Redox tuning of a de novo membrane cytochrome.

Abstract

Many proteins incorporate versatile redox cofactors, such as heme, in order to perform a broad range of different cellular functions, including vital bioenergetic processes such as respiration and photosynthesis. Hemoproteins have therefore been a key target for the design of artificial proteins, not only for use in synthetic applications but also to model and study these complex processes. A variety of hemoproteins have previously been designed based on a ubiquitous 4-helix bundle motif that is shared by critical transmembrane respiratory and photosynthetic complexes. In these natural proteins the bundle motif binds to two heme cofactors to facilitate electron transport. In designed proteins, this mutual fold can be approximated as two antiparallel coiled-coil dimers. Recently, this
approach was used to produce a new synthetic diheme membrane cytochrome known as CytbX. CytbX exhibited many of the properties of natural cytochromes, including the ability to engage in electron transport reactions with small molecules and other proteins. However, the reaction rates, and the potential applications of CytbX in a broader set of electron transport processes, are limited
by the particular redox potentials of the bound heme. This thesis describes attempts to expand the potential utility of CytbX by applying computational design and rational engineering to shift the redox potentials of the heme cofactors. The main focus concerns efforts to introduce His-Met heme ligation that were largely unsuccessful. However, this work led to the introduction of a new protein purification method that was highly beneficial, leading to a five-fold increase in the yield of purified protein. This thesis also clearly demonstrates the ’engineerability’ and overall robustness of the CytbX design, and suggests that this CytbX fold remains a suitable target for future applications in synthetic bioenergetic processes.

Date of Award	5 Dec 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Paul Curnow (Supervisor)