Abstract
CpNifS, a cysteine desulfurase required to supply sulfur for ironsulfur cluster biogenesis in Arabidopsis thaliana chloroplasts, belongs to a class of NifS-like enzymes with low endogenous cysteine desulfurase activity. Its bacterial homologue SufS is stimulated by SufE. Here we characterize the Arabidopsis chloroplast protein CpSufE, which has an N-terminal SufE-like domain and a C-terminal BolA-like domain unique to higher plants. CpSufE is targeted to the chloroplast stroma, indicated by green fluorescent protein localization and immunoblot experiments. Like CpNifS, CpSufE is expressed in all major tissues, with higher expression in green parts. Its expression is light-dependent and regulated at the mRNA level. The addition of purified recombinant CpSufE increased the Vmax for the cysteine desulfurase activity of CpNifS over 40-fold and decreased the KM toward cysteine from 0.1 to 0.043 mm. In contrast, CpSufE addition decreased the affinity of CpNifS for selenocysteine, as indicated by an increase in the KM from 2.9 to 4.17 mm, and decreased the Vmax for selenocysteine lyase activity by 30%. CpSufE forms dynamic complexes with CpNifS, indicated by gel filtration, native PAGE, and affinity chromatography experiments. A mutant of CpSufE in which the single cysteine was changed to serine was not active in stimulating CpNifS, although it did compete with WT CpSufE. The iron-sulfur cluster reconstitution activity of the CpNifS-CpSufE complex toward apoferredoxin was 20-fold higher than that of CpNifS alone. We conclude that CpNifS and CpSufE together form a cysteine desulfurase required for iron-sulfur cluster formation in chloroplasts.
Highlights
Especially the green chloroplasts need to synthesize and maintain a variety of Fe-S proteins with at least five different cluster types [5]
When the in vitro reconstitution activity of purified CpNifS protein was compared with the activity of CpNifS in stromal extracts, it was concluded that CpNifS in stroma is about 50 – 80 times more active [27]
The Cys desulfurase activity of CpNifS is required for Fe-S formation in chloroplast stroma [27]
Summary
Especially the green chloroplasts need to synthesize and maintain a variety of Fe-S proteins with at least five different cluster types [5]. Several of the plastid Fe-S biosynthesis components tentatively identified to date are most related to the bacterial suf cluster genes; other components are unique, [5] Chloroplasts have their own Fe-S assembly systems [13, 14]. Characterization of the chloroplastic Fe-S formation machinery started with the identification of a Cys desulfurase CpNifS [17, 18] and of scaffold proteins CpNfu2 [19, 20, 21] and CpIscA [22]. The depletion of CpNifS led to the loss of Fe-S reconstitution activity of chloroplast stroma [27] This Cys desulfurase of chloroplasts is distinct from the Cys desulfurases NifS and IscS of the Nif and Isc type assembly systems and is more similar in sequence to SufS [18]. We show here that CpSufE forms a complex with CpNifS, stimulating Cys desulfurase activity over 40-fold and enhancing CpNifS-dependent Fe-S reconstitution in vitro
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