Bioenergetics Chemical Reactivity

Chemical Reactivity Most biochemical reactions fall within only a few categories: •Cleavage and formation of C–C bonds •Cleavage and formation of polar covalent bonds (Addition-Elimination Reactions) ‒nucleophilic substitution mechanism ‒addition–elimination mechanism o hydrolysis and condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) •Oxidations-reductions (e– transfers) 1 Chemical Reactivity Rearrangements Isomerizations and Eliminations: (No Change in Oxidation State) Proline racemase Ribulose-5phosphate epimerase Chemical Reactivity Most biochemical reactions fall within only a few categories: •Cleavage and formation of C–C bonds •Cleavage and formation of polar covalent bonds (Addition-Elimination Reactions) ‒nucleophilic substitution mechanism ‒addition–elimination mechanism o hydrolysis and condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3, PO32–) •Oxidations-reductions (e– transfers) 2 Chemical Reactivity Group Transfers • Proton transfer, very common • Methyl transfer, various biosyntheses • Acyl transfer, biosynthesis of fatty acids • Glycosyl transfer, attachment of sugars • Phosphoryl transfer, to activate metabolites O O Z W O ‒ also important in signal transduction Substitution from sp3 phosphorous proceeds via the nucleophilic substitution (usually associative, SN2-like) mechanism. The nucleophile forms a partial bond to the phosphorous center, giving a pentacovalent intermediate or a penta-coordinated transition state. Chemical Reactivity Most biochemical reactions fall within only a few categories: •Cleavage and formation of C–C bonds •Cleavage and formation of polar covalent bonds (Addition-Elimination Reactions) ‒nucleophilic substitution mechanism ‒addition–elimination mechanism o hydrolysis and condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) •Oxidations-reductions (e– transfers) 3 Chemical Reactivity Re-dox Lessons from Quantum Chemistry • Most organic molecules, including the reduced fuels, are in the singlet spin state. – All electrons are paired. • Molecular oxygen is in the triplet spin state. – Two electrons are unpaired. • Direct electron transfer from a singlet reduced species to a triplet-oxidizing species is quantum-mechanically unfavorable. • This is why spontaneous oxidation of biomolecules (carbon oxidation states differ by 2e–) does not occur readily, despite the exergonic reaction thermodynamics. • Cofactors, such as transition metal ions and flavin adenine dinucleotide (FAD), are able to catalyze consecutive singleelectron transfers needed for utilization of O2. Chemical Reactivity Oxidation-Reduction Reactions Reduced organic compounds serve as fuels from which electrons can be stripped off during oxidation. Most energy Re-dox Relative DG°’ of oxidation per carbon (kcal/mol) –196 –168 –125 –68 Least energy 0 4 Chemical Reactivity Oxidation-Reduction Reactions Reduced organic compounds serve as fuels from which electrons can be stripped off during oxidation. Most energy Re-dox Relative DG°’ of oxidation per carbon (kcal/mol) –196 –168 –125 –68 Least energy Chemical Reactivity 0 Re-dox EXAMPLE: Reversible Oxidation of a Secondary Alcohol to a Ketone • Many biochemical oxidation-reduction reactions involve transfer of two electrons. • In order to keep charges in balance, proton transfer often accompanies electron transfer. • In many dehydrogenases, the reaction proceeds by a stepwise transfers of proton (H+) and hydride (:H–). NAD+ NADH + +H+ :H– + H :H– + H+ + NAD+ NADH + H 5 Chemical Reactivity Re-dox Common Redox Cofactors: NADH, NADPH, and FADH2 • Niacin Cofactors allow Hydride (2 e–) Electron Transfers • Flavin Cofactors allow single Electron Transfers Vitamin B3 (niacin) • Measure the change of absorbance at 340 nm • Very useful signal when studying the kinetics of NAD-dependent dehydrogenases Vitamin B2 (riboflavin) • These are commonly called pyridine nucleotides. • They can dissociate from the enzyme after the reaction. • In a typical biological oxidation reaction, hydride from an alcohol is transferred to NAD+, giving NADH. • Permits the use of molecular oxygen as an ultimate electron acceptor – flavin-dependent oxidases • Flavin cofactors are covalently or tightly bound to proteins. Bioenergetics Energetics of Biochemical