Fe-S Cluster Formation of Ferredoxin in Chloroplast Stroma

Abstract

Iron-sulfur proteins are found in a wide range of organisms from bacteria to man. They contain iron-sulfur (Fe-S) clusters which are known to be involved in electron transfer, catalysis, or gene regulation. Ferredoxin (Fd) is one of the major iron-sulfur proteins. In higher plants, Fd consists of an acidic 10-kD polypeptide carrying a [2Fe-2S] cluster chelated by four cysteine residues and acts as a multifunctional electron carrier in plastids. Fd is coded by nuclear DNA and synthesized in cytosol as a larger precursor containing a transit peptide at its amino-terminus, and transported into the chloroplast stroma accompanying with a proteolytic removal of the transit peptide. The formation of the Fe-S cluster of Fd has been shown to occur inside the chloroplast, however, details of its molecular mechanism are unknown. In this study, we analyzed the in vitro Fe-S cluster formation by using chloroplast subfractions and an apo Fd polypeptide synthesized in wheat germ extracts. Upon subfractionation of chloroplasts isolated from spinach leaves, the activity for cluster formation was found exclusively in the soluble stromal fraction. By a size-exclusion chromatography, the activity was recovered in the fractions as a single peak whose molecular size was roughly estimated as 160 kD. We propose that some protein factor(s) in the stromal fraction participates in the Fe-S cluster formation of Fd.