PARENT SESSION
Posters P8D Artificial photosynthesis and biological hydrogen production. Abstracts (759-769)

Conversion of cytochrome b₅₆₂ into a photoactive quinone-binding protein. Sam Hay^*,1, Tom Wydrzynski¹, ¹ Research School of Biological Sciences, Canberra, Australia

ABSTRACT- The primary photosynthetic event in green algae, plants and some photosynthetic bacteria involves a one-electron transfer from a light-activated chlorin complex to a bound quinone molecule. Through protein engineering, we have been able to modify the E. coli cytochrome b562 to mimic this process. First a unique quinone binding site was engineered into the cytochrome by introducing a cysteine within the hydrophobic interior of the protein. Various quinones, such as para-benzoquinone and ubiquinone₀, were then covalently attached to the protein through a cysteine sulphur addition reaction to the quinone ring which binds and reduces the quinone. The cysteine placement was designed to bind the quinone about 10 Angstroms from the edge of the bound porphyrin. To our knowledge this is the first report of a synthetic quinone-binding protein. Fluorescence measurements confirmed that the bound hydroquinone is incorporated towards the proteins hydrophobic interior and is partially solvent-shielded. The bound quinones remain redox-active and can be oxidised and re-reduced. The semiquinone can be generated by a 1-electron reduction at high pH and by binding different quinones to the protein we can modulate this midpoint by >200 mV. We have also made several protein mutants which modulate the redox properties of the bound quinone through specific interactions with the protein. The heme binding site of the modified cytochrome b562 was then reconstituted with a light-activate zinc-protoporphyrin or zinc-chlorin. By using both transient EPR and fast optical techniques, we show that in the variously modified proteins light-induced electron transfer can occur from the porphyrin to the bound oxidised quinone but not the hydroquinone. The electron transfer rates in the chlorin/protein/quinone complexes are in the order of about 10⁸ s^-1 which are comparable to rates in the natural reaction centres. Thus, this system is the first simple and functional protein-based model of a reaction centre.

KEY WORDS: light-induced electron transfer, porphyrin, synthetic quinone-binding protein, artificial reaction centre