Spectral changes of lignin peroxidase during reversible inactivation.

The heme environment of lignin peroxidase (LiP) has been investigated by electronic absorption and electron paramagnetic resonance (EPR) spectroscopy. Native LiP was a pentacoordinate, high-spin ferric iron with a high-spin absorption band at 634 nm and g values at 5.86 and 2.07 in the EPR spectrum. Upon thermal inactivation, calcium ions were released from the enzyme and the Soret absorption decreased and red-shifted about 2 nm, the high-spin absorption band at 634 nm disappeared, and a low-spin absorption band appeared at 532 nm. The EPR spectrum and the temperature dependence of electronic absorption spectra revealed that the heme iron of the thermally inactivated enzyme was a mixture of high- and low-spin states, which was further supported by the changes in the electronic absorption and EPR spectra when cyanide was added to the thermally inactivated enzyme. Addition of various imidazoles or CN- to thermally inactivated enzyme demonstrated that the low-spin heme iron of inactivated enzyme was hexacoordinate with a distal histidine as its sixth ligand, in contrast to the active enzyme, which was pentacoordinate and high-spin. Upon addition of calcium to recover the thermally inactivated LiP, the reactivated enzyme had absorptions at 408, 502, and 634 nm and g values at 5.86 and 2.07 in the EPR spectrum, which demonstrated that the heme iron of the reactivated enzyme was again high-spin and pentacoordinated.