A Fluorescent Readout for the Oxidation State of Electron Transporting Proteins in Cell Free Settings

Pathways involving sequential electron transfer between multiple proteins are ubiquitous in nature. Here, we demonstrate a new class of fluorescent protein-based reporters for monitoring electron transport through such multistage cascades, specifically those involving ferredoxin-like electron transporters. We created protein fusions between mammalian Adrenodoxin (Adx) and plant Ferredoxin (Fdx) with fluorescent proteins of different colors and found that the fluorescence of such fusions is highly sensitive to the redox state of the electron transporter. The increase in fluorescence from the oxidized to the reduced state was inversely proportional to the linker length between the fusion partners. We first used our approach to quantitatively characterize electron transfer from NADPH through Adrenodoxin Reductase (AdR) to Adrenodoxin (Adx). Our data allowed us to build a detailed mathematical model of this mitochondrial electron transfer chain and validate previously proposed mechanisms. Then, we showed that an Adx-GFP fusion could serve as a sensor for the activity of bacterial Type I Cytochrome P450s (CYPs), a very large class of enzymes with important roles in biotechnology. We further showed that fluorescence of a direct fusion between CYP and GFP was sensitive to CYP activity, suggesting that our approach is applicable to an even broader class of proteins, which undergo a redox state change during their work cycle.