Abstract
The active site amino acid residues of lignin peroxidase are homologous to those of other peroxidases; however, in contrast to other peroxidases, no pH dependence is observed for the reaction of ferric lignin peroxidase with H2O2 to form compound I (Andrawis, A., Johnson, K.A., and Tien, M. (1988) J. Biol. Chem. 263, 1195-1198). Chloride binding is used in the present study to investigate this reaction further. Chloride binds to lignin peroxidase at the same site as cyanide and hydrogen peroxide. This is indicated by the following. 1) Chloride competes with cyanide in binding to lignin peroxidase. 2) Chloride is a competitive inhibitor of lignin peroxidase with respect to H2O2. The inhibition constant (Ki) is equal to the dissociation constant (Kd) of chloride at all pH values studied. Chloride binding is pH dependent: chloride binds only to the protonated form of lignin peroxidase. Transient-state kinetic studies demonstrate that chloride inhibits lignin peroxidase compound I formation in a pH-dependent manner with maximum inhibition at low pH. An apparent pKa was calculated at each chloride concentration; the pKa increased as the chloride concentration increased. Extrapolation to zero chloride concentration allowed us to estimate the intrinsic pKa for the ionization in the lignin peroxidase active site. The results reported here provide evidence that an acidic ionizable group (pKa approximately 1) at the active site controls both lignin peroxidase compound I formation and chloride binding. We propose that the mechanism for lignin peroxidase compound I formation is similar to that of other peroxidases in that it requires the deprotonated form of an ionizable group near the active site.
Highlights
The active site amino acid residuoefslignin peroxidase are homologous tothose of otherperoxidases; in contrast to other peroxidases, no pH dependence is observed for the reaction of ferric lignin peroxidase with HzOzto form compound I
An apparent pK, was calculatedat each chloride con- Despite the lack of p H dependence for compound I formacentration; the pKa increased as the chloride concen- tion, the active site amino acids thought to be essential for tration increased
We propose that themechanism for lignin peroxidase compound I formation is similar to thatof other peroxidases in thaitt requires the deprotonated
Summary
Active site ionizable group with a pK, below 2 for chloroperoxidase (Lambeiret al., 1983). Thebinding of cyanide to chloroperoxidase is p H independent; when in the presence of 60 mM NaN03, an apparent pK=a4.5 is detected. The binding of NO; to the protonatedform of chloroperoxidase inhibits cyanide bindingby shifting the acidic pK, to a higher, experimentally detectable value. The results demonstratethat chloride inhibits lignin peroxidase catalysis by binding to the H,O, binding site. The binding of chloride inhibits compound I formation at low pH. An acidic ionization in the active site is revealed. We propose that this ionization affects the reaction of lignin peroxidase with H2O2to form compound I and the binding of chloride to ferric lignin peroxidase
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