2 SUBSTRATE INTERACTION SITES IN LIGNIN PEROXIDASE REVEALED BY SITE-DIRECTED MUTAGENESIS

It has been shown recently that Trp171 of lignin peroxidase (LiP) is h ydroxylated at the CP position [Blodig, W., Doyle, W. A., Smith, A. T. , Winterhalter, K., Choinowski, T., and Piontek, K. (1998) Biochemistr y 37, 8832-8838]. Comparative experiments, carried out on both wild-ty pe fungal and recombinant LIP isoenzyme H8 (LiPH8), indicate that the process of hydroxylation is autocatalytic and that Trp171 may be impl icated in catalysis. The role of this residue has therefore been exami ned using site-directed mutagenesis to obtain recombinant enzymes with Trp171 substituted by Phe or Ser (W171F and W171S LiPH8, respectivel y). The wild-type recombinant enzyme (LiPH8) was analyzed in solution using H-1 NMR spectroscopy and its integrity confirmed prior to the k inetic and spectroscopic characterization of LiPH8 mutants. A charge neutralization mutation in the ''classical heme edge'' substrate acces s channel of LiP, in which Glu146 was substituted by Gly (E146G LiPH8 ), showed substantial activity with respect to veratryl alcohol (VA) o xidation and a marked (2.4 pH units) increase in pK(a) for the oxidati on of a negatively charged difluoroazo dye. More surprisingly, the Trp 171 LiPH8 mutants W171F and W171S LiPH8* were found to have lost all activity with VA as substrate, and compounds I and II were unable to r eact with VA. Both mutants, however, retained substantial activity wit h two dye substrates. These data provide the first direct evidence for the existence of two distinct substrate interaction sites in LiP, a h eme-edge site typical of those encountered in other peroxidases and a second, novel site centered around Trp171 which is required for the ox idation of VA. Stopped-flow kinetic studies showed that all the mutant s examined reacted normally with hydrogen peroxide to give a porphyrin cation radical (compound I). However, the rapid phase of spontaneous compound I reduction (2.3 s(-1)), typical of wildtype LiP, was absent in the Trp171 mutants, strongly suggesting that an electron-transfer p athway must exist within the protein leading from the heme to a surfac e site in close proximity to Trp171. The kinetic competence of such a pathway is dependent on interaction of the enzyme with VA, at or near Trp171.