Crossing between Normal and Inverted Potentials: Design Constraints and Principles for Tuning Between One-and-Two Electron Transfer Reactions

Abstract

Electron bifurcation is a multi-electron biological process that oxidizes a two-electron donor (either a flavin or quinone) by sending one electron thermodynamically uphill and one electron thermodynamically downhill. These electrons are then sent to various cofactors along branches where the electrons eventually enter some final reservoir pool (usually NAD+ or ferredoxin). Since electron bifurcation uses two electrons and a proton, understanding the mechanisms that allow bifurcation to perform is fundamental to synthetic bioinspired constructs for artificial energy conversion and transduction. Chapter 1 focuses on electron bifurcation, flavin chemistry, and general electron transfer theory.In Chapter 2, we formulate general constraints for tuning between one-and-two electron transfer. We find that adding more negative charges promotes potential inversion, thus by definition promoting “natural” two-electron transfer. I use the word “natural” because one could always add enough reducing power and force a second electron, but bifurcators have inverted potentials, meaning that the second electron is more reducing, so it is lost near immediately after the first electron with no extra effort. We also find that positive charges promote normally ordered potentials and that positive charges promote proton-coupled electron transfer processes. We also discuss implications for further experiments to be performed regarding the kinetics of the proton lost during bifurcation to further assess whether solvent or the protein environment (in particular, a neutral arginine residue that can accept a proton) will be the proton acceptor.In Chapter 3, we propose a project utilizing FldA, the first flavodoxin known to stabilize an anionic semiquinone, as a target to utilize our design constraints proposed in chapter 2. Our objective is to introduce residue mutations around the flavin cofactor to induce potential inversion, effectively attempting to reengineer the flavodoxin into a bifurcator. This approach leverages the fact that the initial electron transfer step in the FldA protein involves a proton-coupled electron transfer (PCET), a mechanism shared with electron bifurcation. As flavodoxins typically do not release a proton during their redox cycle, FldA presents an excellent candidate for redox potential modulation due to its inherent characteristics. The idea was to use established theories and methodologies for PCET and single-electron transfer to generate free energy curves and hopefully tune the flavodoxin to have inverted potentials. Unfortunately, the project and methods used were unsuccessful,so the chapter has been formulated as an idea that someone else could potentially take over, if they are interested. Chapter 4 focuses on comparing electron bifurcating enzymes that are known to bifurcate with both FMN (flavin mononucleotide) and FAD (flavin adenine nucleotide). We utilized the previously developed Pathways model to attain net decay couplings and then β values for all low-and-high potential branch cofactor pairs in all available experimen-tally determined bifurcating structures. We compare β values between the pathways and square barrier models (in the absence of a specifically determined prefactor for pathways calculations) to compare flux efficiencies between the two models in the Nfn1 bifurcating protein.Chapter 5 discusses my attempts at de novo flavoprotein and xenon-binding protein design. Through a collaboration at UCSF with Dr. William DeGrado and Dr. Samuel Mann, we tried to design flavoproteins and xenon binding proteins using traditional design methods. The flavin designs did not bind flavin and the xenon designs were never experimentally tested. However, this work represented a significant investment (about 2 years) and thus has been added into the thesis. Chapter 6 has concluding remarks as well as future directions for the work that is discussed in each chapter.