Properties of the Cysteine Residues and the Iron−Sulfur Cluster of the Assimilatory 5‘-Adenylyl Sulfate Reductase from Enteromorpha intestinalis

Abstract

The 5'-adenylyl sulfate (APS) reductase from the marine macrophytic green alga Enteromorpha intestinalis uses reduced glutathione as the electron donor for the reduction of APS to 5'-AMP and sulfite. The E. intestinalis enzyme (EiAPR) is composed of a reductase domain and a glutaredoxin-like C-terminal domain. The enzyme contains a single [4Fe-4S] cluster as its sole prosthetic group. Three of the enzyme's eight cysteine residues (Cys166, Cys257, and Cys260) serve as ligands to the iron-sulfur cluster. Site-directed mutagenesis experiments and resonance Raman spectroscopy are consistent with the presence of a cluster in which only three of the four ligands to the cluster irons contributed by the protein are cysteine residues. Site-directed mutagenesis experiments suggest that the thiol group of Cys250, a residue found only in algal APS reductases, is not an absolute requirement for activity. The other four cysteines that do not serve as cluster ligands, all of which are required for activity, are involved in the formation of two redox-active disulfide/dithiol couples. The couple involving Cys342 and Cys345 has an E(m) value at pH 7.0 of -140 mV, and the one involving Cys165 and Cys285 has an E(m) value at pH 7.0 of -290 mV. The C-terminal portion of EiAPR, expressed separately, exhibits the cystine reductase activity characteristic of glutaredoxins. It is proposed that the Cys342-Cys345 disulfide provides the site for entry of electrons from reduced glutathione and that the Cys166-Cys285 disulfide may serve as a structural element that is essential for keeping the enzyme in the catalytically active conformation.