Biological oxidation I Respiratory chain

Biological oxidation I Respiratory chain

Outline Metabolism Macroergic compound Redox in metabolism Respiratory chain Inhibitors of oxidative phosphorylation

Metabolism Metabolism consists of catabolism and anabolism Catabolism: degradative pathways Usually energy-yielding! Anabolism: biosynthetic pathways energy-requiring!

The ATP Cycle ATP is the energy currency of cells In phototrophs, light energy is transformed into the light energy of ATP In heterotrophs, catabolism produces ATP, which drives activities of cells ATP cycle carries energy from photosynthesis or catabolism to the energyrequiring processes of cells

High energy bonds Phosphoanhydride bonds (formed by splitting out H 2 O between 2 phosphoric acids or between carboxylic and phosphoric acids) have a large negative DG of hydrolysis.

Phosphoanhydride linkages are said to be "high energy" bonds. Bond energy is not high, just DG of hydrolysis. "High energy" bonds are represented by the "~" symbol. ~P represents a phosphate group with a large negative DG of hydrolysis.

Phosphocreatine (creatine phosphate), another compound with a "high energy" phosphate linkage, is used in nerve and muscle for storage of ~P bonds. Phosphocreatine is produced when ATP levels are high. When ATP is depleted during exercise in muscle, phosphate is transferred from phosphocreatine to ADP, to replenish ATP.

Phosphoenolpyruvate (PEP), involved in ATP synthesis in Glycolysis, has a very high DG of P i hydrolysis. Removal of P i from ester linkage in PEP is spontaneous because the enol spontaneously converts to a ketone. The ester linkage in PEP is an exception.

Other examples of phosphate esters with low but negative DG of hydrolysis: the linkage between phosphate and a hydroxyl group in glucose-6-phosphate or glycerol-3- phosphate.

ATP has special roles in energy coupling and P i transfer. DG of phosphate hydrolysis from ATP is intermediate among examples below. ATP can thus act as a P i donor, and ATP can be synthesized by P i transfer, e.g., from PEP. Compound Phosphoenolpyruvate (PEP) Phosphocreatine Pyrophosphate ATP (to ADP) Glucose-6-phosphate Glycerol-3-phosphate DG o of phosphate hydrolysis (kj/mol)

Some other high energy bonds: A thioester forms between a carboxylic acid and a thiol (SH), e.g., the thiol of coenzyme A (abbreviated CoA-SH). Thioesters are ~ linkages. In contrast to phosphate esters, thioesters have a large negative DG of hydrolysis.

The thiol of coenzyme A can react with a carboxyl group of acetic acid (yielding acetyl-coa) or a fatty acid (yielding fatty acyl-coa). The spontaneity of thioester cleavage is essential to the role of coenzyme A as an acyl group carrier. Like ATP, CoA has a high group transfer potential.

Coenzyme A includes b-mercaptoethylamine, in amide linkage to the carboxyl group of the B vitamin pantothenate. The hydroxyl of pantothenate is in ester linkage to a phosphate of ADP-3'-phosphate. The functional group is the thiol (SH) of b-mercaptoethylamine.

High energy (macroergic) compounds exemplifying the following roles: Energy transfer or storage ATP, PP i, polyphosphate, creatinephosphate Group transfer ATP, Coenzyme A Transient signal camp

Oxidation and reduction Oxidation of an iron atom involves loss of an electron (to an acceptor): Fe 2+ (reduced) Fe 3+ (oxidized) + e - Since electrons in a C-O bond are associated more with O, increased oxidation of a C atom means increased number of C-O bonds. Oxidation of C is spontaneous. Increasing oxidation number of C

Redox in Metabolism NAD + collects electrons released in catabolism Catabolism is oxidative - substrates lose reducing equivalents, usually H + ions Anabolism is reductive NAD(P)H provides the reducing power (electrons) for anabolic processes

NAD +, Nicotinamide Adenine Dinucleotide, is an electron acceptor in catabolic pathways. The nicotinamide ring, derived from the vitamin niacin, accepts 2 e - and 1 H + (a hydride) in going to the reduced state, NADH. NADP + /NADPH is similar except for P i. NADPH is e donor in synthetic pathways.

