Current status of the chemiosmotic hypothesis.
The basic postulates that Peter Mitchell proposed back in 1961 have all been well established. To that extent, the hypothesis is now generally accepted, and this was recognized in the award of a Nobel Prize for Chemistry in 1978. Most research in the field is now concerned with molecular mechanism, - how do the various enzymes of the electron transfer chains, ATP synthase, photochemical reaction centers, transport carriers, etc., work, and how are they coupled to proton pumping.
Structure for four key enzymes have now been solved by X-ray crystallography; photochemical reaction centers (in several variants), cytochrome oxidase (bacterial and mitochondrial enzymes), the mitochondrial bc1 complex (from several sources), and F1 ATP-ase. The first three structures provide a nice vindication of the general principles of chemiosmotic coupling as applied to electron transfer; - they all function as Mitchellian proton pumps.
We might add to this list, the structure of bacteriorhodopsin, which acts as a direct proton pump in which the conformational change associated with photoisomerization of retinaldehyde is coupled to the movement of a H+ between an input channel and an output channel that connect the reaction site to aqueous phases on opposite sides of the membrane. The pK changes associated with the photocycle effectively transduce the light energy into a proton gradient.
- The reaction center is just as Mitchell expected. Electrons are passed across the membrane in the photochemical reaction, providing an electrogenic arm, and reduce a H-carrier, ubiquinone.
- The bc1 complex also functions as a Mitchellian proton pump, with an electrogenic arm associated with electron transfer across the membrane through the cyt b chain, and oxidation and reduction of H-carriers at either end of the chain; the detailed mechanism (the modified Q-cycle) is a little trickier than what Mitchell had guessed at in his earliest work, but is based on a later hypothesis, the original Q-cycle, also suggested by Mitchell.
- In cytochrome oxidase, the mechanism of coupling of electron transfer to proton pumping is still not completely clear. It's well established that the stoichiometry is higher than Mitchell had expected, due to a mechanism that pumps 1 H+/e- in excess of the H+ taken up on reduction of water. However, all speculation about the mechanism is in the context of a more or less direct coupling to the electron transfer reactions occuring at the catalytic core of the enzyme.
- The F1 ATP-ase is a wonderful little device, and clearly also operates through coupling to the proton gradient. The earlier structures were missing the bits that provide the coupling mechanism, but recent structures show the Fo, with a structure similar to that anticipated. The rotational mechanism that most workers now accept has evolved from the ideas of Paul Boyer, which Mitchell originally strongly opposed. However, the mechanism accomplishes the same job that Mitchell wanted the enzyme to do.
So what is still to be understood? At the mechanistic level, these devices are all exquisite little machines, and much of the detail is still to be worked out. There are still several major enzymes for which the mechanism is not understood, - most important, the NADH:ubiquinone oxidoreductase. But most of our ignorance now lies in areas of higher organization: How are the complexes organized in the membrane? What pathway does the H+ follow between complexes? Are there special devices for channelling protons (these have been identified in individual enzymes, but at present most workers believe that between enzymes, the protons flow through the water of the aqueous phases). How does the higher order structure (for example, the organization of the chloroplast into grana and stromal lamellae) affect the fluxes? Another area of active work is in the development of physico-chemical descriptions of the coupling of fluxes (various non-equilibrium thermodynamics treatments).
Antony Crofts, University of Illinois at Urbana-Champaign,