Lignin Peroxidase

4.1.1 Properties

Lignin peroxidase is secreted as a series of glycosylated isoenzymes with pis ranging from 3.2 to 4.0 and molecular masses ranging from 38 to 43kDa, with each isoenzyme containing 1 mol heme per mole of protein (Farrell et al. 1989; Gold and Alic 1993; Leisola et al. 1987). It possesses a higher redox potential and a lower pH optimum than that of any other isolated peroxidase or oxidase (Call and Mucke 1997; Hammel et al. 1986; Kersten et al. 1990). Like other peroxidases, LIP is capable of oxidizing most phenolic compounds through the generation of phenoxy radicals. However, due to its exceptionally high redox potential and low pH optimum, it is able to oxidize nonphenolic aromatic substrates, typically not oxidized by other peroxidases including the nonphenolic phenylpropanoid units of lignin (Hammel et al. 1986; Hatakka 1994; ten Have et al. 1998b; Kersten et al. 1990). Stable cation centered radicals formed during the oxidation of nonphenolic aromatic nuclei may serve as redox mediators for LIP-catalyzed oxidations, effectively extending the substrate range. Reactions catalyzed by LIP include benzyl alcohol oxidations, side-chain cleavages, ring-opening reactions, dimethoxylations, and oxidative dechlorinations. The ability of LIP to attack such a variety of linkages suggests that it plays a key role in lignin degradation.

4.1.2 Catalytic Cycle

The catalytic cycle of LIP is similar to that of other peroxidases (Renganathan and Gold 1986; Tien et al. 1986).

Reaction of native ferric enzyme [Fe-LIP; Fe3+, P (porphyrin)] with H2O2 yields LIP-compound I (LIPI) a complex of high valent oxo-iron and porphyrin cation radical (Fe4+ = O, P*+). One-electron-oxidation of a reducing substrate (SH) by LIPI yields a radical cation (S*) and the one-electron-oxidized enzyme intermediate, LIP-compound II (LIPII; Fe4+ = O, P). A single one-electron oxidation of a second substrate molecule returns the enzyme to Fe-LIP completing the catalytic cycle.

However, in the absence of suitable reducing substrate or, at high H2O2 concentrations, LIPII is further oxidized by H2O2 to LIP-compound III (LIPIII; Fe3+ = O2-, P), a species with limited catalytic activity.

Lignin peroxidase is unique from other peroxidases in that it exhibits an unusually high reactivity between LIPII and H2O2 (Cai and Tien 1989; 1992; Wariishi and Gold 1990; Wariishi et al. 1990). Since, LIPIII is inactivated rapidly in the presence of excess H2O2, if it is not rapidly reverted to the native state, the enzyme has a "suicidal" tendency.

4.1.3 Role of Veratryl Alcohol During LIP-catalyzed Oxidation

The LIPIII has been shown to readily return to the native ferric state in the presence of H2O2 and veratryl alcohol (3,4-dimethoxybenzyl alcohol; VA) (Barr and Aust 1994; Cai and Tien 1989; 1992; Wariishi and Gold 1990). Ligninolytic cultures of P. chrysosporium normally produce VA, one of the physiological roles of which is believed to protect LIP from H2O2-dependent inactivation by reverting LIPIII to the native state. This is of particular significance, since during oxidation of certain chemicals such as phenols, LIPIII has been shown to accumulate, indicating that they are either poor substrates for LIPII or they lack the ability to revert LIPIII to the native state (Chung and Aust 1995; Harvey and Palmer 1990). Thus, oxidation of such chemicals is inefficient at high H2O2 concentrations.

VAlH2OJ uncertain s—*

Veratryl alcohol has also been shown to act as a chargetransfer mediator in LIP catalyzed reactions (Goodwin et al. 1995; Harvey et al. 1986). During the catalytic cycle of LIP, VA is oxidized to VA cation radical (VA+*), which in the presence of suitable reducing substrate is reduced back to VA and ready for another LIP catalyzed charge-transfer reaction. The roles played by VA in the catalytic cycle of LIP are highlighted.

Low molecular weight redox mediators such as VA are believed to be essential for degradation of lignin, since theoretical and electron microscopic studies have demonstrated that enzymes as large as peroxidases and LAC cannot have direct contact with lignin, since they appear to be too large for the penetration of the cell wall or the middle lamella (Call and Mucke 1997). Pine decayed by wood rotting fungi was infiltrated with a concentrated culture filtrate of the white rot fungus P. chrysosporium and labeled for LIP by postembedding immunoelectron microscopy. This method demonstrated that enzymatic attack on pine by LIP and presumably also other enzymes of the same size is restricted to the surface of the wood cell wall (Srebotnik and Messner 1988).

Measurements of the stability of VA+* have led to the suggestion that it would be unable to act as a diffusable oxidant without some form of stabilization (Candeias and Harvey 1995; Khindaria et al. 1996). It was suggested that a LIP-VA+* complex acts as the redox partner, with the cation radical being stabilized by a protein microenvironment of acidic character (Khindaria et al. 1996).

Nonphenolic aromatic compounds other than VA, such as 3,4-dimethoxycinnamic acid, 1,2-dimethoxybenzene, and 2-chloro-1,4-dimethoxybenzene have also been shown to be capable of mediating oxidation (Teunissen and Field 1998; Ward et al. 2002). The mediation phenomenon appears to be driven by the difference in the oxidation potential (OP) and site-binding affinity of the mediators (possessing higher OP values and higher affinity) and the target substrates (possessing lower OP values and lower affinity) (Ward et al. 2002).

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