NAD + /NADH The electron transfer reaction may be summarized as : NAD + + 2e + H + NADH. It may also be written as: NAD + + 2e + 2H + NADH + H +

FAD (Flavin Adenine Dinucleotide), derived from the vitamin riboflavin, functions as an e acceptor. The dimethylisoalloxazine ring undergoes reduction/oxidation. FAD accepts 2 e - + 2 H + in going to its reduced state: FAD + 2 e - + 2 H + FADH 2

NAD + is a coenzyme, that reversibly binds to enzymes. FAD is a prosthetic group, that remains tightly bound at the active site of an enzyme.

Oxidation of the coenzyme Q

Respiratory Chain An Overview Electron Transport: Electrons carried by reduced coenzymes are passed through a chain of proteins and coenzymes to drive the generation of a proton gradient across the inner mitochondrial membrane Oxidative Phosphorylation: The proton gradient runs downhill to drive the synthesis of ATP It all happens in or at the inner mitochondrial membrane

Electron Transport Four protein complexes in the inner mitochondrial membrane A lipid soluble coenzyme (UQ, CoQ) and a water soluble protein (cyt c) shuttle between protein complexes Electrons generally fall in energy through the chain - from complexes I and II to complex IV

Sequence of electron carriers in the respiratory chain Coenzyme Q electron shuttle Complex I proton pump Complex II, does not pump protons Cytochrome c electron shuttle Complex III proton pump Complex IV proton pump 27

Complexes of Respiratory chain Complex Name No. of Proteins Prosthetic Groups Complex I Complex II NADH Dehydrogenase Succinate-CoQ Reductase 46 FMN, 9 Fe-S centers 5 FAD, cyt b 560, 3 Fe-S centers Complex III CoQ-cyt c Reductase Complex IV Cytochrome Oxidase 11 cyt b H, cyt b L, cyt c 1, Fe-S Rieske 13 cyt a, cyt a 3, Cu A, Cu B

Electron transfer from NADH to CoQ Path: NADH FMN Fe-S UQ FeS UQ Four H + transported out per 2 e- Complex I NADH-CoQ Reductase

Role of FMN: Since it can accept/donate either 1 or 2 e -, FMN has an important role in mediating electron transfer between carriers that transfer 2 e - (e.g., NADH) and carriers that can only accept 1 e - (e.g., Fe 3+ ).

Complex II Succinate-CoQ Reductase aka succinate dehydrogenase (from TCA cycle!) aka flavoprotein 2 (FP 2 ) - FAD covalently bound four subunits, including 2 Fe-S proteins Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S, 2Fe-2S Path: succinate FADH 2 2Fe 2+ UQH 2 Net reaction: succinate + UQ fumarate + UQH 2

Complex III CoQ-Cytochrome c Reductase CoQ passes electrons to cyt c (and pumps H + ) in a unique redox cycle known as the Q cycle The principal transmembrane protein in complex III is the b cytochrome Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents UQH 2 is a lipid-soluble electron carrier cyt c is a water-soluble electron carrier

Heme is a prosthetic group of cytochromes. Heme contains an iron atom embedded in a porphyrin ring system. The Fe is bonded to 4 N atoms of the porphyrin ring. Hemes in the three classes of cytochrome (a, b, c) differ slightly in substituents on the porphyrin ring system. A common feature is two propionate side-chains.

Complex IV Cytochrome c Oxidase Electrons from cyt c are used in a four-electron reduction of O 2 to produce 2H 2 O Oxygen is thus the terminal acceptor of electrons in the electron transport pathway - the end! Cytochrome c oxidase utilizes 2 hemes (a and a 3 ) and 2 copper sites Complex IV also transports H +

Coupling e - Transport and Oxidative Phosphorylation This coupling was a mystery for many years Many biochemists squandered careers searching for the elusive "high energy intermediate" Peter Mitchell proposed a novel idea - a proton gradient across the inner membrane could be used to drive ATP synthesis Mitchell was ridiculed, but the chemiosmotic hypothesis eventually won him a Nobel prize

Peter Mitchell Proposed chemiosmotic hypothesis revolutionary idea at the time 1961 1978 proton motive force 2005-2006 1920-1992

ATP Synthase

subunit ATP synthase Moving unit (rotor) is c ring and Remainder is stationary (stator) c ring subunit a subunit binds to outside of ring Exterior column has 1 a subunit 2 b subunits, and the subunit subunit F 0 contains the proton channel ring of 10-14 c subunits F 1 subunit has 5 types of polypeptide chains ( 3, b 3,,, ), displays ATPase activity b subunit and b are members of P-loop family

The Chemiosmotic Theory of oxidative phosphorylation, for which Peter Mitchell received the Nobel prize: Coupling of ATP synthesis to respiration is indirect, via a H + electrochemical gradient.

