Mn-II is not a productive substrate for wild-type or recombinant lignin peroxidase isozyme H2

The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter was used t o drive the homologous expression of the lignin peroxidase (LiP) isozyme H2 gene in primary metabolic cultures of Phanerochaete chrysosporium. The mol ecular mass, pI, and optical absorption spectra of purified recombinant LiP H2 (rLiPH2) were essentially identical to those of wild-type LiPH2 (wtLiPH2 ). wtLiPH2 was prepared by growing cells in the absence of Mn-II, condition s under which P. chrysosporium manganese peroxidase (MnP) is not expressed, ensuring that wtLiPH2 was not contaminated with MnP, The kinetics of verat ryl alcohol (VA) oxidation were essentially identical for rLiPH2 and wtLiPH 2. The rLiPH2, wtLiPH2, and wild-type LiP isozyme H8 (wtLiPH8) enzymes were used to reexamine previous claims that LiPH2 can oxidize Mn-II at a rate s ufficient to promote catalytic turnover of the enzyme, Our results demonstr ate that rLiPH2, wtLiPH2, and LiPH8 do not turn over under steady-state con ditions, when Mn-II is the sole reducing substrate. Furthermore, transient- state kinetic analyses show that the reduction rate of the catalytic interm ediate, LiP compound I, by VA was at least 2 x 10(3)-fold higher than the r ate of reduction in the presence of Mn-II. No reduction of LiP compound II was observed in the presence of Mn-II. In contrast to previous claims, thes e data strongly suggest that Mn-II is not a productive substrate for LiPH2 or LiPH8, (C) 2000 Academic Press.