Redox properties of human manganese superoxide dismutase and active-site mutants
Redox properties of human manganese superoxide dismutase and active-site mutants
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DOI：
10.1021/bi010792p
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发表时间：
2001-09-04
期刊：
影响因子：
2.9
通讯作者：
Silverman, DN
中科院分区：
文献类型：
--
作者：
Lévêque, VJP;Vance, CK;Silverman, DN
由于寻找合适的电子介体的问题，人锰超氧化物歧化酶(MnSOD)的氧化还原电位一直难以确定。我们发现，铁氰化物和五氰基氨基高铁酸盐可以作为电子媒介，尽管平衡很慢，半衰期接近6h。中点电位值是通过让酶和介体平衡38h和在酶和介体中加入亚硫酸盐的还原滴定来确定的。中点电位的总值为393+/-29 mV。为了阐明His30和Tyr34在人MnSOD活性位点中的作用，我们还测定了定点突变体His30Asn(H30N)和Tyr34Phe(Y34F)的氧化还原特性，并与野生型酶进行了比较。晶体结构表明，每个突变都会中断活性中心的氢键网络，并且每个突变都会导致超氧化物歧化的最大催化速度比野生型降低10倍。本研究表明，在实验不确定度范围内，H30N和Y34F人MnSOD对活性中心金属的氧化还原电位影响很小。电化学测得H30N的氧化还原电位为365+/-28 mV，Y34F-MnSOD的氧化还原电位为435+/-30 mV。这些结果表明，His30和Tyr34的作用更多地是支持催化，可能是质子运输，而不是氧化还原电位的调节。
The redox potential of human manganese superoxide dismutase (MnSOD) has been difficult to determine because of the problem of finding suitable electron mediators. We have found that ferricyanide and pentacyanoaminoferrate can be used as electron mediators, although equilibration is very slow with a half-time near 6 h. Values of the midpoint potential were determined both by allowing enzyme and mediators to equilibrate up to 38 h and by reductive titration adding dithionite to enzyme and mediator. An overall value of the midpoint potential was found to be 393 +/- 29 mV. To elucidate the role of His30 and Tyr34 in the active site of human MnSOD, we have also measured the redox properties of the site-specific mutants His30Asn (H30N) and Tyr34Phe (Y34F) and compared them with the wild-type enzyme. Crystal structures have shown that each mutation interrupts a hydrogen bond network in the active site, and each causes a 10-fold decrease in the maximal velocity of catalysis of superoxide dismutation as compared with wild type. The present study shows that H30N and Y34F human MnSOD have very little effect, within experimental uncertainty, on the redox potential of the active-site metal. The redox potentials determined electrochemically were 365 +/- 28 mV for H30N and 435 +/- 30 mV for Y34F MnSOD. These results suggest that the role of His30 and Tyr34 is more in support of catalysis, probably proton transport, and not in the tuning of the redox potential.