Light-Driven Electron Transport in a Synthetic Membrane Flavocytochrome

Abstract

Most biological energy originates from the light-harvesting processes of photosynthetic organisms,
which relies on the excitation of protein-bound chromophores and subsequent electron transport
across membranes to generate a proton gradient for ATP synthesis. Inspired by this natural
phenomenon, this thesis presents the design of a minimal, synthetic light-activated transmembrane
redox protein. The design is based around the de novo membrane cytochrome known as CytbX, which
binds two b-type hemes within a four-helix bundle architecture. Here, photoactivity is engineered into
CytbX using the F1 flavin-tag system. This introduces a short peptide tag sequence that enables the
covalent attachment of flavin mononucleotide (FMN), a redox-active photosensitiser, via enzymatic
flavinylation catalysed by AbpE. Four CytbXF1 variants were generated by inserting the F1 tag
sequence at distinct topological positions. In each case, the structural feasibility of tag insertion was
assessed using AlphaFold3 and Chai-1, and backbone stability and FMN–heme distances evaluated
via molecular dynamics simulations. The constructs were successfully expressed, purified, and
flavinylated in vitro and with preliminary success in vivo. Photoreduction experiments demonstrated
light-induced flavin→heme electron transfer in detergent micelles and proteoliposomes, as well as
transmembrane electron transport from the attached flavin to a diffusible electron acceptor
(flavin→heme→ferricyanide). This work represents the first use of the F1 tag system in a synthetic
membrane protein and provides a foundational platform for engineering modular light-active
transmembrane electron transport systems. These findings contribute to further advancing the field of
synthetic bioenergetic protein design and offer a step towards harnessing the power of photosynthetic
systems for bioengineering.

Date of Award	20 Jan 2026
Original language	English
Awarding Institution	University of Bristol
Supervisor	Paul Curnow (Supervisor) & J L R Anderson (Supervisor)