Citrate Cycle: Energy conversion through redox reactions, a cycle with eight renewable reactants, and regulation Bioc 460 Spring Lecture 28 (Miesfeld) If the citrate cycle is an engine, what is the fuel, the energy output, and the exhaust? Compound 1080 contains fluoracetate, a poison that blocks the activity of aconitase The Citrate Cycle is spinning out of control!

The primary function of the citrate cycle is to convert energy available from the oxidization acetyl-CoA into 3 moles of NADH, 1 mole of FADH 2 and 1 mole of GTP during each turn of the cycle. The citrate cycle is a "metabolic engine" in which all eight of the cycle intermediates are continually replenished to maintain a smooth-running energy conversion process. The fuel for this metabolic engine is acetyl- CoA, the exhaust is two molecules of CO 2, and the energy output is redox energy used in the electron transport system for ATP synthesis. Oxaloacetate is both the product of reaction 8, and the reactant for reaction 1, which means that flux through the pathway is continuously monitored by resetting the level of available substrate after each turn of the cycle. Two other regulatory mechanisms at play in the citrate cycle are product inhibition and feedback control of key enzymes. Key Concepts in Citrate Cycle

The citrate cycle is considered the "hub" of cellular metabolism because it not only links the oxidation of metabolic fuels (carbohydrate, fatty acids and proteins) to ATP synthesis, but it also provides shared metabolites for numerous other metabolic pathways.

Pathway Overview The eight reactions of the citrate cycle oxidize acetyl-CoA to generate 2 CO 2, and in the process, reduce 3 NAD + and 1 FAD. In addition, 1 GTP is produced by substrate level phosphorylation which is converted to ATP by nucleotide kinase.

All of the enzymes in the citrate cycle, electron transport chain and oxidative phosphorylation reside in the mitochondrial matrix where pyruvate is converted to acetyl-CoA by the enzyme pyruvate dehydrogenase.

The "currency exchange" for redox energy and ATP synthesis in the mitochondria electron transport chain is ~2.5 ATP/ NADH. Oxidation of 2 FADH 2 molecules by the electron transport chain results in only ~3 molecules of ATP (~1.5 ATP/FADH 2 ) because of differences in where these two coenzymes enter the electron transport chain. Based on this ATP currency exchange ratio, and the one substrate level phosphorylation reaction, each turn of the cycle produces ~10 ATP for every acetyl-CoA that is oxidized.

Hans Krebs Elucidated the Citrate Cycle Hans Krebs, a biochemist who fled Nazi Germany for England in 1933, first described the citrate cycle in The citrate cycle is sometimes called the Krebs cycle, the citric acid cycle, or the tricarboxylic acid cycle, although we will refer to it as the citrate cycle because citrate is the first product of the pathway. The unprotonated form of citric acid is citrate which is the predominant species at physiological pH (the pK a values of the three carboxylate groups are 3.1, 4.7 and 6.4).

What does the citrate cycle accomplish for the cell? –Transfers 8 electrons from acetyl-CoA to the coenzymes NAD + and FAD to form 3 NADH and 1 FADH 2 which are then re- oxidized by the electron transport chain to produce ATP by the process of oxidative phosphorylation. –Generates 2 CO 2 as “waste products” and uses substrate level phosphorylation to generate 1 GTP which is converted to ATP by nucleoside diphosphate kinase. –Supplies metabolic intermediates for amino acid and porphyrin biosynthesis. Pathway Questions

What is the overall net reaction of citrate cycle? Acetyl-CoA + 3 NAD + + FAD + GDP + Pi + 2 H 2 O → CoA + 2 CO NADH + 2 H + + FADH 2 + GTP ΔGº’ = kJ/mol Pathway Questions Is the citrate cycle a favorable reaction? Of course it is, you are alive aren’t you!

Pathway Questions What are the key regulated enzymes in citrate cycle? Pyruvate dehydrogenase – not a citrate cycle enzyme but it is critical to flux of acetyl-CoA through the cycle; this multisubunit enzyme complex requires five coenzymes, is activated by NAD +, CoA and Ca 2+ (in muscle cells), and inhibited by acetyl-CoA, ATP and NADH. Citrate synthase – catalyzes the first reaction in the pathway and can be inhibited by citrate, succinyl-CoA, NADH and ATP; inhibition by ATP is reversed by ADP.

