Dr. Aga Syed SameerCSIR Lecturer

Department of Biochemistry,

Medical College,

Sher-I-Kashmir Institute of Medical Sciences,

Bemina, Srinagar, Kashmir, 190018. India.

Biological

Oxidation

CHEMI-OSMOTIC THEORY

ATP SynthesisInhibitors of ETCUncouplers of ETCShuttle Systems

Electron Transference

NADH→FMN →FeS →CoQ

Succinate →FAD →FeS →CoQ

CoQ →FeS →Cytb →Cytc1 →Cytc

Cytc →Cytaa3 →O2

Oxidative Phosphorylation

The process of Phosporylation of ADP to ATP; coupled with the flow of via mitochondrial ETC

Dependency on ETC is because of Proton Pumps, which generate the H+ gradient across IMM by utilizing the energy of e-s flow

In addition, It also serves as proton pump; transporting H+s from inner chamber to Inner Membrane Space of Mitochondria

So, OP is cumulative function of ETC and ATP Synthase enzyme

ATP Synthase

F1-Fo ParticlesConsists of Head Piece – F1 and Base piece – Fo

attached with stalk

Fo component spans the IMM and forms an H+

channel or pore

F1 component catalyzes the phosphorylationof ADP to ATP by allowing the flow of H+

ions from Inter Membrane Space to Matrix via proton channel of Fo component

Thus, energy of proton gradient is utilized for the creation of ATP from ADP and Inorganic P

ATP Synthase

Chemiosmotic Theory

Proposed by Peter Mitchell in 1961

Flow of e-s through mitochondrial ETC and ATP synthesis are coupled by proton gradient that develops across the Inner Mitochondrial Membrane

Energy released during the transport of e-s from RE’s to O2 result in the efflux of H+ ions from Mitochondrial Matrix across IMM to Inner Membrane Space; Resulting in the generation of electro chemical proton gradient across IMM – 1.4pH unit

This electro chemical proton gradient serves as the potential source of energy for ATP synthesis

P:O RatioNumber of moles of inorganic phosphate (Pi)

utilized for ATP generating per gram atom of oxygen (half mole of O2) consumed

For NADH:

Complex I, II, IV are used for oxidation

10H+ are accumulated across IMM

Phosphorylation of 3 moles of ADP to ATP P:O Ratio = 3

For FADH2:

Complex III, IV are used for oxidation

6H+ are accumulated across IMM

Phosphorylation of 2 moles of ADP to ATP P:O Ratio = 2

Inhibitors

Complex I: Rotenones; Barbiturates (Amytal); Piericidin A; Chloropromazine; Alkylguanidines

Complex II: Malonate; Carboxin

Complex III: AntimycinA; BAL; Napthoquinone

Complex IV: Cyanide; H2S; CO; Sodium Azide

Other InhibitorsOligomycin: Antibiotic, which binds to Fo

subunit of ATP Synthase and blocks re-entry of protons into matrix thus preventing ATP synthesis

DiCyclohexylCarboDiimide (DCCD) : bonds covalently to a glutamic acid residue of the c subunit of Fo, proton flow through Fo is blocked and ATP synthase activity is inhibited

Atractyloside: Glycoside of plant Thistle, prventsOXPHOS by inhibiting Adenine Nucleotide Transporter/translocase (ANT) in IMM. Hence, blocks the supply of ADP for ATP synthase

UncouplersThey disrupt the tight

coupling between electron transport and the ATP synthase

2,4-dinitrophenolDicumarol, and Carbonyl Cyanide-p-

Trifluoromethoxyphenylhydrazone(fluorocarbonyl-cyanide phenylhydrazone or FCCP): 100x effective than DNP

UncouplersAdipose tissue in polar hibernating animals

contains so many mitochondria that it is called brown adipose tissue for the color imparted by the mitochondria.

The inner membrane of brown adipose tissue mitochondria contains an endogenous protein called thermogenin (literally, “heat maker”), or uncoupling protein, that creates a passive proton channel through which protons flow from the cytosol to the matrix.

IonophoresThey are lipophilic molecules that are capable of

binding and carrying ions across the biological membranes (by increasing the permeability of the membrane) and hence dissipating the proton gradient across IMM to inhibit OXPHOS

Valinomycin: Transports K ions

Nigercin: Transports K ions

Gramacidin: binds carries Na & K ions, and

Shuttle-Systems Most of the NADH used in electron transport is

produced in the mitochondrial matrix space, an appropriate site because NADH is oxidized by Complex I on the matrix side of the inner membrane

NADH produced in cytosol, if not oxidized to regenerate NAD, the glycolytic pathway would cease to function due to NAD limitation

Eukaryotic cells have a number of shuttle systems that harvest the electrons of cytosolic NADH for delivery to mitochondria without actually transporting NADH across the inner membrane

Malate-Aspartate

Liver

Heart

Kidneys

Malate-Aspartate

Glycero-PhosphateSkeletal Muscles

Brain

Questions?