Chemiosmotic theory - respiration: Spontaneous e transfer through complexes I, III, & IV is coupled to non-spontaneous H + ejection from the matrix. H + ejection creates a membrane potential (DY, negative in matrix) and a ph gradient (DpH, alkaline in matrix).

Chemiosmotic theory - F 1 F o ATP synthase: Non-spontaneous ATP synthesis is coupled to spontaneous H + transport into the matrix. The ph and electrical gradients created by respiration are the driving force for H + uptake. H + return to the matrix via F o "uses up" ph and electrical gradients.

ATP-ADP Translocase ATP must be transported out of the mitochondria ATP out, ADP in - through a "translocase" ATP movement out is favored because the cytosol is "+" relative to the "-" matrix But ATP out and ADP in is net movement of a negative charge out - equivalent to a H + going in So every ATP transported out costs one H + One ATP synthesis costs about 3 H + Thus, making and exporting 1 ATP = 4H +

What is the P/O Ratio? i.e., How many ATP made per electron pair through the chain? e - transport chain yields 10 H + pumped out per electron pair from NADH to oxygen 4 H + flow back into matrix per ATP to cytosol 10/4 = 2.5 for electrons entering as NADH For electrons entering as succinate (FADH 2 ), about 6 H + pumped per electron pair to oxygen 6/4 = 1.5 for electrons entering as succinate

Shuttle Systems for e - Most NADH used in electron transport is cytosolic and NADH doesn't cross the inner mitochondrial membrane What to do? "Shuttle systems" effect electron movement without actually carrying NADH Glycerophosphate shuttle stores electrons in glycerol-3-p, which transfers electrons to FAD Malate-aspartate shuttle uses malate to carry electrons across the membrane

Respiratory chain = oxidative phosphoryltion + electron transport

Inhibitors of Oxidative Phosphorylation Rotenone inhibits Complex I - and helps natives of the Amazon rain forest catch fish! Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe 3+ ) of a 3 Oligomycin are ATP synthase inhibitors

Uncouplers Uncoupling e- transport and oxidative phosphorylation Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient Uncouplers are hydrophobic molecules with a dissociable proton They shuttle back and forth across the membrane, carrying protons to dissipate the gradient

Uncouplers and Inhibitors There are six distinct types of poison which may affect mitochondrial function: 1. Respiratory chain inhibitors (e.g. cyanide, antimycin, rotenone and TTFA) block respiration in the presence of either ADP or uncouplers. 2. Phosphorylation inhibitors (e.g. oligomycin) abolish the burst of oxygen consumption after adding ADP, but have no effect on uncouplerstimulated respiration.

3. Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP) abolish the obligatory linkage between the respiratory chain and the phosphorylation system which is observed with intact mitochondria. 4. Transport inhibitors (e.g. atractyloside, bongkrekic acid, NEM) either prevent the export of ATP, or the import of raw materials across the the mitochondrial inner membrane. 5. Ionophores (e.g. valinomycin, nigericin) make the inner membrane permeable to compounds which are ordinarily unable to cross. 6. Krebs cycle inhibitors (e.g. arsenite, aminooxyacetate) which block one or more of the TCA cycle enzymes, or an ancillary reation.

Inhibitors of respiratory chain Name Function Site of action retenone e transport inhibitor Complex I amytal e transport inhibitor Complex I antimycin A e transport inhibitor Complex III cyanide e transport inhibitor Complex IV carbon monoxide e transport inhibitor Complex IV azide e transport inhibitor Complex IV 2,4-initrophenol uncoupling agent transmembrane H+ carrier pentachlorophenol uncoupling agent transmembrane H+ carrier oligomycin inhibits ATP-ase OSCP protein