CHEMIOSMOTIC HYPOTHESIS 4th Semester CC8 TH Peter Mitchell (1920 - 1992)

Nobel Prize for Chemistry in 1978 (1)Electrons pass from NADH or FADH 2 to O 2 , the terminal electron acceptor, through a chain of carriers in the inner membrane (FMN, Fe-S center, Heme group Fe, CoQ); (2) As electrons move through the electron-transport chain, H + are pumped out across the inner membrane, and form Proton motive force ; (3)The pH and electrical gradient resulting from transport of protons links oxidation to phosphorylation. Experimental Evidences

• Addition of acid to the external media ,thus establishing a proton gradient, also leads to ATP synthesis • Oxidative phosphorylation (OP) requires an intact mitochondrial membrane. OP thus does not occur in soluble preparation or in mem fragment lacking well- defined inside & outside mem. • Uncouplers such as DNP are amphipathic & increase permeability of mitochondria to protons, thus reducing electrochemical potential & short- circuiting the anisotropic ATP synthase system, thus preventing ATP formation. • Ionophores, like valinomycin, help to pass potassium ions, whereas gramicidin induce the entry not only of potassium but also sodium & other monovalent cations through inner membrane, which prevent OP. Chemiosmotic hypothesis

Mitchell proposed chemiosmotic hypothesis in 1961, which was further elaborated in 1966. According to chemiosmotic hypothesis, ATP synthesis is coupled to electrochemical gradient which is created across inner mitochondrial membrane due to asymmetric distribution of protons across the membrane with protons accumulating in the inter membrane space. The electrochemical gradient results in proton motive force (PMF). Proton motive force is created due to: i) Chemical potential gradient created across the inner mitochondrial membrane due to difference in concentration of protons, i.e., Δ pH. ii) Voltage gradient across the inner mitochondrial membrane due to charge separation with positively charged ions accumulating in the intermembrane space, i.e., ΔE. Basic Requirements

• An Anisotropic(direction oriented) proton translocating respiratory chain • Coupling membrane ,which is impermeable except via diffusion exchange system • An anisotropic ATPase complex Mechanism

Electron transport occurs through various electron carriers of ETC present in inner mitochondrial membrane. The energy released during this electron transport is used in translocating protons across the inner mitochondrial membrane. The inner membrane of mitochondria is impermeable to protons. Energy of electrons is conserved in the form of differential distribution of protons across the membrane. As a result of accumulation of protons in the inter membrane space, a difference in pH and voltage across the inner mitochondrial membrane is created.. According to second law of thermodynamics, any differential distribution of ions is a source of energy because it will lead to increased entropy of the system. The storage of energy in this form is called electrochemical gradient Protons flow back in response to PMF through ATP synthase (also called Complex V), which is localized in the inner mitochondrial membrane. The energy of the protons flowing down the electrochemical potential is coupled with ATP synthesis. The relationship between the above said components of electrochemical gradient and PMF is expressed as: PMF = Δ E – 59 Δ pH (pHi - pHo) Where Δ E is the transmembrane electric potential gradient and Δ pH is pH difference between pH of the inside of mitochondria (pHi) and pH of the intermembrane space (pHo). Transmembrane pH difference of 1 unit at 250C is equivalent to a membrane potential of 59 mV. So, 59 is the proportionality constant value Molecular basis of phosphorylation: ATP synthase The structure of the ATP synthase

F 1 particle is the catalytic subunit; The F 0 particle attaches to F 1 and is embedded in the inner membrane. F 1 : 5 subunits in the ratio 3 a :3 b :1 g :1 d :1 e Electron Transport Chain

During electron transport, energy released is used to transport H+ across the inner mitochondrial membrane to create an electrochemical gradient

Protons complex I =4 , Complex III=4 ,Complex IV=2 . total =10 Fig. 16-19