Biochemistry Energy Metabolism Part 1 PDF

BIOCHEMISTRY Energy Metabolism Part 1 Dr. Santos, Ricardo BIOLOGIC OXIDATION Continued….. OXIDATION The iron on the left...

BIOCHEMISTRY Energy Metabolism Part 1 Dr. Santos, Ricardo BIOLOGIC OXIDATION Continued….. OXIDATION The iron on the left side is reduced. If you reduce something, it will become  Losing or removal of electrons oxidized  State: Oxidized So from Fe++ → Fe+++ What does this mean? It means that Iron gave its electron and by giving its  Dehydrogenation, you lose Hydrogen electron it gains a proton (adding another +). Note that at first it has 2  Increase valence because of losing electrons (-) “positive (+) signs” and after the reaction it has “3 + signs,” denoting that it lost an electron, making Iron “less negative” through giving its electron (-) REDUCTION  Gaining of electrons Copper on the left is in the oxidized state, it can oxidize Iron. Copper now  State: Reduced will be in the reduced state  Hydrogenation, you gain Hydrogen So from Cu++ → Cu+  Decrease valence because of accepting electrons (-) What does this mean? It means that Copper accepted an electron (-) and by accepting the electron it becomes “more negative” which is denoted by the OXIDIZING AGENT removal of a “+ sign”  Oxidizes a substrate REDOX REACTIONS  Also called oxidant; electron acceptor  Must be in the oxidized state  Converted to the reduced form REDUCING AGENT Picture Above:  Reduces a substrate Look at the topmost reaction. If the arrow is going to the right, the product  Also called reductant; electron donor is reduced because the reduced form of oxygen is water (O gained the  Must be in the reduced state electrons of H and became H2O). We call this reaction reduction half reaction  Converted to the oxidized state Oxygen has not gained the 2 H+ and 2e-. If they are still separated by + signs Redox Reactions occur when electrons are transferred between an electron this means that it is still in its oxidized form (loss state). But when they come donor (reducing agent) and an electron acceptor (oxidizing agent). Redox together as one molecule (H2O), it becomes the reduced form (gained state) reactions are usually coupled, that’s why it’s called REDOX reactions Look at the second reaction. NADH has the H already and has gained the electrons (not actually seen in the formula but it is there) which means that Example: it is in its gained state (reduced form). It then releases the electrons and  Iron (metal) commonly found in the body becomes a loss state or oxidized form. This is an oxidation half reaction  It can be in reduced or oxidized form TYPES OF REDOX REACTIONS 1. Electrons are transferred in the form of hydrogen atoms SH2 + FAD S + FADH2 Reductant Oxidant Oxidized Reduced How was electron transferred to FAD? It is through the hydrogen atoms. FADH2 carries 2 electrons and 2 protons 2. Electron can be transferred as the hydride ion (H:) NAD+ + H: - NADH + H+ REDUCED OXIDIZED Has lesser valence Has greater valence Note that the reductant here is SH2 and the oxidant is the NAD+. Has more electrons Has less electrons NAD+ is the coenzyme of a pyridine-linked dehydrogenase. The In the gain state In the loss state dehydrogenase will remove the electrons from SH2 and pass it on Ferrous Ferric to NAD+ in the form of H: - (hydride ion). H: - contains 2 electrons and 1 proton that is the reason why the proton (H+) is written separately 3. Direct transfer of electron from donor to acceptor Fe2+ + Cu2+ Fe3+ + Cu1+ Picture Above: Redox reaction Reductant Oxidant Oxidized Reduced (From left to right) This was already explained on the example earlier . This shows Iron is in the reduced state (reductant) → It will give electrons to Copper that electrons were directly transferred (oxidant) which is in a loss state (cupric) → Iron gets oxidized and Copper is now reduced (cuprous) 4. Direct reaction between a reductant (AH) and molecular oxygen AH + O2 + BH2 A-OH + H2O + B #GrindNation “Strength In Knowledge” BESHYWAP 1  BIOCHEMISTRY Energy Metabolism Part 1 Dr. Santos, Ricardo STANDARD REDOX POTENTIAL (SRP) B. Dehydrogenase  