Chapter 42 Techniques to Isolate O2-Sensitive Proteins. [4Fe-4S]-FNR as an Example

Abstract

Many key enzymes in biological redox reactions require metal centers or cofactors for optimum activity and function. While the metal centers provide unique properties for protein structure and function, some also render protein activity sensitive to environmental O2 and cause experimental challenges to isolation and biochemical analysis. Iron-sulfur (Fe-S) clusters represent an important class of such metal centers and Fe-S proteins are widely distributed in nature. Here, we utilize FNR, a regulatory Fe-S protein from Escherichia coli, as an example to describe the techniques essential to purifying O2-labile proteins and summarize various approaches for their biochemical analysis. These methods can be readily adapted to purify other O2-labile proteins and advance our understanding of this interesting class of proteins. © 2009 Elsevier Inc. All rights reserved.