Sinclair R, Hallam S, Chen M, Chance B, Powers L
National Center for the Design of Molecular Function, Utah State University, Logan 84322-4630, USA.
Biochemistry. 1996 Nov 26;35(47):15120-8. doi: 10.1021/bi961064i.
The globins and peroxidases, while performing completely different chemistry, share features of the iron heme active site: a protoporphyrin IX prosthetic group is linked to the protein by the proximal histidine residue. X-ray absorption spectroscopy provides a method to determine the local structure of iron heme active sites in proteins. Our previous studies using X-ray absorption spectroscopy revealed a significant difference in the Fe-N epsilon bond length between the peroxidases and the globins [for a review, see Powers, L. (1994) Molecular Electronics and Molecular Electronic Devices, Vol. 3, p 211 CRC Press Inc., Boca Raton, FL]. Globins typically have an Fe-N epsilon distance close to 2.1 A while the Fe-N epsilon distance in the peroxidases is closer to 1.9 A. We have proposed [Sinclair, R., Powers, L., Bumpus, J., Albo, A., & Brock, B. (1992) Biochemistry 31, 4892] that strong hydrogen bonding to the proximal histidine is responsible for the shorter bond length in the peroxidases. Here we use site-specific mutagenesis to eliminate the strong proximal hydrogen bonding in cytochrome c peroxidase and to introduce strong proximal hydrogen bonding in myoglobin. Consistent with our hypothesis, elimination of the Asp235-His175 hydrogen bond in CcP results in elongation of Fe-N epsilon from approximately 1.9 to approximately 2.1 A. Conversely, introduction of a similar strong proximal hydrogen bond in myoglobin shortens Fe-N epsilon from approximately 2.1 to approximately 1.9 A. These results correlate well with other biochemical data.
珠蛋白和过氧化物酶虽然进行完全不同的化学反应，但它们的铁血红素活性位点具有共同特征：原卟啉IX辅基通过近端组氨酸残基与蛋白质相连。X射线吸收光谱法提供了一种确定蛋白质中铁血红素活性位点局部结构的方法。我们之前使用X射线吸收光谱法的研究揭示了过氧化物酶和珠蛋白之间Fe-Nε键长的显著差异[综述见Powers, L. (1994)《分子电子学与分子电子器件》，第3卷，第211页，CRC出版社，佛罗里达州博卡拉顿]。珠蛋白的Fe-Nε距离通常接近2.1 Å，而过氧化物酶中的Fe-Nε距离更接近1.9 Å。我们曾提出[Sinclair, R., Powers, L., Bumpus, J., Albo, A., & Brock, B. (1992)《生物化学》31, 4892]，与近端组氨酸的强氢键作用导致过氧化物酶中键长较短。在此，我们使用定点诱变来消除细胞色素c过氧化物酶中强近端氢键，并在肌红蛋白中引入强近端氢键。与我们的假设一致，消除CcP中Asp235-His175氢键会导致Fe-Nε从约1.9 Å延长至约2.1 Å。相反，在肌红蛋白中引入类似的强近端氢键会使Fe-Nε从约2.1 Å缩短至约1.9 Å。这些结果与其他生化数据相关性良好。