It measures the tendency of a biologic system to release or accept  Transfer hydrogen from one substrate to another BUT NOT to electrons oxygen  Expressed in volts (Eo) Functions: GENERAL RULE: o Transfer of hydrogen from one substrate to another in The more negative the Eo , the better the reductant; the better is a coupled oxidation-reduction reaction; enables the substance as an electron donor; the lower its affinity for reducing equivalents oxidative processes to occur in electrons the absence of oxygen o Components of the ETC (electron transport chain) The more positive the Eo , the better the oxidant; the better is the substance as an electron acceptor; the higher its affinity of redox pair for electrons REDOX POTENTIAL IN MAMMALIAN OXIDATION SYSTEMS Types of Dehydrogenases: 1. Pyridine-linked dehydrogenases use derivatives of niacin as coenzymes o Nicotinamide adenine dinucleotide (NAD+)–linked dehydrogenase; involved in oxidative pathways of metabolism that generates ATP e.g. Glycolysis, Krebs cycle and ETC o Nicotinamide adenine dinucleotide phosphate (NADP)–linked dehydrogenase; involved in reductive synthesis e.g. fatty acid and steroid synthesis Picture Above: (Discussion explanation) The form on the left side before the slash ( / ) is the oxidized form. And the 2. Flavin linked dehydrogenases form on the right after the slash is the reduced form. So oxygen is the o These are FMN and FAD which are more tightly bound oxidized form. On the other hand water is the reduced form. o Involved in electron transport in the respiratory chain and therefore ATP generation So applying the rule in SRP which is the better reductant? NADH or reduced o Examples are NADH, succinate, acyl-CoA, glycerol 3- lipoate? Answer: NADH because it is more negative (-0.32) than reduced phosphate dehydrogenases lipoate (-0.29). So the flow of electrons would be from NADH, it will transfer o Also involved in dehydrogenation of reduced lipoate electrons to oxidized lipoate. So oxidized lipoate will become reduced lipoate (by dihydrolipoyl DH), an intermediate in the oxidative after the reaction. What happens to NADH? NADH becomes oxidized to NAD+ decarboxylation of pyruvate and α-ketoglutarate What is the best oxidant or electron acceptor of all the substances on the 3. Cytochromes except cytochrome oxidase table? Answer: Oxygen. Oxygen become reduced to water if it accepts an o They are iron-containing hemoproteins electron. Oxygen inhaled from the atmosphere is converted to water o Iron atoms oscillates between oxidized (Fe3+, ferric) molecules inside our body. and reduced (Fe2+, ferrous) state o Involved as carriers of electrons from flavoproteins to TYPES OF OXIDOREDUCTASES cytochrome oxidase A. Oxidase o Occur in the respiratory chain such as Cytochromes b, c1, c and a  Transfers electrons directly to oxygen  e.g. xanthine oxidase, cytochrome oxidase Cytochrome oxidase (Cyt. a3, Cyt. aa3); hemoprotein; contains 2 hemes (prosthetic group), 2 Fe, 2 Cu; terminal component of the respiratory chain; inhibited by carbon monoxide, cyanide and hydrogen sulfide Continued next page….. Flavoprotein oxidases- contain Flavin mononucleotide (FMN) or Flavin dinucleotide (FAD) as prosthetic groups; derivatives of vitamin B2 or riboflavin; e.g. L-amino acid oxidase (FMN-linked; found in kidney) #GrindNation “Strength In Knowledge” BESHYWAP 2  BIOCHEMISTRY Energy Metabolism Part 1 Dr. Santos, Ricardo C. Hydroperoxidases OXYGEN TOXICITY  Use hydrogen peroxide or an organic peroxide as substrate;  Is due to the conversion of oxygen superoxide found in peroxisome  Transfer of a single electron to oxygen generates superoxide anion free radical 1. Peroxidases o Formed when reduced flavins (FMNH2) are reoxidized o Hydrogen peroxidase is reduced to water by univalently by O2 substances that act as electron acceptors  Superoxide Dismutase protects aerobic organisms against oxygen o Found in leukocytes, platelets, RBC, milk toxicity. It catalyzes the removal of superoxide by the following o Glutathione peroxidase protects the RBC membrane reaction: against oxidation