Light-harvesting bioconjugates as chloroplast mimics

Abstract

Cells are highly complex bio-nanoreactors comprised of a complex medium where multiple multistep reactions occur simultaneously across the cell. To prevent interference and degradation of these catalytic pathways, cells compartmentalise. Compartmentalisation achieves control of synthetic pathways at specific sites in the cell (organelles). In nature, compartmentalisation is demonstrated in plants by the organelle, chloroplast, which is responsible for photosynthesis. Photosynthesis is one of the most important biological reactions, responsible for sustaining life.

In this thesis, the development of light harvesting bioconjugates as artificial chloroplasts is reported. Donor-acceptor light activated bioconjugates based on ruthenium(II)-bisterpyridine complexes (Ru(II)) is covalently linked to a redox metalloprotein cytochrome c (cyt c), which was prepared using maleimide-cysteine chemistry. These bioconjugates are capable of undergoing electron transfer upon photoactivation using ~480 nm light.

In order to compartmentalise the Ru(II)-cyt c bioconjugates to construct artificial organelles, polymersomes (polymer vesicles) were formed based on a novel method developed in this project based on the polyelectrolyte, polystyrene-b-poly(acrylic acid) (PS-b-PAA). Polymersomes could be induced in the presence of positively charged biomolecules including cyt c and green fluorescent protein. In the presence of simple salts or negatively charged biomolecules such as calmodulin, micelles form.

A primitive synthetic chloroplast capable of converting light energy into chemical energy by generating a proton gradient upon photo-excitation was developed. An artificial photosynthetic-respiratory hybrid was achieved by replacing chlorophyll (photosensitiser) with Ru(II)-cyt c. This light-harvesting bioconjugate as well as its natural electron acceptor, cytochrome c oxidase, is encapsulated in the synthetic polymersome membrane of the diblock copolymer PS-b-PAA, reminiscent of a photosynthetic membrane construct. The successful self-assembly of this complex light-harvesting enzyme cascade within membranes of polymeric vesicles were characterised using confocal laser-scanning microscopy (CLSM), TEM and cryo-TEM. Upon light-activation, cytochrome c oxidase (reduced) is capable of pumping protons across the membrane to generate an electrochemical gradient which is detected using a fluorescent pH dye encapsulated within the aqueous core of the polymersome.