Khindaria A, Yamazaki I, Aust S D
Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
Biochemistry. 1995 Dec 26;34(51):16860-9. doi: 10.1021/bi00051a037.
Lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium catalyzes the H2O2-dependent oxidation of veratryl alcohol (VA), a secondary metabolite of the fungus, to veratryl aldehyde (VAD). The oxidation of VA does not seem to be simply one-electron oxidation by LiP compound I (LiPI) to its cation radical (VA.+) and the second by LiP compound II (LiPII) to VAD. Moreover, the rate constant for LiPI 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 insufficient to account for the turnover rate of LiP (8 +/- 0.4 s-1) at 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 ferrocyanide, with VA. However, when LiPII was formed by reduction of LiPI with VA, an oxalate-sensitive burst phase was observed during its reduction with VA. This was explained by the presence of LiPII, formed by reduction of LiPI with VA, in two different states, one that reacted faster with VA than the other. Activity during the burst was sensitive to preincubation 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 LiPII. This was supported by an anomalous, oxalate-sensitive, LiPII visible 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 microM at steady state (10 microM LiP, 250 microM H2O2, and 2 mM VA) and increased to 8.9 microM when measured after the reaction was acid quenched. Therefore, we assumed the presence of two states of VA.+ bound to LiPII, one ESR-active and one ESR-silent. The ESR-silent species, which could be detected after acid quenching, would be responsible for the burst phase. Both of the VA.+ species disappeared in the presence of 5 mM oxalate. The ESR-active species reached a maximum (3.5 microM) at 0.5 mM VA under steady state. From these studies, a mechanism for VA oxidation by LiP is proposed in which a complex of LiPII and VA.+ reacts with an additional molecule of VA, leading to veratryl aldehyde formation.
来自白腐真菌黄孢原毛平革菌的木质素过氧化物酶（LiP）催化真菌的次生代谢产物藜芦醇（VA）依赖H₂O₂氧化为藜芦醛（VAD）。VA的氧化似乎并非简单地由LiP化合物I（LiPI）将其一电子氧化为阳离子自由基（VA⁺），再由LiP化合物II（LiPII）将其氧化为VAD。此外，VA还原LiPI的速率常数（3×10⁵ M⁻¹ s⁻¹）肯定足够，但VA还原LiPII的速率常数（5.0±0.2 s⁻¹）不足以解释pH 4.5时LiP的周转速率（8±0.4 s⁻¹）。发现草酸盐可将LiP的周转速率降至5 s⁻¹，但对LiP与H₂O₂或LiPI和LiPII（后者由亚铁氰化物还原LiPI与VA形成）的速率常数没有影响。然而，当用VA还原LiPI形成LiPII时，在用VA还原LiPII的过程中观察到一个草酸盐敏感的爆发期。这可以解释为，用VA还原LiPI形成的LiPII存在两种不同状态，其中一种与VA反应的速度比另一种快。爆发期的活性对LiPI与VA的预孵育敏感，以0.54 s的半衰期衰减，可能是由于VA⁺和LiPII的不稳定中间复合物。这得到了VA稳态氧化过程中观察到的异常的、草酸盐敏感的LiPII可见吸收光谱的支持。爆发期的一级速率常数为8.3±0.2 s⁻¹，快到足以解释pH 4.5时LiP的稳态周转速率。因此，得出结论，草酸盐通过与结合在LiPII上的VA⁺反应降低了LiP的周转。通过电子自旋共振光谱（ESR）测定，稳态时（10 μM LiP、250 μM H₂O₂和2 mM VA）VA⁺的浓度为2.2 μM，酸淬灭反应后测定时增加到8.9 μM。因此，我们假设结合在LiPII上的VA⁺存在两种状态，一种具有ESR活性，一种无ESR活性。酸淬灭后可检测到的无ESR活性物种将导致爆发期。在5 mM草酸盐存在下，两种VA⁺物种均消失。稳态下，在0.5 mM VA时，具有ESR活性的物种达到最大值（3.5 μM）。基于这些研究，提出了LiP氧化VA的机制，即LiPII和VA⁺的复合物与另一个VA分子反应，导致藜芦醛的形成。
