MnII Is Not a Productive Substrate for Wild-Type or Recombinant Lignin Peroxidase Isozyme H2

Abstract

The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter was used to drive the homologous expression of the lignin peroxidase (LiP) isozyme H2 gene in primary metabolic cultures of Phanerochaete chrysosporium. The molecular mass, pI, and optical absorption spectra of purified recombinant LiPH2 (rLiPH2) were essentially identical to those of wild-type LiPH2 (wtLiPH2). wtLiPH2 was prepared by growing cells in the absence of MnII, conditions under which P. chrysosporium manganese peroxidase (MnP) is not expressed, ensuring that wtLiPH2 was not contaminated with MnP. The kinetics of veratryl alcohol (VA) oxidation were essentially identical for rLiPH2 and wtLiPH2. The rLiPH2, wtLiPH2, and wild-type LiP isozyme H8 (wtLiPH8) enzymes were used to reexamine previous claims that LiPH2 can oxidize MnII at a rate sufficient to promote catalytic turnover of the enzyme. Our results demonstrate that rLiPH2, wtLiPH2, and LiPH8 do not turn over under steady-state conditions, when MnII is the sole reducing substrate. Furthermore, transient-state kinetic analyses show that the reduction rate of the catalytic intermediate, LiP compound I, by VA was at least 2 × 103-fold higher than the rate of reduction in the presence of MnII. No reduction of LiP compound II was observed in the presence of MnII. In contrast to previous claims, these data strongly suggest that MnII is not a productive substrate for LiPH2 or LiPH8.