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Abstract: Ribosomal function is dependent on multiple proteins. The ABCE1 ATPase, a unique ABC superfamily member that bears two Fe4S4 clusters, is crucial for ribosomal biogenesis and recycling. Here, the ATPase activity of the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe–S clusters) and holo- (with full complement of Fe–S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe–S clusters and Mg2+. Typically ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a negative allosteric effector that modulates ATP affinity of PabABCE1. Physiological [Mg2+] inhibits the PabABCE1 ATPase (K i of ∼1μM) for both apo- and holo-PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent ATP K m of apo-PabABCE1 increases >30-fold from ∼30μM to over 1mM with Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge (φ) to being dependent on φ with cellular [Mg2+]. These findings uncover intricate overlapping effects by both [Mg2+] and the status of Fe–S clusters that regulate ABCE1’s ATPase activity with implications to ribosomal function. [Copyright &y& Elsevier]