Kanbi Lalji D, Antonyuk Svetlana, Hough Michael A, Hall John F, Dodd Fraser E, Hasnain S Samar
Faculty of Applied Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, UK.
J Mol Biol. 2002 Jul 5;320(2):263-75. doi: 10.1016/S0022-2836(02)00443-6.
The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65 A resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant ( approximately 800 mV) is elevated compared to that of the native protein ( approximately 670 mV at pH 3.2) while that of the Ser86Asp mutant ( approximately 623 mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.
分别以1.82埃和1.65埃的分辨率展示了锈蓝蛋白的Met148Leu和Ser86Asp突变体的晶体结构。与天然蛋白质晶体形式中存在的一个分子相比，这两种结构在不对称单元中均有两个分子。这为研究锈蓝蛋白中的分子内电子转移途径提供了机会。Met148Leu突变体的氧化还原电位（约800毫伏）相对于天然蛋白质（在pH 3.2时约670毫伏）有所升高，而Ser86Asp突变体的氧化还原电位（在pH 3.2时约623毫伏）则降低。讨论了Ser86Asp突变对1型铜位点附近氢键的影响，并因此研究了其在决定酸稳定性中的作用。Met148Leu的1型铜位点模拟了在铜蓝蛋白和真菌漆酶结构域II中发现的那些结构和生化特征。此外，天然锈蓝蛋白的铜蓝蛋白核心和1型铜位点与抗坏血酸氧化酶和亚硝酸还原酶中发现的那些非常相似。鉴于有关锈蓝蛋白和真菌漆酶的额外结构数据，基于结构的系统发育树已被重新审视。我们确认锈蓝蛋白与亚硝酸还原酶结构域2、漆酶结构域3以及铜蓝蛋白结构域2、4和6属于同一类别。
