ENGINEERED REDOX SWITCH FOR CONTROLLED MOLECULAR COMPOSITION OF PHENAZINES

Abstract

Biological systems communicate through chemically encoded molecules, whereas electronic systems largely use electron flow (currents) for communication, creating a fundamental barrier to continuous information flow between the two. This dissertation addresses this gap by exploiting redox chemistry, the addition or removal of electrons, as a shared communication modality between biological components like proteins and components like electrodes. In particular, we use phenazine composition as a redox-sensitive messenger that can be written by biology and read by electronics. In the Pseudomonas aeruginosa phenazine pathways, phenazine-1-carboxylic acid (PCA) is a critical junction molecule that can be converted into pyocyanin (PYO) or 1-hydroxyphenazine (1-OHPHZ). This branching point provides an opportunity to switch the metabolic flux by controlling the enzymatic steps required to produce either phenazine. First, to investigate redox control of these pathways, we examined the kinetic mechanism of PhzM, an S-adenosylmethionine (SAM)-dependent methyltransferase in the PYO biosynthesis pathway that commits PCA to PYO. Multisubstrate kinetics and product inhibition revealed that the enzyme must undergo a compulsory order bi-bi mechanism, with SAM binding before PCA. Bioinformatic analysis, structural modeling, and alanine scanning identified the residues involved in substrate recognition. Furthermore, we confirmed the previously proposed transient interaction between the two terminal PYO synthases, PhzM and PhzS, using crosslinking label transfer. Next, we introduced the potential for a redox-mediated disulfide bond into the structure of PhzM and assessed its redox-switching ability. A library of intra-chain cysteine pairs was screened in E. coli strains with defined cytoplasmic redox environments. A single variant-A116C/G123C-showed particularly promising switching potential with similar activity to wildtype when reduced and substantially less activity when oxidized. Molecular dynamics and circular dichroism spectroscopy suggest that oxidation of the variant’s cysteine thiols perturbs the local structure and disrupts the residues required for substrate orientation. Lastly, we attempted to demonstrate the redox-induced phenazine pathway redirection of A116C/G123C PhzM by chemically manipulating the cytoplasmic redox conditions in vivo. We combined electrochemical methods with the chemical oxidants and remarkably, found profound in phenazine composition in response to oxidizing inputs. This transduction of chemical perturbation into electronic information can be suitable for interpretation using microelectronic circuits.