Purification of three iron-sulfur proteins from the iron-protein fragment of mitochondrial NADH-ubiquinone oxidoreductase

Abstract

A fragment containing non-heme Fe and acid-labile sulfide but little flavin can be solubilized from the [bovine heart] mitochondrial NADH-ubiquinone oxidoreductase complex with chaotropic agents. This Fe-protein fragment has was resolved with detergents and ammonium sulfate fractionation into Fe and aicd-labile sulfide containing fractions, here called ISP-I and ISP-(II + III). ISP-I consists predominantly of a single polypeptide of MW 75,000 ISP-(II + III) consists predominantly of 3 polypeptides in equimolar concentrations with MW of 49,000, 30,000, and 13,000. Treatment of the latter with sodium trichloroiracetate followed by ammonium sulfate fractionation results in separation of the 49,000 MW polypeptide from the 2 smaller subunits. Both of these subfractions (ISP-II and ISP-III, respectively) contain non-heme Fe. The 3 iron-sulfur proteins were characterized by their absorption spectra and Fe and acid-labile sulfide contents. On the basis of the distribution of Fe among the fractions obtained from chaotropic resolution of the NADH-ubiquinone oxidoreductase cmplex, a minimum of 6 or 7 Fe-S centers are present in this enzyme.