Reactions: 6 Bioenergetics Energetics of Some Chemical Reactions: • Hydrolysis reactions tend to be strongly favorable (spontaneous). – Recall that being thermodynamically favorable is not the same as being kinetically rapid. • Isomerization reactions have smaller free-energy changes. – isomerization between enantiomers: DG° = 0 • Complete oxidation of reduced compounds (redox reactions) is strongly favorable. – This is how heterotrophs (sometimes called chemotrophs) obtain most of their energy. – In biochemistry, the oxidation of reduced fuels with O2 is stepwise and controlled. Bioenergetics Big Picture: Flow of Electrons Generally exergonic ADP ATP Separate pools of redox cofactors Generally endergonic 7 Bioenergetics Reduction Potential determines the Flow of Electrons • Reduction potential (E) – affinity for electrons; higher E, higher affinity – electrons transferred from lower to higher E DE°’ = E°’ (e− acceptor) – E°’ (e− donor) (that which is reduced) (that which is oxidized) DE°’ = (RT/ nF )ln (Keq) = ƒ DG°’ /nF DG°’ = –n F DE°’ – For negative DG, need positive DE E°’(acceptor) > E°’(donor) Bioenergetics Reduction Potential determines the Flow of Electrons TABLE 13-7a Standard Reduction Potentials of Some Biologically Important HalfReactions Half-reaction 1/ 2 E'˚ (V) O2 + 2H+ + 2e– à H2O 0.816 Fe3+ + e– à Fe2+ 0.771 NO3– + 2H+ + 2e– à NO2– + H2O 0.421 Cytochrome f (Fe3+) + e– à cytochrome f (Fe2+) Fe(CN)63– (ferricyanide) + e– à Fe(CN)64– Cytochrome a3 (Fe3+) + e– à cytochrome a3 (Fe2+) O2 + 2H+ + 2e– à H2O2 0.36 0.35 0.295 Cytochrome a (Fe3+) + e– à cytochrome a (Fe2+) 0.29 Cytochrome c (Fe3+) + e– à cytochrome c (Fe2+) 0.254 Cytochrome c1 (Fe3+) + e– à cytochrome c1 (Fe2+) 0.22 Cytochrome b (Fe3+) + e– à cytochrome b (Fe2+) 0.077 Ubiquinone + 2H+ + 2e– à ubiquinol 0.045 Fumarate2– + 2H+ + 2e– à succinate2– 2H+ + 2e– à H2 (at standard conditions, pH 0) 1x10-7 M 0.365 0.031 0.000 Crotonyl-CoA + 2H+ + 2e– à butyryl-CoA –0.015 Oxaloacetate2– –0.166 + 2H+ + 2e– à malate2– Source: Data mostly Loach, –in Handbook of Biochemistry and Molecular Biology, 3rd edn (G. D. Fasman, ed.), Physical –+ Pyruvate 2H +from 2e–R.àA.lactate –0.185 and Chemical Data, Vol. 1, p. 122, CRC Press, 1976. aThis is the value for free+ FAD; FAD bound to a specific flavoprotein (e.g., succinate dehydrogenase) has a different E'˚ that–0.197 Acetaldehyde + 2H + 2e– à ethanol depends on its protein environment. FAD + 2H+ + 2e– à FADH2 –0.219a Glutathione + 2H+ + 2e– à 2 reduced glutathione S + 2H+ + 2e– à H2S Lipoic acid + 2H+ + 2e– à dihydrolipoic acid –0.23 –0.243 –0.29 NAD+ + H+ + 2e– à NADH –0.320 NADP+ + H+ + 2e– à NADPH –0.324 Acetoacetate + 2H+ + 2e– à β-hydroxybutyrate –0.346 α-Ketoglutarate + CO2 + 2H+ + 2e– à isocitrate 2H+ + 2e– à H2 (at pH 7) Ferredoxin (Fe3+) + e– à ferredoxin (Fe2+) –0.38 –0.414 –0.432 8 Bioenergetics Reduction Potentials with higher affinity at bottom Hates e– Loves e– Bioenergetics EXAMPLE: NAD+ NADH + +H+ :H– + H :H– + H+ + NAD+ NADH + H DE°’ = E°’ (reduction) – E°’ (oxidation) = –0.32 V – (–0.19 V) = –0.13 V DG°’ = –n F DE°’ = –(2)(23.06kcalV-1mol-1)(–0.13 V) = +6 kcalmol-1 This is why we always ran the reaction the other way! 9 Bioenergetics EXAMPLE: DG°’ = –678 kcal mol-1 What is the DE°’ for the complete oxidation of glucose? DG°’ = –n F DE°’ –678 kcal mol-1 = –(24)(23.06 kcal V-1mol-1)(x V) DE°’ = +1.25 V What is the reduction potential (E°’ ) for Glc à CO2? DE°’ = E°’ (reduction) – E°’ (oxidation) 1.+1.25 V = +0.82 V – (E°’ (Glc oxidation)) E°’ (Glc oxidation) = – 0.43 V Summary We learned that: • The rules of thermodynamics and organic chemistry still apply to living systems • Reactions are favorable when the free energy of products is much lower than the free energy of reactants • Biochemical phosphoryl transfer reactions are favorable when: – the phosphate donors are destabilized by electrostatic repulsion – the reaction products are often stabilized by resonance • Unfavorable reactions can be made possible by chemically coupling a highly favorable reaction to the unfavorable reaction • Oxidation-reduction reactions commonly involve transfer of electrons from reduced organic compounds to specialized redox cofactors – Reduced cofactors can be used in biosynthesis, or may serve as a source of energy for ATP synthesis. 10