Plant adenosine 5 '-phospho sulfate reductase is a novel iron-sulfur protein

Adenosine 5'-phosphosulfate reductase (APR) catalyzes the two-electron redu ction of adenosine 5'-phosphosulfate to sulfite and AMP, which represents t he key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minor and Arabidopsis thaliana were overexpressed in Escherichi a coli and isolated as yellow-brown proteins. UV-visible spectra of these r ecombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of ir on and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor s ignal at g = 2.01. Therefore, Mossbauer spectra of Fe-57-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S](2+) cluster. This cluster was unusual because onl y three of the iron sites exhibited the same Mossbauer parameters. The four th iron site gave, because of the bistability of the fit, a significantly s maller isomer shift or larger quadrupole splitting than the other three sit es. Thus, plant assimilatory APR represents a novel type of adenosine 5'-ph osphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which i s clearly different from the dissimilatory adenosine 5'-phosphosulfate redu ctases found in sulfate reducing bacteria.