Ferredoxins are small iron-sulfur cluster-containing proteins that participate in a wide range of chemical redox reactions. They are present in all domains of life and play an important role in electron transfer and cellular redox processes, acting as redox partners for multiple reactions (1). We hypothesize that novel ferredoxins play key roles in microbial bioenergetics, particularly in Archaea, where they participate in energy-converting pathways such as the reductive TCA cycle (2) and the reverse Embden-Meyerhof pathway (3). In this work, a new bioinformatic view of ferredoxin structure-function relationships will be presented, along with the latest efforts to identify and characterize new ferredoxin family members.
We employed Sequence Similarity Networks (SSN) and Genome Neighborhood Networks (4,5) to analyze the diversity of 2x[4Fe-4S] ferredoxins, using structurally and electrochemically characterized bacterial ferredoxins as references. Here we present progress on the recombinant overexpression of selected ferredoxins in E. coli, along with the biophysical characterization of the proteins by cyclic voltammetry, EPR spectroscopy, and X-ray crystallography to examine their redox properties and structural features. Our long-term goal is the development of a comprehensive database of ferredoxin properties.
SSN analysis presented here reveals numerous uncharacterized ferredoxins, despite the extensive historical study of these iron-sulfur cluster proteins. The bioinformatic relationships between these sub-families of sequences will be described, along with predictions regarding novel structural and/or functional roles.
This study provides a bioinformatic and experimental framework to uncover novel ferredoxins and their roles in microbial metabolism. By elucidating their structural and electrochemical properties, ferredoxins may pave the way to new biological processes that engage in useful transformations of carbon. A predictive model for ferredoxin functionality could revolutionize their use in synthetic biology and biotechnology.