Interaction of bacteriophage lambda protein phosphatase with Mn(II): Evidence for the formation of a [Mn(II)](2) cluster

The interaction of bacteriophage lambda protein phosphatase with Mn2+ was s tudied using biochemical techniques and electron paramagnetic resonance spe ctrometry. Reconstitution of bacteriophage lambda protein phosphatase in th e presence of excess MnCl2 followed by rapid desalting over a gel filtratio n column resulted in the retention of approximately 1 equiv of Mn2+ ion bou nd to the protein. This was determined by metal analyses and low-temperatur e EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. Th e Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in th e absence of added MnCl2. The EPR spectrum of the mononuclear species disap peared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)(2) cluster. EPR spectr a of the dinuclear (Mn2+)(2) cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependen t studies indicated that these features arose from an excited state. Titrat ions of the apoprotein with MnCl2 provided evidence of one Mn2+ binding sit e with a micromolar affinity and at least one additional Mn2+ site with a 1 00-fold lower affinity. The dependence of the phosphatase activity on Mn2concentration indicates that full enzyme activity probably requires occupat ion of both Mn2+ sites. These results are discussed in the context of dival ent metal ion activation of this enzyme and possible roles for Mn2+ activat ion of other serine/threonine protein phosphatases.