Reversible alkaline inactivation of lignin peroxidase involves the releaseof both the distal and proximal site calcium ions and bishistidine co-ordination of the haem

Phanerochaete chrysosporium lignin peroxidase isoenzyme H2 (LiP H2) exhibit s a transition to a stable, inactive form at pH 9.0 with concomitant spectr oscopic changes. The Soret peak intensity decreases some 55% with a red shi ft from 408 to 412 nm; the bands at 502 nm and 638 nm disappear and the pea k at 536 nm increases. The EPR spectrum changes from a signal typical of hi gh spin ferric haem to an exclusively low spin spectrum with g = 2.92, 2.27 , 1.50. These data indicate that the active pentacoordinated haem is conver ted into a hexaco-ordinated species at alkaline pH. Room temperature near-I R MCD data coupled with the EPR spectrum allow us to assign the haem co-ord ination of alkali-inactivated enzyme as bishistidine. Re-acidification of t he 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 CaCl2 results in the re covery of the initial activity together with the spectroscopic characterist ics of the native ferric enzyme. Incubating with 50 mM CaCl2 at a pH betwee n 6.0 and 9.0 can also re-activate the enzyme. Divalent metals other than C a2+ do not result in restoration of activity. Experiments with Ca-45 indica te that two tightly bound calcium ions per enzyme monomer are lost during i nactivation and reincorporated during subsequent re-activation, consistent with the presence of two structural Ca2+ ions in LiP H2. It is concluded th at both the structural Ca2+ ions play key roles in the reversible alkaline inactivation of LIP H2.