13. ATP synthase was first identified by dissociation and reconstitution studies abundant knoblike protruding structures were observed on the matrix side of the inner mitochondrial membrane by EM (Racker in 1960) The inside-out submitochondrial particles with the knobs are capable of both electron transferring and aTP synthesis When the protruding Fi part was removed by agitation, electron transferring could still occur, but neither proton gradient nor ATP are produced
13. ATP synthase was first identified by dissociation and reconstitution studies • Abundant knoblike protruding structures were observed on the matrix side of the inner mitochondrial membrane by EM (Racker in 1960). • The inside-out submitochondrial particles with the “knobs” are capable of both electron transferring and ATP synthesis. • When the protruding F1 part was removed by agitation, electron transferring could still occur, but neither proton gradient nor ATP are produced
ATP synthesis reappeared when FI was reconstituted back(the solubilized F, alone can catalyze ATP hydrolysis, thus was originally named as F,ATPase) f was the first essential factor identified for oxidative phosphorylation
• ATP synthesis reappeared when F1 was reconstituted back (the solubilized F1 alone can catalyze ATP hydrolysis, thus was originally named as F1ATPase). • F1 was the first essential factor identified for oxidative phosphorylation
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b Intact mitochondrion membrane esicles, the inner membrane is inverted the vesicle contain the electron carriers, are able to carry out el transfer to O but nnot phosphorylate ADP reconstitution The reconstituted to electron transfer
14. Isotope exchange experiments revealed that the G for AtP synthesis on purified F, is close to zero. When solubilized f(act as a ATPase)was incubated with aTP in the presence of O-labeled H,O, three or four 8O atoms were incorporated into the Pi, indicating that aTP formation/hydrolysis are readily reversible and multiple rounds of aTP formation and hydrolysis occurred on the enzyme Measurement of Kd values: ATP has a much higher affinity than adp to the enzyme(10-12 M vs 10-5 M) The proton gradient was thus proposed to drive the release of ATP from the enzyme surface
14. Isotope exchange experiments revealed that the G`0 for ATP synthesis on purified F1 is close to zero! • When solubilized F1 (act as a ATPase) was incubated with ATP in the presence of 18O-labeled H2O, three or four 18O atoms were incorporated into the Pi , indicating that ATP formation/hydrolysis are readily reversible and multiple rounds of ATP formation and hydrolysis occurred on the enzyme! • Measurement of Kd values: ATP has a much higher affinity than ADP to the enzyme (10-12 M vs 10–5 M). • The proton gradient was thus proposed to drive the release of ATP from the enzyme surface
The 180 exchange experiment ATP+HoO The AGo for AtP Readily rever sibl reactions lr I I synthesis on purified ADP Fi is close to zero 18 O (Paul Boyer) 18O-P=180 O Enzyme (F1)
The 18O exchange experiment : The G` 0 for ATP synthesis on purified F1 is close to zero! (Paul Boyer) Readily reversible reactions Keq = 2.4 G`0 = 0
80 60 P ADP+Pi ATP 40 ES [E·ATP] (in solution) 20 E·ADP+P1 长 0 E+S Reaction coordinate ypical ATP synthase enzyme Release of afp from atP synthase was proposed to be the major energy barrier for ATP synthesis
Release of ATP from ATP synthase was proposed to be the major energy barrier for ATP synthesis
15. ATP synthase comprises a proton channel (fo and a atPase (fu The Fi part consists of nine subunits of five types C3B368. The knoblike Fi portion is a hexamer of alternating a and B subunits(arranged like the segments of an orange), which sits atop the single rod-shaped y subunit The Fo portion consists three types of subunits ab 210-12 The c subunits, each forming two transmembrane helices, form a donut-shaped ring in the plane of the membrane
15. ATP synthase comprises a proton channel (Fo ) and a ATPase (F1 ) • The F1 part consists of nine subunits of five types: a3b3 gde. • The knoblike F1 portion is a hexamer of alternating a and b subunits (arranged like the segments of an orange), which sits atop the single rod-shaped g subunit. • The Fo portion consists three types of subunits: ab2 c10-12. • The c subunits, each forming two transmembrane helices, form a donut-shaped ring in the plane of the membrane
The leg-and-foot-shaped yE subunits stands firmly on the ring of c subunits The two b subunits of Fo seem to connect to the aB hexamer via the s subunit of f The proton channel is believed to lie between the a subunit and the ring of c subunits X-ray crystallography revealed that the three B subunits of f assumes three different conformations with bound ADP, ATP analog, or empty respectively (John Walker, 1994, Nature, 370: 621-628)
• The leg-and-foot-shaped ge subunits stands firmly on the ring of c subunits. • The two b subunits of Fo seem to connect to the ab hexamer via the d subunit of F1 . • The proton channel is believed to lie between the a subunit and the ring of c subunits. • X-ray crystallography revealed that the three b subunits of F1 assumes three different conformations, with bound ADP, ATP analog, or empty respectively (John Walker, 1994, Nature, 370:621-628)!
The ten c subunits of f The yeast FoF structure) The y subunit of F The ATP synthase I comprises a proton adkrdasaisn channel (f ) and a t ATPase(F1) (n H+
The ATP synthase comprises a proton channel (Fo ) and a ATPase (F1 ) The ten c subunits of Fo The g subunit of F1 a b (The yeast FoF1 structure)