O2. - + O2. - + 2H+ → H2O2 + O2 ELECTRON TRANSPORT CHAIN (RESPIRATORY CHAIN)  Found in the inner mitochondrial membrane  Collects and transports reducing equivalents and directs them to their final reaction with oxygen to form water  The final acceptor of electrons is oxygen 2. Catalase  The components of the ETC are contained in 4 large protein o Uses hydrogen peroxide as electron donor and complexes referred to as Complex I, II, III and IV electron acceptor to form water  3 complexes serve as proton pumps which are Complex I, III and IV o Found in blood, bone marrow, mucous membrane, kidney and liver  Complex II is not a proton pump o Destroy H2O2 formed by oxidases  Coupled with oxidative phosphorylation Picture on Left: D. Oxygenases This is a diagrammatic  Catalyze the direct transfer and incorporation of oxygen into a representation of the substrate molecule mitochondrion showing the outer mitochondrial Two Kinds of Oxygenases membrane and the inner a. Dioxygenase mitochondrial membrane with o Incorporates both atoms of molecular oxygen into invaginations forming cristae. the substrate The electron transport o E.g. homogentisate, L-tryptophan and hydroxyan assembly containing the thranilate dioxygenases different electron carriers are b. Monooxygenase in the inner mitochondrial o Incorporates only one atom of molecular oxygen into membrane. What are the the substrate electron carriers? NAD, FMN, o Also called mixed-function oxidases, hydroxylases Coenzyme Q, and cytochromes o Products formed are hydroxylated b, c, a, & a3. All of these o Cytochrome P450 electron carriers are contained in the four protein complexes Cytochrome P450 (complex I to IV). Side by side o Are monooxygenases important for the detoxification the ETC is another complex of many drugs and hydroxylation of steroids (very which is Complex V, an important in metabolism of drugs inside our body) enzyme called ATP synthase. o Heme-containing enzymes Complex V is the site of ATP o NADH and NADPH donate reducing equivalents for the synthesis by a process known reduction of these cytochromes as Oxidative Phosphorylation. o Reduced cytochromes are oxidized by other substrates The ETC is coupled or linked to oxidative phosphorylation or CYP FAMILIES IN HUMANS complex V. CYP1 Drug and steroid (especially estrogen) metabolism Is Complex V part of the ETC? CYP2 Drug and steroid metabolism NO, because it is outside the Drug and steroid (including testosterone) CYP3 ETC. In the mitochondrial metabolism CYP4 Arachidonic acid or fatty acid metabolism matrix, the 2 most important CYP5 Thromboxane A2 synthesis oxidative pathways of Bile acid biosynthesis 7-alpha hydroxylase of steroid metabolism occurs there, CYP7 which are TCA cycle (Krebs nucleus CYP11 Steroid biosynthesis Cycle) and ß-oxidative CYP24 Vitamin D degradation pathway for the oxidation of CYP51 Cholesterol biosynthesis fatty acids. #GrindNation “Strength In Knowledge” BESHYWAP 3  BIOCHEMISTRY Energy Metabolism Part 1 Dr. Santos, Ricardo OVERVIEW OF CELLULAR RESPIRATION 3. Complex III: Q-Cytochrome C oxidoreductase  Passes electrons from CoQ to cytochrome C 4. Complex IV: Cytochrome C oxidase  Transfer electrons to O2 reducing the latter to H2O  The final acceptor of electrons in ETC is O2  Note: last is oxidase because the final acceptor is oxygen MAGNIFIED VIEW OF THE ELECTRON TRANSPORT ASSEMBLY IN THE INNER MITOCHONDRIAL MEMBRANE Picture Above: Cellular Respiration Oxidation of the nutrients (glycerol, fatty acids, glucose, amino acids) take place in the mitochondria. Oxidation of the aforementioned nutrients generate ATP. A common intermediate metabolite in the metabolism of these nutrient is acetyl-CoA which enters the Krebs cycle when it reacts with oxaloacetate catalyzed by the enzyme citrate synthase to form Citrate. From Citrate it will form isocitrate, alpha-ketoglutarate, succinate and malate. Electron Transport Assembly Explained in Detail: These formed substances are reduced intermediates that will feed electrons to the ETC (respiratory chain) when they are acted upon by their corresponding dehydrogenases. The