Metal-based redox catalysts that efficiently convert energy to or from stored fuel is a key part of the transition to sustainable global energy. Metal-substituted heme proteins have been shown to perform rapid oxygen or proton reduction, demonstrating their potential towards redox catalysis, but their initial exploration has largely been through synthetic modification of heme proteins. The biosynthetic generation of a substituted holocytochrome c would be a first step towards efficient generation of proteins with these metal-substituted hemes. Holocytochrome c is a protein that has a covalently attached heme group at a CXXCH amino acid motif and performs electron transfer in bacteria as well as in the mitochondrial electron transport chain. A truncated from of cobalt-substituted holocytochrome c, unlike its naturally occurring iron-heme containing counterpart, can catalyze the reduction of water into diatomic hydrogen. To synthesize a large quantity of the desired coenzyme, the cobalt-substitution has previously been performed synthetically after recombinant expression of the iron-heme protein in E. coli. Cytochrome c production in E. coli requires co-expression of a cytochrome c heme lyase, the naturally existing enzyme is responsible for heme attachment to form holocytochrome c. In this project expression vectors for cytochrome c and its heme lyase have been modified to express axial ligand mutants of cytochrome c as well as mutants of heme lyase with decreased substrate -specificity for the heme attachment motif. The cytochrome c and heme lyase mutants, upon purification, will be quantitatively analyzed to find the tolerance of the mutated heme lyase towards heme attachment motifs within cytochrome c and metal-ion substitution within the heme substrate. Success would represent a step towards the goal of providing a method to cost-effectively generate a biocatalyst that could aid in efficient production of fuels from sustainable energy sources utilizing a modification to the heme protein biosynthesis pathway.

Creation of mutant holocytochrome c expression vectors in E. coli

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.