OXIDATIVE PHOSPHORYLATION This complex mechanical art form can be viewed as a metaphor for the molecular apparatus underlying electron transport and ATP synthesis by oxidative phosphorylation.

A PROTON GRADIENT POWERS THE SYNTHESIS OF ATP The transport of electrons from NADH or FADH 2 to O 2 via the electron-transport chain is exergonic process: NADH + ½O 2 + H +  H 2 O + NAD + FADH 2 + ½O 2  H 2 O + FAD +  G o ’ = kcal/mol for NADH kcal/mol for FADH 2 How this process is coupled to the synthesis of ATP (endergonic process)? ADP + P i  ATP + H 2 O  G o ’=+7.3 kcal/mol

Proposed by Peter Mitchell in the 1960’s (Nobel Prize, 1978) Chemiosmotic theory: electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane Mitchell’s postulates for chemiosmotic theory 1.Intact inner mitochondrial membrane is required 2.Electron transport through the ETC generates a proton gradient 3. ATP synthase catalyzes the phosphorylation of ADP in a reaction driven by movement of H + across the inner membrane into the matrix The Chemiosmotic Theory

As electrons flow through complexes of ETC, protons are translocated from matrix into the intermembrane space. The free energy stored in the proton concentration gradient is tapped as protons reenter the matrix via ATP synthase. As result ATP is formed from ADP and P i Overview of oxidative phosphorylation

An artificial system demonstrating the basic principle of the chemiosmotic hypothesis Synthetic vesicles contains bacteriorhodopsin and mitochondrial ATP synthase. Bacteriorhodopsin - protein that pumps protons when illuminated. When the vesicle is exposed to light, ATP is formed.

ATP Synthase Two units, F o and F 1 (“knob-and- stalk”; “ball on a stick”) F 1 contains the catalytic subunits where ADP and P i are brought together for combination. F 0 spans the membrane and serves as a proton channel. Energy released by collapse of proton gradient is transmitted to the ATP synthesis.

F 1 contains 5 types of polypeptide chains -  3  3  F o - a 1 b 2 c (c subunits form cylindrical, membrane- bound base) F o and F 1 are connected by a  stalk and by exterior column (a 1 b 2 and  The proton channel – between c ring and a subunit.

there are 3 active sites, one in each  subunit c-  unit forms a “rotor” a-b-  -  3  3 unit is the “stator” passage of protons through the F o channel causes the rotor to spin rotation of the  subunit inside the  3  3 hexamer causes domain movements in the  - subunits, opening and closing the active sites

Each  subunit contains the catalytic site. At any given time, each site is in different conformation: open (O), loose (L) or tight (T). O conformation binds ADP and P i The affinity for ATP of T conformation is so high that it converts ADP and P i into ATP.

1. ADP and P i bind to an open site 2. Passage of protons causes each of three sites to change conformation. 3. The open conformation (containing the newly bound ADP and P i ) becomes a loose site. The loose site filled with ADP and Pi becomes a tight site. The ATP containing tight site becomes an open site. 4. ATP released from open site, ADP and P i form ATP in the tight site Binding-Change Mechanism of ATP Synthase

Experimental observation of ATP synthase rotation Fluorescent protein arm (actin) attached to  subunits      subunits bound to a glass plate Arm seen rotating when ATP added (observed by microscopy)