3 H+

ADP + Pi ATP + HOH

F1

F0

ATP synthesis by the membrane bound proton turbine ATP synthase

2 e-

3 OH-

Proton pump, same efficiency in ATP synthesis and hydrolysis

Coupling membrane conserve the H+ formed by pumping of H+ by the electron transport chain in the membrane during oxidation. H+ is used for ATP synthesis by the pump.

Mechano-electric process of the turbine convert H+ to chemical bond.

3H+/ATP , Mg++, cofactor, no acylphosphate intermediate.

F1- catalytic sector, 5 subunits: 3, 400 kD

F0 - membrane sector, 3 subunits: a, 2b, 9c, 150 kD

6 ATP binding site one on each of the and subunits

The 3 active sites are on the 3 subunits

MF1 (DCCD)red

yellowblue

: magenta green

1E79 (pdb)

EF1Focyan pink

MF1 red, EF1 green: yellow c orange

AMPPNP Thr Mg HOH Glu

O O O O- O O O-

AR-O-P-O-P-O- P-OH AR-O-P-O-P-O-P-O-

O- O- O- H O- O- O- OH H

Mg++ O O Mg++ O O

C Glu 188 C Glu 188

ADP Pi Pentacovalent intermediate.

O O O-

ATP AR-O-P-O-P-O-P-O-

O- O- O- H-O-H

Mg++ O O-

C Glu 188

Mode of action of catalysis

Mg++ induces more positive P

Nucleophylic attack by O- of ADP on the electrophyl P in Pi

Formation of pentacovalent P as a transition state intermediate in the formation of ATP

OH- leaving group facilitated by general acid/base catalysis of Glu188

ADP-VO5 transition state inhibitor

F1-bound ADP-VO5 is trigonal bipyramide pentacoordinate vanadium

Km(Pi) 500 M

Km(Vi) 500 M

Ki(ADPVi) 12.4 M

Kd(ADPVi) 0.5 nM

Transition state inhibitor: AMPP.VO5 bound to Mg.Thr.Ser

A

DP Pi

ATP

ADP Pi

AD

P Pi

ATP

ATP

AD

P PiAD

P Pi

ATP

AD

P Pi

ADP Pi

H+

HOH

ATP

Binding-Change mechanism

Binding-Change rotation mechanism

Each site sequentially participate in catalysis, 3 strokes for a full cycle

Negative cooperativity in ATP binding between the 3 sites transmitted by the subunit

ATP tightly bind to one site. Keq = 1 of tightly bound ATP - ADP Pi

Therefore energy is not needed for formation of unhydride bond in the formation of ATP

Energy in a form of conformational change is needed for release of tightly bound products

H is transmitted indirectly by the subunit. It forms a shaft that is connected to the turbine in the membrane causing conformational changes in the three active sites during rotation.

Evidence for the hypothesis

Single site KmATP =1.5x10-12 M, V=1x10-3 s-1, G= 0 Kcal/mole

3 sites KmATP =1.0x10-3 M, V=1x103 s-1, G= -12 Kcal/mole

Km- low for single site because of tight binding of ATP but high to three sites due to negative cooperativty

V- slow in single site mode because of slow release of ADP but fast in 3 sites mode

Keq=1 from analysis of the concentrations of bound ATP, ADP and Pi at a single site

Asymmetry in structure of the three active sites as seen from crystallography, empty, bound ATP and bound ADP

Different interactions between and each of the three subunites

ATP Synthase1. F1 catalytic sector

2. Fo membrane proton turbine

1bmf (PDB)

E (empty) TP (bound ATP)

DP:- active site subunit +ADP subunit

E188

HOH

T163Mg

ATP

active site

FoF1

Fo

Helixes of subunit C in Fo

Proton turbine

Rotor- 9 c subunits arranged in a ring, each has two transmembrane hilexes single an essential Asp61 in the middle of the membrane

Stator- one a subunit in the membrane, an essential Agr210, is attached to F1 by two extended b subunits.

Shaft- subunit attached to c with the aid of d and e, extended through a tunnel opened in the middle of F1. Transmit the rotation of the turbine to the catalytic subunits causing conformational changes.

Arg 210 in a subunit has permanent + charge (high pKa) located between two Asp61 in c.

Synthesis: H+ come through the half channel protonat unchanged Asp61-

The protonated AspH is repulsed by Arg+ counter clockwise into the lipid.

Protonated AspH approach Arg+. The + charge lower the pKa and deprotonate Asp. The proton move out through the half channel.

Asp61Arg+210

Rotation of subunit

Rotation of subunit

Rotation

Histage engineered at the n-terminal of subunits in complex with and attached to glass slide coated with Ni-nitriloacetate.

At the tip -S107C mutated, linked to biotin-maleimide, avidin attach to C107 and flourescence biotintelated 2 m actin filament is attached to the avidin on the .

Movement recorded by CCD vidio camera only when ATP added, NaN3 inhibited.

F1 10 nm

1 nm

ATPase rate 52 s-1 (17 RPS)

Rotaion 4 RPS (load)

Torque 40 pN nm-1

Nickel dots array. Two methods, electron beam (e-beam) and nanoimprint lithography, were used to create nanofabricated substrates capable of precisely and accurately positioning individual biological motors. In the first method, a standard silicon wafer was coated with two layers of electron beam-sensitive polymer poly(methyl methacrylate) (PMMA) resist, with the lower layer being slightly more sensitive to the e-beam exposure. Arrays were patterned into the bilayer using e-beam lithography, followed by development. A 5 nm tantalum (Ta) adhesion layer and a 10 nm metallic nickel (Ni2+) layer were deposited using electron-beam physical vapor deposition. Lift-off was performed using a 1:1 solution of methylene chloride:acetone for 15 min followed by ultrasonic agitation for 10 s. Regular arrays of 60-600 nm size dots on 1 m pitch with circular profiles were confirmed by scanning electron microscopy. Arrays were stored under appropriate

conditions (e.g., vacuum) to prevent oxidation of the Ni surface prior to experimental use.

Figure 1 Schematic diagram (A) and CCD photomicrograph (B) of fluorescent microspheres attached to the subunit of individual F1-ATPase molecules immobilized on nickel dot arrays(C) that were created using electron beam lithography. Dots are 50-250 nm diameter, 5-15 nm high.

Fig. 1. Schematic diagram of the F1-ATPase biomolecular motor-powered nanomechanical device. The device consisted of (A) a Ni post (height 200 nm, diameter 80 nm), (B) the F1-ATPase biomolecular motor, and (C) a nanopropeller (length 750 to 1400 nm, diameter 150 nm). The device (D) was assembled using sequential additions of individual components and differential attachment chemistries.

Nickel propellers were made by lithography. The Ni was coated by biotinylated His rich peptide. The biotin attached to avidin connected to biotinylated subunit of F1. His tag F1 was attached to Ni posts on the slide.

Fig. 2. Image sequence (viewed left to right) of nanopropellers being rotated anticlockwise at 8.3 rps (A) and 7.7 rps (B) by the F1-ATPase biomolecular motor. Observations were made using 100x oil immersion or 60x water immersion and were captured with a CCD video camera (frame rate 30 Hz). The rotational velocity ranged from ~0.8 to 8.3 rps, depending on propeller length. Data were recorded for up to 30 min; however, propellers rotated for almost 2.5 hours while ATP was maintained in the flow cell

Fig. 3. Time course of F1-ATPase subunit rotation. Each line represents data from a rotating nanopropeller. Solid lines, propellers 750 nm long; dashed lines, propellers 1400 nm long; dotted lines, propellers 1400 nm long in the presence of NaN3