Pathway Questions What are the key regulated enzymes in citrate cycle? Isocitrate dehydrogenase - catalyzes the oxidative decarboxylation of isocitrate by transferring two electrons to NAD + to form NADH, and in the process, releasing CO 2, it is activated by ADP and Ca 2+ and inhibited by NADH and ATP. α-ketoglutarate dehydrogenase - functionally similar to pyruvate dehydrogenase in that it is a multisubunit complex, requires the same five coenzymes and catalyzes an oxidative decarboxylation reaction that produces CO 2, NADH and succinyl-CoA; it is activated by Ca 2+ and AMP and it is inhibited by NADH, succinyl-CoA and ATP.

Pathway Questions What are examples of citrate cycle in real life? Citrate is produced commercially by fermentation methods using the microorganism Aspergillus niger. Every year almost a half of million tonnes (5 x 10 8 kg) of citrate are produced worldwide by exploiting the citrate synthase reaction. Purified citrate is a food additive

The complete oxidation of glucose to CO 2 and H 2 O is summarized by the reaction: Glucose (C 6 H 12 O 6 ) + 6O 2 → 6CO 2 + 6H 2 O ΔGº’ = -2,840 kJ/mol ΔG = -2,937 kJ/mol Four of the CO 2 molecules are produced in the Citrate Cycle, but what reaction generates the other two CO 2 ?

Eight Reactions of the Citrate Cycle In the first half of the cycle, the two carbon acetate group of acetyl-CoA is linked to the four carbon oxaloacetate substrate to form a six carbon citrate molecule. Citrate is then converted to isocitrate to set up two decarboxylation reactions yielding two NADH and the high energy four carbon cycle intermediate succinyl-CoA. In the second half of the cycle, oxaloacetate is regenerated from succinyl-CoA by four successive reactions that lead to the formation of one GTP (ATP), one FADH 2, and one NADH.

Reaction 1: Condensation of oxaloacetate and acetyl-CoA by citrate synthase to form citrate This reaction commits the acetate unit of acetyl-CoA to oxidative decarboxylation Reaction follows an ordered mechanism: Oxaloacetate binds, inducing a conformational change in the enzyme that facilitates: - acetyl-CoA binding - formation of the transient intermediate, citryl-CoA - rapid hydrolysis that releases CoA-SH and citrate

Reaction 2: Isomerization of citrate by aconitase to form isocitrate This is a reversible two step isomerization reaction. The intermediate, cis-aconitate, is formed by a dehydration reaction that requires the participation of an iron-sulfur cluster (4Fe-4S) in the enzyme active site. H 2 O is added back to convert the double bond in cis-aconitate, to a single bond with a hydroxyl group, on the terminal carbon.

Aconitase is one of the targets of fluorocitrate Fluorocitrate is derived from fluoroacetate. Fluoroacetate-containing plants, such as acacia found in parts of Australia and Africa, are so deadly that Australian sheep herders have reported finding sheep with their heads still in the bush they were feeding on when they died. Fluoracetate is the active ingredient in the poison compound 1080 used to kill rodents and livestock predators. Sometimes, the poison is used indiscriminately, causing animal deaths.

Reaction 3: Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase to form α-ketoglutarate, CO 2 and NADH First of two decarboxylation steps in the citrate cycle First reaction to generate NADH used for energy conversion reactions in the electron transport system Catalyzes an oxidation reaction that generates the transient intermediate oxalosuccinate In the presence of the divalent cations Mg 2+ or Mn 2+, oxalosuccinate is decarboxylated to form α-ketoglutarate

Reaction 4: Oxidative decarboxylation of by α-ketoglutarate dehydrogenase to form succinyl-CoA, CO 2 and NADH Second oxidative decarboxylation reaction and also produces NADH. α-Ketoglutarate dehydrogenase complex utilizes essentially the same catalytic mechanism we have already described for the pyruvate dehydrogenase reaction. Includes the binding of substrate to an E1 subunit (α-ketoglutarate dehydrogenase), followed by decarboxylation and formation of a TPP-linked intermediate.

