be calculated by the following formula: ΔG = RT ln (C2)/C1) + ZFΔψ where C1 and C2 are the concentrations of the two ions (C2>C1), Z is the absolute value of the electrical charge (1 for a proton) and Δψ is the transmembrane difference in potential measured in volts. CHEMIOSMOTIC MODEL FOR ATP SYNTHESIS Electron transport sets up a proton-motive force. Energy of proton-motive force drives synthesis of ATP by ATP Synthase In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient (∆pH) and an electrical gradient (∆ψ) (combined, the proton-motive force). The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix ONLY through proton-specific channels (Fo). The proton-motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the F1 complex associated with Fo of ATP Synthase. COUPLING OF ELECTRON TRANSPORT AND ATP SYNTHESIS ATP synthesis requires electron transport But electron transport also requires ATP synthesis (a) Addition of ADP and Pi alone results in little or no increase in either respiration (black line) or ATP synthesis (red line). When succinate is added, respiration begins immediately and ATP is synthesized. Addition of cyanide (CN-), which blocks electron transfer between cytochrome oxidase (Complex IV) and O2, inhibits both respiration and ATP synthesis. (b) Mitochondria provided with succinate respire and synthesize ATP only when ADP and Pi are added. Subsequent addition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. Dinitrophenol (DNP) is an uncoupler, allowing respiration to continue without ATP synthesis. UNCOUPLED MITOCHONDRIA IN BROWN ADIPOSE TISSUE PRODUCE HEAT Heat generation by uncoupled mitochondria. UCP1, The uncoupling protein in the mitochondria of brown adipose tissue causes the energy conserved by proton pumping to be dissipated as heat by providing an alternative route for protons to reenter the mitochondrial matrix. A futile cycle in which creatine is phosphorylated by creatine kinase (CK), using ATP and producing ADP transported by ATP/ADP carrier (AAC) also generates heat. AGENTS THAT INTERFERE WITH OXIDATIVE PHOSPHORYLATION AND ATP SYNTHESIS (Complex IV) (Complex III) (Complex I) MITOCHONDRIAL ATP SYNTHASE COMPLEX Contains two functional units: – F1 Soluble complex in the matrix Individually catalyzes the hydrolysis of ATP (functions as an ATPase) – Fo Integral membrane complex Transports protons from IMS to matrix, dissipating the proton gradient Energy transferred to F1 to catalyze phosphorylation of ADP ATP SYNTHASE COMPLEX ATP Synthase has 2 functional domains: Fo and F1 ATP synthase is a large enzyme complex associated with the inner membrane of the mitochondria. This complex catalyzes the synthesis of ATP from ADP and Pi driven by the flow of protons across the membrane. ATP synthase has two distinct components: F1, a peripheral membrane protein on the matrix side, and Fo which is integral to the inner membrane. ATP synthesis F1 Fo has the composition ab2cn with n ranging from 8 to 17 depending on the species. c ring = arrangement of c subunits into two concentric circles. The c subunits rotate together as a unit F1 has the composition α3β3γδε each of the three β subunits has one catalytic site for ATP synthesis each β subunit has a different conformation: β-ATP, β-ADP and β-empty ATP SYNTHASE COMPLEX Diagram of the FoF1 complex, deduced from biochemical and crystallographic studies. The two b subunits (b2) of Fo associate firmly with the α and β subunits of F1, holding them fixed relative to the membrane. In Fo, the membrane-embedded cylinder of c subunits is attached to the shaft made up of F1 subunits γ and ε. C17 As protons flow through the membrane from the P side to the N side through Fo, the cylinder and γ shaft rotate, and the β subunits of F1 change conformation as the γ subunit associates with each in turn. THE F1 CATALYZES ATP FORMATION ADP + PI ATP F1 Hexamer arranged in three αβ dimers Dimers can exist in three different conformations: – Open: empty – Loose: binding ADP and Pi – Tight: catalyzes ATP formation and binds product COUPLING PROTON TRANSLOCATION TO ATP SYNTHESIS Proton translocation causes a rotation of the Fo subunit and the central shaft g This