reduced intermediates (isocitrate, alpha-ketoglutarate, succinate, malate) will undergo dehydrogenation and some of the electrons carried by the hydrogen will enter the ETC and will be used to reduce oxygen to water. And some of those protons will be pumped out into the intermitochondrial membrane space and will be used to phosphorylate ADP to form ATP by a process known as oxidative phosphorylation. What are formed during cellular respiration? Water & ATP What are the by-products during TCA (not shown in diagram)? Carbon dioxide What happens to carbon dioxide? It is eliminated by the lungs COMPLEX I: Coming from the mitochondrial matrix are the different reduced substrates such as isocitrate, alpha-ketoglutarate, succinate, and malate from the Krebs PROTEIN COMPLEXES IN THE ETC cycle; Fatty acyl CoA and Beta hydroxyl-fatty acyl CoA from Beta oxidation. When the reduced substrate is acted upon by their corresponding dehydrogenase (e.g. isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, fatty acyl CoA dehydrogenase) the enzyme will extract electrons from the reduced substrate and transfer it to NAD+ and the reduced substrate after losing the electron becomes oxidized. NAD+ upon accepting the electrons in the form of hydride ions, is reduced to NADH. So the carriers of electrons to complex I is NADH. NADH is then acted upon by NADH dehydrogenase whose prosthetic group is FMN (Flavin mononucleotide). So the enzyme (NADH dehydrogenase) will transfer electrons from NADH to FMN, and NADH will be reoxidized to NAD and FMN will be reduced to FMNH2. FMNH2 will be reoxidized back to FMN when it transfer the electrons to CoQ (Coenzyme Q or ubiquinone) and CoQ will be reduced to CoQH2. What happens in Complex I? Energy is released as electrons transfer from 1. Complex I : NADH-Q oxidoreductase or NADH dehydrogenase 1 electron carrier to another. That energy released in complex I will be used  Electrons are transferred from NADH to coenzyme Q to pump protons from the matrix to the intermembrane space. Complex I is (ubiquinone) a proton pump. 2. Complex II: Succinate-Q reductase or FADH coenzyme Q reductase or Succinate dehydrogenase  FAD-linked dehydrogenase oxidizes a substrate (e.g Continued next page….. succinate) and passes electrons to CoQ (ubiquinone) #GrindNation “Strength In Knowledge” BESHYWAP 4  BIOCHEMISTRY Energy Metabolism Part 1 Dr. Santos, Ricardo COMPLEX II: COMPLEX IV: Succinate, acyl CoA and glycerol 3-PO4 are the reduced substrates that can There are 2 cytochromes in the above diagram, cytochrome a and enter complex II but succinate is the reduced substrate that is most cytochrome a3. Cytochrome a3 is cytochrome oxidase, it transfers common/most abundant that will feed electrons to complex II. Succinate will electrons directly to oxygen. The electron acceptor of the cytochrome a and be acted upon by succinate dehydrogenase and the enzyme will extract a3 is Ferric iron (Fe3+) and the electron donor is Ferrous iron (Fe2+). So the electrons from succinate and transfer it to FAD. Succinate will be oxidized to Fe2+ of cytochrome a3 (cytochrome oxidase) will transfer electrons directly fumarate and FAD is reduced to FADH2. The electrons are transferred to FAD to oxygen forming water (H2O). in the form of hydrogen atoms. FADH2 then transfer electrons to CoQ, Complex IV is also a proton pump. Note that the site of proton pumps are reducing it to CoQH2. also the site of energy conservation because the energy that is released in So FADH2 is the carrier of electrons in complex II. CoQ (coenzyme Q) these complexes (complex I, III, & IV) are used to pump protons from the serves as a common acceptor of electrons coming from complex I and II. matrix to the intermembrane space. And the proton gradient that is Complex II is NOT a proton pump. CoQ also links complex I and II to complex generated will be used to phosphorylate ADP to ATP through oxidative III phosphorylation. OTHER COMPONENTS OF THE ETC  Flavoproteins - Components of complexes I and II - FMN is the prosthetic group of NADH dehydrogenase in complex I - FAD is the prosthetic group of