Reaction 5: Conversion of succinyl-CoA to succinate by succinyl-CoA synthetase in a substrate level phosphorylation reaction that generates GTP The available free energy in the thioester bond of succinyl-CoA (ΔGº' = kJ/mol) is used in the succinyl-CoA synthetase reaction to carry out a phosphoryl transfer reaction (ΔGº' = kJ/mol), in this case, a substrate level phosphorylation reaction, that produces GTP (or ATP). Nucleoside diphosphate kinase interconverts GTP and ATP by a readily reversible phosphoryl transfer reaction: GTP + ADP ↔ GDP + ATP (ΔGº' = 0 kJ/mol).

Reaction 6: Oxidation of succinate by succinate dehydrogenase to form fumarate This coupled redox reaction directly links the citrate cycle to the electron transport system through the redox conjugate pair FAD/FADH 2 which is covalently linked to the enzyme succinate dehydrogenase, an inner mitochondrial membrane protein. Oxidation of succinate results in the transfer of 2 e- to the FAD moiety, which in turn, passes the two electrons to the electron carrier coenzyme Q in complex II of the electron transport system.

Reaction 6: Oxidation of succinate by succinate dehydrogenase to form fumarate Is FAD oxidized or reduced in this redox reaction? Is succinate the reductant or the oxidant in this reaction?

Reaction 7: Hydration of fumarate by fumarase to form malate Fumarase the reversible hydration of the C=C double bond in fumarate to generate the L-isomer of malate. Fumarate and malate are citrate cycle intermediates that enter and exit the cycle from several different interconnected pathways.

Reaction 8: Oxidation of malate by malate dehydrogenase to form oxaloacetate Oxidation of the hydroxyl group of malate to form oxaloacetate in a coupled redox reaction involving NAD + /NADH. The change in standard free energy for this reaction is unfavorable (ΔGº' = kJ/mol), but the actual  G for this reaction is favorable. In order for this unfavorable  Gº’ to allow for a favorable  G, the metabolite concentrations need to be far from equilibrium. Based on what you know about the citrate cycle, what do you think explains the favorable  G in terms of [metabolite] in this case?

Bioenergetics of the citrate cycle Glycolysis + pyruvate dehydrogenase reaction + citrate cycle = net reaction: Glucose + 2 H 2 O + 10 NAD FAD + 4 ADP + 4 Pi → 6 CO NADH + 6 H FADH ATP ??

The reducing power of NADH and FADH 2 can be converted to ATP equivalents using the currency exchange ratio. ~2.5 ATP/NADH ~1.5 ATP/FADH 2 This yields ~28 ATP based on 3 NADH and 1 FADH 2 Anoter 4 ATP are synthesized by substrate phosphorylation, generates a maximum of ~32 ATP. The complete oxidation of glucose by the pyruvate dehydrogenase complex and the citrate cycle leads to the production of 6 CO 2 molecules as “waste”.

Radioactive 14 C-acetyl CoA In the first turn of the cycle, both carbons are incorporated into oxaloacetate. It isn't until the second turn of the cycle that the first carbon is released as CO 2 by the isocitrate dehydrogenase reaction. The second carbon is not lost as CO 2 until the α-ketoglutarate reaction in the fourth turn of the cycle. Intermediates must be continually regenerated in a cyclic pathway to maintain metabolic flux.

Three main control points in the cycle: Citrate synthase Isocitrate dehydrogenase α-ketoglutarate dehydrogenase

Pyruvate dehydrogenase is activated by CoA-SH to stimulate acetyl-CoA production. Pyruvate carboxylase in turn, is stimulated by acetyl-CoA to maintain OAA for citrate synthesis. If carbohydrate is limiting, (low pyruvate), then acetyl-CoA is converted to ketone bodies (ketogenesis) or citrate is shuttled to the cytosol.