causes a conformational change within all the three ab pairs The conformational change in one of the three pairs promotes condensation of ADP and Pi into ATP ATP SYNTHESIS BY ROTATIONAL CATALYSIS Rotational Catalysis: mechanism by which the flow of protons through Fo causes the c ring to rotate and, in turn, trigger the subunit conformational changes in F1: (3 H+ are needed to form one ATP) Protons enter ATP synthase through Fo pore u As they stream by, proton motor force causes gamma subunit (green) to rotate 120o. This rotation causes a conformational change at all 3 active sites on F1 beta subunits u These sites alternate catalyzing ATP synthesis: - ADP + Pi - loose binding; - ATP - tightly bound; - empty - very low affinity for ATP u The conformational change is cooperative, and ATP will not be released from one site until ADP + Pi are bound at next ATP SYNTHASE MECHANISM https://www.youtube.com/watch?v=eVT2LAQU07I TRANSPORT OF ADP AND PI INTO THE MATRIX (1 H+ is needed to transport Pi) Adenine nucleotide and phosphate translocases: - - - - - Transport systems of the inner mitochondrial membrane carry ADP and Pi into the matrix and newly synthesized ATP into the cytosol. The adenine nucleotide translocase is an antiporter that moves ADP into the matrix and ATP out. The effect of replacing ATP4– with ADP3– in the matrix is the net efflux of one negative charge, which is favored by the charge difference across the inner membrane (outside positive). At pH 7, Pi is present as both HPO42– and H2PO4–; the phosphate translocase is specific for H2PO4–. There is no net flow of charge during symport of H2PO4– and H+, but the relatively low proton concentration in the matrix favors the inward movement of H+. Thus, the proton-motive force is responsible both for providing the energy for ATP synthesis and for transporting substrates (ADP and Pi) into and product (ATP) out of the mitochondrial matrix. All three of these transport systems can be isolated as a single membrane-bound complex (ATP synthasome) SHUTTLE SYSTEMS INDIRECTLY CONVEY CYTOSOLIC NADH INTO MITOCHONDRIA FOR OXYDATION: 1/ THE MALATE-ASPARTATE SHUTTLE Glycolysis NADH formed in the cytoplasm CANNOT itself cross the inner mitochondrial membrane. To complex1 It is necessary to shuttle the electrons into the matrix in order for NADH to be reoxidized to NAD+. The malate-aspartate shuttle (the most active shuttle mechanism) depends on two transporters that allow for the exchange of malate/aspartate across the otherwise impermeable inner membrane. Because electrons from NADH are shuttled from the IMS and retrieved as NADH in the matrix (thereby using complex I)à 2.5 ATP are thus generated as a pair of electrons passes from NADH to O2 (see later). This shuttle for transporting reducing equivalents from cytosolic NADH into the mitochondrial matrix is used in liver, kidney, and heart. 1 NADH in the cytosol (intermembrane space) passes two reducing equivalents to oxaloacetate, producing malate. 2 Malate crosses the inner membrane via the malate–α-ketoglutarate transporter. 3 In the matrix, malate passes two reducing equivalents to NAD+, and the resulting NADH is oxidized by the respiratory chain; the oxaloacetate formed from malate cannot pass directly into the cytosol. 4 Oxaloacetate is first transaminated to aspartate, and 5 aspartate can leave via the glutamate-aspartate transporter. 6 Oxaloacetate is regenerated in the cytosol, completing the cycle. SHUTTLE SYSTEMS INDIRECTLY CONVEY CYTOSOLIC NADH INTO MITOCHONDRIA FOR OXIDATION: 2/ THE GLYCEROL 3-PHOSPHATE SHUTTLE This alternative mean of moving reducing equivalents from the cytosol to the mitochondrial matrix operates in adipose tissue, skeletal muscle and the brain. In the cytosol, dihydroxyacetone phosphate accepts two reducing equivalents from NADH in a reaction catalyzed by cytosolic glycerol 3phosphate dehydrogenase. An isozyme of glycerol 3-phosphate dehydrogenase bound to the outer face of the inner membrane then transfers two reducing equivalents from glycerol 3-phosphate in the intermembrane space to ubiquinone. Note that this shuttle does not involve membrane transport systems. Because this shuttle