succinate dehydrogenase in complex II  Iron-sulfur proteins (non-heme, Fe-S) - Found in complexes I, II, and III COMPLEX III: - Take part in single electron transfer reaction Cytochrome b is the representative cytochrome in the illustration above. - Iron atom undergoes oxidoreductions between ferrous and There are other cytochromes in complex III that can be used as carriers. ferric CoQH2 (reduced substrate) will transfer its electron to the Ferric iron (Fe3+) of cytochrome b. CoQH2 will then be oxidized to CoQ upon transferring ELECTRON CARRIERS electrons. Fe3+ will be reduced to Ferrous iron (Fe2+). Fe2+ will now serve as NAD FAD the electron donor (reducing agent) and will transfer the electron to Ferric Derived from: Niacin (Vitamin B3) Riboflavin (Vitamin B2) iron (Fe3+) of cytochrome c. So Fe2+ of cytochrome b will be converted back Active portion: Nicotinamide ring Isoalloxazine ring to Fe3+ , while Fe3+ of cytochrome c will be reduced to Ferrous iron (Fe2+) Carries 2 electrons but Carries 2 electrons and 2 You can see in the diagram that cytochrome c links complex III to complex Carrier of: only one H+ H+ IV. Complex III is also a proton pump. NADH + H+ FADH2 Reduced NADH – derived from NAD-linked dehydrogenases, like:  Isocitrate, α-ketoglutarate, & malate dehydrogenases of the Krebs cycle  Pyruvate dehydrogenase which links glycolysis to the TCA cycle  Hydroxyacyl CoA dehydrogenase of the fatty acid beta oxidative pathway Reduced FADH2 – derived from FAD-linked dehydrogenases, like:  Succinate dehydrogenase of the TCA cycle  Alpha-glycerophosphate shuttle dehydrogenase  Fatty acyl CoA dehydrogenase of beta oxidation Continued next page….. #GrindNation “Strength In Knowledge” BESHYWAP 5  BIOCHEMISTRY Energy Metabolism Part 1 Dr. Santos, Ricardo FLOW OF ELECTRONS IN EACH COMPLEXES TYPES OF CARRIERS - Substrates (e.g. malate, α-ketoglutarate, isocitrate) are acted upon by NAD-linked dehydrogenases that Carrier Prosthetic Group # Electrons/H+ transferred reduces NAD → NADH2 FAD - This is coupled with the transfer of 4 H+ across the Flavoprotein 2 FMN inner mitochondrial membrane into the Cytochromes Heme 1 Complex I intermembrane space Contains long HC - NADH2 is acted upon by NADH dehydrogenase Ubiquinone tails of Isoprene 2 which transfers the electrons to FMN initially then (UQ or coenzyme Q) units to a series of Fe-S centers and finally CoQ forming Iron-Sulfur proteins Fe-sulfur center 1 CoQH2 - Substrates (e.g. succinate, acyl CoA, & glycerol 3- PO4) are acted upon by FAD-linked REFERENCES dehydrogenases that reduced FAD → FADH2 Complex II - FADH2 is reoxidized in the ETC via Fe-S centers to  Biochemistry Manual (2018) CoQ (is reduced to CoQH2)  Dr. Santos Recordings - CoQ links Complexes I and II to Complex III  Shawn Juan Trans on Energy Metabolism - No proton translocation occurs at this site  PPT Notes - Q cycle couples electron transfer to transport 4 protons (H+) in Complex III - Complex III is made up of cytochromes c1, bL , bH and a Rieske Fe-S (Fe atoms are linked to two histidine- Complex III SH groups) - CoQH2 is reoxidized to CoQ in Complex III when electrons are transferred to cytochrome c - Cytochrome c links Complex III to Complex IV - The final acceptor of electrons in the ETC is O2 - Electrons are passed initially to CuA → Cyt a → Cyt a3 → CuB → ½ O2 (is reduced to 1 H2O) - Of the 8 H+ removed from the matrix (Complexes I and III), 4 are used to form 2 water molecules and 4 are pumped into the intermembrane space Complex IV - Transfer of one electron to O2 forms superoxide - Transfer of two electrons to O2 forms hydrogen peroxide - Transfer of two electrons to ½ O2 forms one water molecule - Transfer of four electrons to ½ O2 forms two water molecules ELECTRON TRANSPORT COMPLEXES Mass Enzyme Complex # of Subunits Prosthetic Groups (kDa) Complex I: NADH 850 42 FMN, Fe-S dehydrogenase Complex II: Succinate 140 5 FAD, Fe-S dehydrogenase Complex III: Cytochrome c 250 11 Hemes, Fe-S oxidoreductase Cytochrome c 13 1 Heme Complex IV: Cytochrome c 150 13 Hemes; Cu oxidase #GrindNation “Strength In Knowledge” BESHYWAP 6