Oxidative Phosphorylation

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Oxidative phosphorylation

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a. Synthesis of ATP (phosphorylation) which is driven by oxidation of carbohydrates, fats, and proteins, coupled to reduction of oxygen (oxidative)
b. Occurs only in mitochondria
c. Accomplished by 5 enzymes complexes of the mitochondrial inner membrane and coenzyme Q

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Complex I

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1. NADH DH, or NADH-coenzyme Q oxidoreductase

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Complex II

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1. Succinate DH, or succinate-coenzyme Q oxidoreductase

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Complex III

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1. Coenzyme Q-cytochrome c oxidoreductase

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Complex IV

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1. Cytochrome c oxidase

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Complex V

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1. ATP synthase, or F1F0-ATP synthase

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Conezyme Q

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1. Ubiquinone
2. A lipid soluble electron transfer molecule
3. Reduced and oxidized forms

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Oxidations of substrate molecules within mitochondria

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a. Result in production of reduced cofactors
b. NADH from several steps of TCA cycle, from pyruvate DH, or from products of FA oxidation
c. FADH2 from TCA cycle, G3P DH, or from porducts of fatty acid oxidation

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Re-oxidation of cofactors results in reduction of Coenzyme Q to QH2

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a. NADH feeds into complex I
b. G3P DH is a membrane boud flavoprotein enzyme which directly reduces CoQ
c. FADH2 from fatty acyl CoA DH transfers electrons to electron transferring flavoprotein (ETF)

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ETF

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a. Substrate for membrane-bound ETF-coenzyme Q oxidoreductase which reduces coenzyme Q

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Complex III

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a. Re-oxidizes coenzyme Q
b. Subsequently reduces cytochrome c

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Cytochrome c

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a. Globular hydrophilic protein
b. Serves as a mobile electron carrier like coenzyme Q
c. Remains loosely associated with the outer surface of inner mitochondrial membrane
d. Can diffuse from complex III and transfer electrons to complex IV

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Complex IV

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a. Re-oxidizes cytochrome c and reduces O2 to H2O

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ETC and proton-motive force

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a. Produces ATP from ADP and Pi

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Oxidation of NADH coupled to reduction of O2

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i. (½)O2+NADH+H+→H2o+NAD+
ii. Calculation of ΔE’0 = 0.816-(-.320)=1.136 volts
iii. Calculation of ΔG’=-nF ΔE’0=-2*23kcal/volt*1.136 volts=-52.256 kcal

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Free energy change is conserved as a proton-motive force

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i. Associated with redox reactions carried out by complexes I, III, and IV
ii. H+ ions pumped out of mitochondira
iii. Generates a very deep H+ ion concentration gradient across the mitochondrial inner membrane

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Two components of proton-motive force

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1. No counter ion deposited in mitochondrial matrix as H+ is pumped out→ matrix negative relative to cytosol
2. ΔpH→ isolated mitochondria may achieve 1 pH unit

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Mitochondrial ATP synthase

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a. Complex V, F1F0-ATP synthase
b. Large, multi-subunit enzyme which utilizes the proton-motive force to generate ATP from ADP and Pi
c. F1 domain is peripheral, F0 domain is integral to inner membrane

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Proton flow through F0

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a. Rotor domain→ c subunits
b. Rotates, generating catalytic activity of ATP synthesis

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Stoichiometry of oxidative phophorylation-- P/O ratio

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i. Describes number of ATP molecules which can be produced by one pair of electrons flowing down respiratory chain

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Free energy available from complex IV may have been overestimated

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i. NADH P/O=2.5
ii. Succinate P/O=1.5

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Rate control of P/O ratio

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i. Regulated by availability of ADP “respiratory control”
ii. Rate measured by measuring rate of O2 consumption of mitochondria

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State 4 respiration

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i. O2 consumption is low because→
ii. → Due to sufficient substrates, Pi and O2, with no work being done, proton-motive force is maximal

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Addition of ADP promotes

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i. Synthesis of ATP from ADP and Pi at expense of proton-motive force
ii. Rate of O2 consumption increases (state 3) until all of the added ADP has been phosphorylated to ATP and the maximal proton-motive force has been re-established

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Rotenone

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i. Inhibits complex I
ii. Due to chain nature of electron transport, all respiration is inhibited
iii. May be partially overcome by adding substrate which enters chain after complex I

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Antimycin A

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i. Inhibits complex III
ii. Inhibition cannot be overcome by succinate, but can be overcome by artificial substrate of complex IV→ ascorbate

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Azide, cyanide, and CO

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i. Each of these inhibit complex IV
ii. Inhibition of complex IV cannot be overcome

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Oligomycin

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i. Inhibits ATP synthase
ii. Since respiration is coupled to ATP synthesis, oligomycin will result in slow respiration which cannot be stimulated by addition of ADP

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Uncouplers

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i. Promote maximal rates of respiration
ii. No ATP is synthesized either in presence or absence of oligomycin

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UCP, thermogenin

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1. Family of proteins expressed in brown adipose tissue
2. Uncoupling of oxidative phosphorylation and respiration results in the generation of heat
3. UCPS are expressed in human→ mostly in infants, barely in adults

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Biological oxidation by CYP

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a. Catalyze hydroxylation of a number of different compounds
b. RH+O2+NADPH+H+→ ROH+H2O+NADP+
c. Currently classified as monooxygenases or mixed function oxidases
d. Substrates are more lipophilic, and products have been made more hydrophilic

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Prosthetic group in CYP

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i. Contains Fe2+
ii. Involved in redox cycle of enzyme

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Steroid substrate for CYP

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a. Hydroxylation reactions of cholesterol, bile acids and salts, and steroid hormones
b. P450 enzymes found in adrenal tissues
c. Very often mitochondrial membrane enzymes

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Endogenous substrates of CYP

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Steroids, eicosanoids, fatty acids, retinoids

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Exogenous substrates of CYP

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i. Members of P450 usually ER enzymes of liver
ii. 50% of common drugs used by humans are metabolized by CYP proteins

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Phase 1 metabolism of drugs

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1. Addition of -OH→ more hydrophilic

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Phase 2 metabolism

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1. Addition of -OH makes them a substrate for further metabolism
2. Often involves addition of a hydrophilic group to hydroxyl group→ glucuronic acid, sulfate

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CYP isoforms

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a. 150 isoforms, 57 genes in humans
b. Family denoted by number (CYP1)
c. Sub-family=capital letter CYP1A
d. Next number signifies individual CYP member=CYP1A1

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Most CYPs are inducible

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a. Stabilization of mRNA
b. Enzyme stabilization
c. Others