bypasses the NADH dehydrogenase complex (Complex I), and delivers electrons to FAD then QH2, this shuttle yields only 1.5 ATP per electron pair (see later). ATP YIELD FROM COMPLETE OXYDATION OF GLUCOSE - ATP production in ATP synthase requires 3 H+ per turn through ATP synthase (per ATP) and 1 H+ needed to transport ADP and Pi into the matrix and ATP into cytosol. - Therefore 4 H+ are needed in total to make one ATP molecule. - NADH pumps 4+4+2 = 10 protons (Complex I, III, IV). Thus 10/4 = 2.5 ATP are made per NADH (or per pair of electrons). FAD pumps 4+2 = 6 protons (Complex III, IV). Thus 6/4 = 1.5 ATP per FADH2 molecule (or per pair of electrons). Difference in total yield depends on which shuttle system is used to move NADH from cytosol (produced in glycolysis) to mitochondrial matrix: - Malate-aspartate shuttle used more commonly (5 ATP) total 32 ATP. - Glycerol 3-phosphate shuttle used in the skeletal muscle and brain (3 ATP) total 30 ATP; - *If the malate/aspartate shuttle is used to transfer reducing equivalents into the mitochondrion, the yield is 5 ATP. If the glycerol 3-phosphate shuttle is used, the yield is 3 ATP. ATP SYNTHASE CATALYZE REACTIONS IN BOTH DIRECTIONS (ATP SYNTHESIS AND ATP HYDROLYSIS) A large proton gradient can supply the energy to drive ATP synthesis by ATP synthase (red arrows) However, ATP synthase can function in the reverse direction, and becomes an ATPase (black arrows): named F-type ATPase F-type ATPases catalyze the uphill transmembrane passage of protons, driven by ATP hydrolysis. They are ATP driven proton pumps. REGULATION OF OXIDATIVE PHOSPHORYLATION Primarily regulated by substrate availability – NADH and ADP/Pi – Due to coupling, both substrates required for electron transport AND ATP synthesis Inhibition of oxidative phosphoryation leads to accumulation of NADH – Causes feedback inhibition cascade up to PFK-1 in glycolysis Inhibitor of F1 (IF1), an 84 AA protein that: – Binds to ATP synthase inhibiting it in both directions – Prevents hydrolysis of ATP during low oxygen (Example: heart attack or stroke à hypoxia à electron transfer to O2 stalls à proton pumping by ETC slows à proton motive force collapses à reverse functioning of ATP synthase as an ATPase à dangerous drop in ATP à IF1 inhibits the synthase) – Only active at lower pH (high [H+] due to lactate formation), encountered when electron transport it stalled (i.e., low oxygen) REGULATION OF ATP PRODUCING PATHWAYS ATP-Producing Pathways Are Coordinately Regulated The major catabolic pathways have interlocking and concerted regulatory mechanism that allow them to function together in an economical and self-regulating manner. The relative concentrations of ATP, ADP and AMP control not only the rates of electron transfer and oxidative phosphorylation pathways but also the rate of the citric acid cycle, pyruvate oxidation and glycolysis pathways. When ATP consumption increases (low ATP), ADP and AMP increase and those pathways speed up. When ATP production increases (high ATP) and ADP and AMP decrease, then those pathways slow down. In addition to these effects, increased levels of NADH and acetylCoA also inhibit oxidation of pyruvate to acetyl-CoA and a high [NADH/[NAD+] ratio also inhibits the dehydrogenase reactions of the citric acid cycle pathway. REGULATION OF ATP PRODUCING PATHWAYS ATP-Producing Pathways Are Coordinately Regulated Interlocking regulation of glycolysis, pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation by the relative concentrations of ATP, ADP, and AMP, and by NADH. § High [ATP] (or low [ADP] and [AMP]) produces low rates of glycolysis, pyruvate oxidation, acetate oxidation via the citric acid cycle, and oxidative phosphorylation. § All four pathways are accelerated when the use of ATP and the formation of ADP, AMP, and Pi increase. § The interlocking of glycolysis and the citric acid cycle by citrate, which inhibits glycolysis, supplements the action of the adenine nucleotide system. In addition, increased levels of NADH and acetyl-CoA also inhibit the oxidation of pyruvate to acetyl-CoA, and a high [NADH]/[NAD+] ratio inhibits the dehydrogenase reactions of the citric acid cycle