VERATRYL ALCOHOL OXIDATION BY LIGNIN PEROXIDASE

Citation
A. Khindaria et al., VERATRYL ALCOHOL OXIDATION BY LIGNIN PEROXIDASE, Biochemistry, 34(51), 1995, pp. 16860-16869
Citations number
50
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
34
Issue
51
Year of publication
1995
Pages
16860 - 16869
Database
ISI
SICI code
0006-2960(1995)34:51<16860:VAOBLP>2.0.ZU;2-N
Abstract
Lignin peroxidase (LiP) from the white rot fungus Phanerochaete chryso sporium catalyzes the H2O2-dependent oxidation of veratryl alcohol (VA ), a secondary metabolite of the fungus, to veratryl aldehyde (VAD). T he oxidation of VA does not seem to be simply one-electron oxidation b y LiP compound I (LiPI) to its cation radical (VA(.+)) and the second by LiP compound II (LiPII) to VAD. Moreover, the rate constant for LiP I reduction by VA (3 x 10(5) M(-1) s(-1)) is certainly sufficient, but the rate constant for LiPII reduction by VA (5.0 +/- 0.2 s(-1)) is in sufficient to account for the turnover rate of LiP (8 +/- 0.4 s(-1)) a t pH 4.5. Oxalate was found to decrease the turnover rate of LiP to 5 s(-1), but it had no effect on the rate constants for LiP with H2O2 or LiPI and LiPII, the latter formed by reduction of LiPI with ferrocyan ide, with VA. However, when LiPII was formed by reduction of LiPI with VA, an oxalate-sensitive burst phase was observed during its reductio n with VA. This was explained by the presence of LiPII, formed by redu ction of LiPI with VA, in two different states, one that reacted faste r with VA than the other. Activity during the burst was sensitive to p reincubation of LiPI with VA, decaying with a half-life of 0.54 s, and was possibly due to an unstable intermediate complex of VA(.+) and Li PII. This was supported by an anomalous, oxalate-sensitive, LiPII visi ble absorption spectrum observed during steady state oxidation of VA. The first order rate constant for the burst phase was 8.3 +/- 0.2 s(-1 ), fast enough to account for the steady state turnover rate of LiP at pH 4.5. Thus, it was concluded that oxalate decreased the turnover of Lip by reacting with VA(.+) bound to LiPII. The VA(.+) concentration measured by electron spin resonance spectroscopy (ESR) was 2.2 mu M at steady state (10 mu M LiP, 250 mu M H2O2, and 2 mM VA) and increased to 8.9 mu M when measured after the reaction was acid quenched. Theref ore, we assumed the presence of two states of VA(.+) bound to LiPII, o ne ESR-active and one ESR-silent. The ESR-silent species, which could be detected after acid quenching, would be responsible for the burst p hase. Both of the VA(.+) species disappeared in the presence of 5 mM o xalate. The ESR-active species reached a maximum (3.5 mu M) at 0.5 mM VA under steady state. From these studies, a mechanism for VA oxidatio n by LiP is proposed in which a complex of LiPII and VA(.+) reacts wit h an additional molecule of VA, leading to veratryl aldehyde formation .