Biophysics

bc1 complex

FRET

Only occurs between 1-10nm

Non-radiative energy transfer

The emission and absorption spectra of the donor and acceptor chromophore must overlap

Depends on the angle between the two molecules

Greater than 10nm effects usually ignored by the theory of FRET become more prominent

At distances less than 1nm complex formation is more likely and the Ideal Dipole Approximation (IDA), that FRET is based on, breaks down

Theory shows that the FRET efficiency is proportional to the sixth power of the distance between the two molecules

Used to measure protein-protein interactions

R0 is the distance at which the FRET efficiency is 50% and ranges between 2-7nm

ATPsynthase :

Attached fluorescent dyes to ATP synthase molecule, via sulphide bonds, on the gamma domain and the b2 domain

Reconstituted into liposome

Cannot use fluorescent proteins because they are too big

Distance between the donor dye and acceptor dye will vary as the protein rotates during ATP synthesis

Should produce 3 distinct FRET efficiency values

Single liposome with single ATP synthase

Liposome moves slowly (several 100ms), so spends longer in the detection volume of a laser confocal microscope.

Bursts of emission detected

In hydrolysis conditions (adding ATP) ATP synthase turned in one direction (1>2>3>1) - measured ratio of donor fluorescence over acceptor fluorescence - produced three distinct levels of FRET efficiency

ATP synthesis conditions () protein turned in the opposite direction

Experiments showed that the transition between the different conformational states was very fast - sub ms

Bifurcated mechanism

Oxidation of quinone derivative (QH2) at Q0 site

cytochrome c2 complex

four transmembrane cytochrome b

BH (high potential) b-type haem

BL (low potential) b-type haem

Rieske domain 2Fe2S

First electron transferred to Fe-S and then cytochrome c2

second electron transferred to BL then BH and then to the Q1 site where it reduces a ubiquinone (Q) to form a semi-quinone radical (SQ)

Process repeated to produce QH2 at the Q1 site

Two consecutive turnovers of the enzyme

Not understood why the second electron does not also follow the energetically stable pathway

2H+ released on one side of the membrane per e- for every QH2 oxidised

However, this must happen or no charge is translocated across the membrane so electric potential is formed which would be energetically disastrous

Many mechanisms proposed over the years

'Double occupancy' model

Transfer of the second electron to the BL haem can be attributed to the instability of the SQ intermediate formed after the transfer of the first electron to the 2Fe2S. This is aided by a second Q molecule at the Q0 site that acts as an immediate acceptor of the second electron.

'Proton-gated charge transfer' model

Also envisages a second Q molecule at the Q0 site, but also a conformation change (catalytic switch) that prevents the Fe2S2 accepting the second electron

However, none of the models mechanistically correlate with the experimental data, so conformational changes at the Q0 site have also been suggested

Various X-ray crystal structures have been solved with the 2Fe2S either near the cytochrome c1 (c1 position), in the b position (intermediate position) or at the Q0 site

None of these structures satisfy the spectroscopic and kinetic data for cytochrome bc1

At the b position the Fe2S2 cluster is too far away from the cytochrome c1 to allow for the fast electron transfer observed in the kinetics.

At the c1 position the Fe2S2 cluster is too far away from Q0 site to produce the close interaction observed by electron paramagnetic resonance (EPR) spectroscopy

Evidence for 2Fe2S motion

crystallisation studies using inhibitors that mimic reaction intermediate showed that the 2Fe2S domain remains in a fixed position at the Q0 site

Use of other inhibitors that do no directly interact with the 2Fe2S, but displace QH2 show that the reisk domain is released from it's fixed position

In different structures the anchor of the FeS domain remains fixed meaning that conformational changes of the flexible region (acts as a hinge) linking the anchor to the cluster domain are the reason for the rotation of the FeS cluster.