4] Measuring nitric oxide and superoxide: Rate constants for aconitase reactivity

Abstract

This chapter discusses the sensitive measurement of intracellular oxidant concentrations. The usefulness of measuring aconitase activity, the role of the disassembly/reassembly of the [4Fe- 4S] cluster of the cytosolic aconitase in iron metabolism suggests a regulatory function for the oxidizing metabolites formed inside cells. The citric acid cycle enzyme aconitase is a dehydratase that catalyzes the reversible interconversion of citrate to isocitrate through the intermediate cis -aconitate. The active enzyme contains a cubane [4Fe-4S] 2+ cluster. Cytosolic aconitase is identical to the iron-responsive element-binding protein (IREBP), which regulates the biosynthesis of ferritin and the transferrin receptor by binding to untranslated regions of the messenger RNAs (mRNAs) for these proteins. The IRE-BP has aconitase activity when its [4Fe-4S] cluster is intact, and acquires mRNA-binding activity, with a concomitant loss of aconitase activity, when the iron-sulfur cluster is disassembled. Aconitase activity can be measured spectrophotometrically as the formation of cis -aconitate from isocitrate, by following the increase in absorbance at 240 nm. If the rate constants for inactivation and reactivation are known, intracellular O 2 – concentrations can be estimated using determined aconitase activities.