Oxidation-reduction properties of two enzymes involved in reductive sulfate assimilation in plants

Abstract

Assimilation of sulfate by plants involves reaction of sulfate with ATP to form 5¿-adenylylsulfate (APS), followed by the two-electron reduction of APS to sulfite by glutathione, catalyzed by APS reductase. The subsequent six-electron reduction of sulfite to sulfide, catalyzed by sulfite reductase, utilizes reduced ferredoxin as the electron donor. Arabidopsis thaliana has three isoforms of APS reductase. The activity of the APR1 form is redox-state controlled via a regulatory disulfide. A second APR1 disulfide is the likely electron acceptor at the active site. APR1 becomes more active, through an increase in Vmax and decrease in the Km for APS, when the regulatory cysteines are oxidized to a disulfide. Oxidative activation plays an important role in the plant¿s response to oxidative stress. The Em value for this regulatory disulfide, ¿330 mV (at pH 8.5), is equal to that for glutathione at this pH. Sulfite reductase contains one siroheme and one [4Fe-4S] cluster as prosthetic groups. It is likely that a sulfur from the [4Fe-4S] cluster serves as an axial ligand to the iron of the siroheme. We have carried out the first the redox titrations of a ferredoxin-dependent sulfite reductase, using the wild-type enzyme from maize, and measured Em values (at pH 7.5) of -205 mV and ¿410 mV for the siroheme and [4Fe-4S] cluster, respectively. The effects of cyanide and phosphate binding to the siroheme and of site-specific mutations (R193A and R193E) that alter substrate specificity on the redox properties of the enzyme have also been investigated.