The Effect of Conserved Histidine on the Proximity of Fe-S Clusters in Adenosine-5′-Phosphosulfate Reductases from Pseudomonas aeruginosa and Enteromorpha intestinalis

Abstract

This study investigates the impact of conserved histidine (His) residue mutations on the adenosine 5′-phosphosulfate (APS) reductase enzymes Pseudomonas aeruginosa APR (PaAPR) and Enteromorpha intestinalis APR (EiAPR), focusing on the effects of His-to-alanine (Ala) and His-to-arginine (Arg) substitutions on enzyme activity, iron–sulfur [4Fe-4S] cluster stability, and APS binding affinity. Using recombinant His-tagged wild-types (WTs) and variants expressed in Escherichia coli, analyses revealed that both PaAPR and EiAPR enzymes exhibit a distinct absorption peak associated with their [4Fe-4S] clusters, which are critical for their catalytic functions. Notably, the His-to-Ala variants displayed reduced enzymatic activities and lower iron and sulfide contents compared to their respective WTs, suggesting alterations in the iron–sulfur cluster ligations and thus affecting APS reductase catalysis. In contrast, His-to-Arg variants maintained similar activities and iron and sulfide contents as their WTs, highlighting the importance of a positively charged residue at the conserved His site for maintaining structural integrity and enzymatic function. Further kinetic analyses showed variations in Vmax and Km values among the mutants, with significant reductions observed in the His-to-Ala variants, emphasizing the role of the conserved His in enzyme stability and substrate specificity. This study provides valuable insights into the structural and functional significance of conserved His residues in APS reductases, contributing to a better understanding of sulfur metabolism and its regulation in bacterial and plant systems. Future investigations into the structural characterization of these enzymes and the exploration of other critical residues surrounding the [4Fe-4S] cluster are suggested to elucidate the complete mechanism of APS reduction and its biological implications.