酿酒酵母Fet3p中1型铜位点的光谱学与反应活性：多铜氧化酶中结构与反应活性的相关性

Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: correlation of structure with reactivity in the multicopper oxidases.

作者信息

Machonkin T E, Quintanar L, Palmer A E, Hassett R, Severance S, Kosman D J, Solomon E I

机构信息

Department of Chemistry, Stanford University, Stanford, California 94305, USA.

出版信息

J Am Chem Soc. 2001 Jun 13;123(23):5507-17. doi: 10.1021/ja003975s.

DOI:10.1021/ja003975s

PMID:11389633

Abstract

Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomyces cerevisiae. Fet3p is functionally homologous to ceruloplasmin (Cp) in that both are ferroxidases. However, by sequence homology Fet3p is more similar to fungal laccase, and both contain a type 1 Cu site that lacks the axial methionine ligand present in the functional type 1 sites of Cp. To determine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circular dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-depleted mutants. The spectroscopic features of the type 1 Cu site of Fet3p are nearly identical to those of fungal laccase, indicating a very similar three-coordinate geometry. We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics. From poised potential redox titrations, the E degrees of the type 1 Cu site is 427 mV, which is low for a three-coordinate type 1 Cu site. The kinetics of reduction of the type 1 Cu sites of four different multicopper oxidases with two different substrates were compared. The type 1 site of a plant laccase (Rhus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky organic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.029 and 0.013 s(-)(1), respectively). On the other hand, the type 1 site of a fungal laccase (Coprinus cinereus) is reduced very rapidly by both substrates (k(obs) > 23 s(-)(1)). In contrast, both Fet3p and Cp are rapidly reduced by Fe(II) (k(obs) > 23 s(-)(1)), but only very slowly by 1,4-hydroquinone (10- and 100-fold more slowly than plant laccase, respectively). Semiclassical theory is used to analyze the origin of these differences in reactivity in terms of type 1 Cu site accessibility to specific substrates.

摘要

Fet3p是一种最近从酿酒酵母中分离出来的多铜氧化酶。Fet3p在功能上与铜蓝蛋白（Cp）同源，因为二者都是铁氧化酶。然而，从序列同源性来看，Fet3p与真菌漆酶更为相似，且二者都含有一个1型铜位点，该位点缺少Cp功能型1型位点中存在的轴向甲硫氨酸配体。为了确定Fet3p的1型铜位点的电子结构对铁氧化酶机制的贡献，我们研究了野生型Fet3p以及1型和2型铜缺失突变体的吸收光谱、圆二色光谱、磁圆二色光谱、电子顺磁共振光谱和共振拉曼光谱。Fet3p的1型铜位点的光谱特征与真菌漆酶的几乎相同，表明其具有非常相似的三配位几何结构。我们还通过氧化还原滴定和停流动力学研究了1型铜位点的反应活性。从平衡电位氧化还原滴定可知，1型铜位点的E°为427 mV，这对于一个三配位的1型铜位点来说是较低的。比较了四种不同的多铜氧化酶的1型铜位点与两种不同底物的还原动力学。植物漆酶（漆树）的1型位点被Fe(II)和一种大分子有机底物1,4 - 对苯二酚还原的速度适中较慢（底物用量为6当量时，k(obs)分别为0.029和0.013 s⁻¹）。另一方面，真菌漆酶（灰盖鬼伞）的1型位点被两种底物还原的速度都非常快（k(obs) > 23 s⁻¹）。相比之下，Fet3p和Cp都能被Fe(II)快速还原（k(obs) > 23 s⁻¹），但被1,4 - 对苯二酚还原的速度非常慢（分别比植物漆酶慢10倍和100倍）。半经典理论被用于根据1型铜位点对特定底物的可及性来分析这些反应活性差异的起源。