Rate law for the linearisation of the incomplete cuboidal Fe3S4+ active site in aconitase at high pH

Abstract

The conversion of the inactive form of aconitase, with an incomplete cuboidal (Fe_ametal depleted) Fe₃S₄⁺ active site, to the linearised Fe(µ-S)₂Fe(µ-S)₂Fe⁺ form at high pH (9.5–10.6) has been studied kinetically. A uniphasic process requiring up to 4 h, with an [H⁺]^–2 rate-law dependence was indicated. Care is required in controlling the pH with a protein of such high molecular weight. In this work buffer concentrations of at least 30 mM were required to avoid pH variations during runs. Even at these concentrations initial pH changes of up to 0.08 were observed on mixing the protein with buffer. Rate constants were found to be independent of amounts of 3-cyclohexylaminopropane-1-sulfonic acid, glycine or the more ionic carbonate buffers. It was not possible to monitor the reverse process involving re-formation of the incomplete cube in the pH range studied.