Cardoso Luciano A, Ferreira Sérgio T, Hermes-Lima Marcelo
Oxyradical Research Group, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF 70910-900, Brazil.
Programa de Bioquímica e Biofisica Celular, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21944-590, Brazil.
Comp Biochem Physiol A Mol Integr Physiol. 2008 Nov;151(3):313-321. doi: 10.1016/j.cbpa.2007.03.025. Epub 2007 Mar 30.
Glutathione reductase (GR) carries out the enzymatic reduction of glutathione disulfide (GSSG) to its reduced form (GSH) at the expense of the reducing power of NADPH. Previous studies have shown that GR from several species is progressively inactivated in the presence of NADPH, but that the mechanism of inactivation (especially in the presence of metals) has not been fully elucidated. We have investigated the involvement of iron ions in the inactivation of yeast (Saccharomyces cerevisiae) GR in the presence of NADPH. Even in the absence of added iron, inactivation of GR was partly blocked by the iron chelators, deferoxamine and ortho-phenanthroline, suggesting the involvement of trace amounts of contaminating iron in the mechanism of inhibition. Exogenously added antioxidants including ethanol, dimethylsulfoxide and 2-deoxyribose did not protect GR against NADPH-induced inactivation, whilst addition of exogenous Fe(II) (but not Fe(III)) potentiated the inactivation. Moreover, removal of oxygen from the medium led to increased inhibition of GR, whereas pre-incubation of the Fe(II)-containing medium for 30 min under normoxic conditions prior to the addition of GR abolished the enzyme inactivation by NADPH. Under these pre-incubation conditions, Fe(II) is fully oxidized to Fe(III) within 1 min. Furthermore, GR that had been previously inactivated in the presence of Fe(II) plus NADPH could be partially reactivated by treatment with ortho-phenanthroline and deferoxamine. In contrast, Fe(III) had no effect on GR reactivation. Together, these results indicate that yeast GR is inactivated by a reductive mechanism mediated by NADPH and Fe(II). According to this mechanism, GR is diverted from its normal redox cycling by the generation of an inactive reduced enzyme form in which both the FAD and thiol groups at the active site are likely in a reduced state.
谷胱甘肽还原酶（GR）利用烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的还原力，将氧化型谷胱甘肽（GSSG）酶促还原为还原型谷胱甘肽（GSH）。先前的研究表明，几种物种的GR在NADPH存在下会逐渐失活，但失活机制（尤其是在金属存在的情况下）尚未完全阐明。我们研究了铁离子在NADPH存在下对酵母（酿酒酵母）GR失活的影响。即使在未添加铁的情况下，铁螯合剂去铁胺和邻菲罗啉也能部分阻止GR的失活，这表明微量的污染铁参与了抑制机制。包括乙醇、二甲基亚砜和2-脱氧核糖在内的外源性抗氧化剂并不能保护GR免受NADPH诱导的失活，而添加外源性亚铁离子（Fe(II)，而非铁离子（Fe(III)））会增强失活作用。此外，从培养基中去除氧气会增强对GR的抑制作用，而在添加GR之前，在常氧条件下将含Fe(II)的培养基预孵育30分钟，则可消除NADPH对该酶的失活作用。在这些预孵育条件下，Fe(II)在1分钟内会完全氧化为Fe(III)。此外，先前在Fe(II)加NADPH存在下失活的GR，经邻菲罗啉和去铁胺处理后可部分恢复活性。相比之下，Fe(III)对GR的再活化没有影响。这些结果共同表明，酵母GR是通过由NADPH和Fe(II)介导的还原机制失活的。根据这一机制，GR因生成一种无活性的还原酶形式而偏离其正常的氧化还原循环，在这种形式中，活性位点的黄素腺嘌呤二核苷酸（FAD）和硫醇基团可能都处于还原状态。
