This complex mechanical art form can be viewed as a metaphor for the molecular apparatus underlying electron transport and ATP synthesis by oxidative phosphorylation. OXIDATIVE PHOSPHORYLATION
A PROTON GRADIENT POWERS THE SYNTHESIS OF ATP The transport of electrons from NADH or FADH2 to O2 via the electron-transport chain is exergonic process: NADH + ½O2 + H+ H2O + NAD+ FADH2+½O2 H2O + FAD+ Go’ = -52.6 kcal/mol for NADH -36.3 kcal/mol for FADH2 How this process is coupled to the synthesis of ATP (endergonic process)?ADP + Pi ATP + H2OGo’=+7.3 kcal/mol
The Chemiosmotic Theory • 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 • Intact inner mitochondrial membrane is required • 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
+ + + + + + - - - - Overview of oxidative phosphorylation 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 Pi.
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, Fo and F1(“knob-and-stalk”; “ball on a stick”) F1 contains the catalytic subunits where ADP and Pi are brought together for combination. F0spans the membraneand serves as a proton channel. Energy released by collapse of proton gradient is transmitted to the ATP synthesis.
F1contains 5 types of polypeptide chains -a3b3gde • Fo - a1b2c10-14(csubunits form cylindrical, membrane-bound base) • Fo and F1 are connected by agestalk and by exterior column (a1b2 andd) • The proton channel – between c ring and a subunit.
there are 3 active sites, one in eachbsubunit • c-e-gunit forms a “rotor” • a-b-d-a3b3unit is the “stator” • passage of protons through the Fo channel causes the rotor to spin • rotation of thegsubunit inside thea3b3hexamer causes domain movements in theb-subunits, opening and closing the active sites
Each b 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 Pi The affinity for ATP of T conformation is so high that it converts ADP and Pi into ATP.
Binding-Change Mechanism of ATP Synthase 1. ADP and Pi 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 Pi) 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 Pi form ATP in the tight site
Experimental observation of ATP synthase rotation • Fluorescent protein arm (actin) attached togsubunits • a3b3subunits bound to a glass plate • Arm seen rotating when ATP added (observed by microscopy)