Phanerochaete chrysosporium lignin peroxidase isoenzyme H2 (LiP H2) exhibits a transition to a stable, inactive form at pH 9.0 with concomitant spectroscopic changes. The Söret peak intensity decreases some 55% with a red shift from 408 to 412 nm; the bands at 502 nm and 638 nm disappear and the peak at 536 nm increases. The EPR spectrum changes from a signal typical of high spin ferric haem to an exclusively low spin spectrum with g=2.92, 2.27, 1.50. These data indicate that the active pentaco-ordinated haem is converted into a hexaco-ordinated species at alkaline pH. Room temperature near-IR MCD data coupled with the EPR spectrum allow us to assign the haem co-ordination of alkali-inactivated enzyme as bishistidine. Re-acidification of the alkali-inactivated enzyme to pH 6 induces further spectroscopic changes and generates an irreversibly inactivated species. By contrast, a pH shift from 9.0 to 6.0 with simultaneous addition of 50 mM CaCl(2) results in the recovery of the initial activity together with the spectroscopic characteristics of the native ferric enzyme. Incubating with 50 mM CaCl(2) at a pH between 6.0 and 9.0 can also re-activate the enzyme. Divalent metals other than Ca(2+) do not result in restoration of activity. Experiments with (45)Ca indicate that two tightly bound calcium ions per enzyme monomer are lost during inactivation and reincorporated during subsequent re-activation, consistent with the presence of two structural Ca(2+) ions in LiP H2. It is concluded that both the structural Ca(2+) ions play key roles in the reversible alkaline inactivation of LiP H2.