Khindaria A, Yamazaki I, Aust S D
Biotechnology Center, Utah State University, Logan 84322-4705, USA.
Biochemistry. 1996 May 21;35(20):6418-24. doi: 10.1021/bi9601666.
Lignin peroxidase (LiP) catalyzes the H2O2-dependent oxidation of veratryl alcohol (VA) to veratryl aldehyde, with the enzyme-bound veratryl alcohol cation radical (VA.+) as an intermediate [Khindaria et al. (1995) Biochemistry 34, 16860-16869]. The decay constant we observed for the enzyme generated cation radical did not agree with the decay constant in the literature [Candeias and Harvey (1995) J. Biol. Chem. 270, 16745-16748] for the chemically generated radical. Moreover, we have found that the chemically generated VA.+ formed by oxidation of VA by Ce(IV) decayed rapidly with a first-order mechanism in air- or oxygen-saturated solutions, with a decay constant of 1.2 x 10(3) s-1, and with a second-order mechanism in argon-saturated solution. The first-order decay constant was pH- independent suggesting that the rate-limiting step in the decay was deprotonation. When VA.+ was generated by oxidation with LiP the decay also occurred with a first-order mechanism but was much slower, 1.85 s-1, and was the same in both oxygen- and argon-saturated reaction mixtures. However, when the enzymatic reaction mixture was acid-quenched the decay constant of VA.+ was close to the one obtained in the Ce(IV) oxidation system, 9.7 x 10(2) s-1. This strongly suggested that the LiP-bound VA.+ was stabilized and decayed more slowly than free VA.+. We propose that the stabilization of VA.+ may be due to the acidic microenvironment in the enzyme active site, which prevents deprotonation of the radical and subsequent reaction with oxygen. We have also obtained reversible redox potential of VA.+/VA couple using cyclic voltammetery. Due to the instability of VA.+ in aqueous solution the reversible redox potential was measured in acetone, and was 1.36 V vs normal hydrogen electrode. Our data allow us to propose that enzymatically generated VA.+ can act as a redox mediator but not as a diffusible oxidant for LiP-catalyzed lignin or pollutant degradation.
木质素过氧化物酶（LiP）催化依赖过氧化氢将藜芦醇（VA）氧化为藜芦醛，酶结合的藜芦醇阳离子自由基（VA.+）作为中间体[Khindaria等人（1995年）《生物化学》34卷，16860 - 16869页]。我们观察到的由酶产生的阳离子自由基的衰变常数与文献[Candeias和Harvey（1995年）《生物化学杂志》270卷，16745 - 16748页]中化学产生的自由基的衰变常数不一致。此外，我们发现通过Ce(IV)氧化VA形成的化学产生的VA.+在空气或氧气饱和溶液中以一级反应机制快速衰变，衰变常数为1.2×10³ s⁻¹，在氩气饱和溶液中以二级反应机制衰变。一级衰变常数与pH无关，这表明衰变中的限速步骤是去质子化。当用LiP氧化产生VA.+时，衰变也以一级反应机制发生，但要慢得多，为1.85 s⁻¹，并且在氧气和氩气饱和的反应混合物中相同。然而，当酶促反应混合物用酸淬灭时，VA.+的衰变常数接近在Ce(IV)氧化系统中获得的衰变常数，即9.7×10² s⁻¹。这强烈表明与LiP结合的VA.+比游离的VA.+更稳定且衰变更慢。我们提出VA.+的稳定可能是由于酶活性位点中的酸性微环境，这阻止了自由基的去质子化以及随后与氧气的反应。我们还使用循环伏安法获得了VA.+/VA电对的可逆氧化还原电位。由于VA.+在水溶液中的不稳定性，可逆氧化还原电位是在丙酮中测量的，相对于标准氢电极是1.36 V。我们的数据使我们能够提出酶促产生的VA.+可以作为氧化还原介质，但不能作为LiP催化的木质素或污染物降解的可扩散氧化剂。
