Wen Xin, Bren Kara L
Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, USA.
Biochemistry. 2005 Apr 5;44(13):5225-33. doi: 10.1021/bi047556+.
Proteins in the cytochrome c (cyt c) family with His-Met heme axial ligation display diverse heme electronic structures as revealed by the NMR spectra of their oxidized (paramagnetic) forms. These variations in electronic structure are thought to result primarily from differences in heme axial Met orientation among cyt c species. The factors determining Met orientation in cyts c, however, remain poorly understood. An additional layer of complexity was revealed with the recent finding that the axial Met in Hydrogenobacter thermophilus cytochrome c(552) (Ht cyt c(552)) is fluxional, sampling two conformations rapidly on the NMR time scale, resulting in an unusual compressed range of heme substituent hyperfine shifts [Zhong, L., Wen, X., Rabinowitz, T. M., Russell, B. S., Karan, E. F., and Bren, K. L. (2004) Proc.Natl. Acad. Sci. U.S.A. 101, 8637-8642]. In this work, the (1)H NMR hyperfine shift pattern of Ht cyt c(552) is drastically altered by making the conservative heme pocket mutation Gln64Asn. The mutant (Ht Q64N) displays a pattern of heme hyperfine shifts with a remarkable resemblance to that of structurally homologous Pseudomonas aeruginosa cyt c(551), which has Asn at position 64 and a single heme axial Met conformation. NMR analysis reveals that Asn64 in Ht Q64N is positioned to interact with the axial Met61, whereas the Gln64 in wild-type Ht cyt c(552) is not. It also is found that the heme axial Met is not fluxional in Ht Q64N and has an orientation similar to that in P. aeruginosa cyt c(551). These results indicate that peripheral interactions with the axial Met play an important role in determining axial Met orientation and heme electronic structure in cyts c.
细胞色素c（cyt c）家族中具有组氨酸-甲硫氨酸血红素轴向配体的蛋白质，其氧化（顺磁性）形式的核磁共振光谱显示出多样的血红素电子结构。这些电子结构的变化被认为主要是由于不同细胞色素c物种之间血红素轴向甲硫氨酸取向的差异所致。然而，决定细胞色素c中甲硫氨酸取向的因素仍知之甚少。最近发现嗜热氢杆菌细胞色素c(552)（Ht cyt c(552)）中的轴向甲硫氨酸具有流动性，在核磁共振时间尺度上快速采样两种构象，导致血红素取代基超精细位移的范围异常压缩，这揭示了另一层复杂性[钟，L.，文，X.，拉比诺维茨，T. M.，拉塞尔，B. S.，卡兰，E. F.，和布伦，K. L.（2004年）美国国家科学院院刊101，8637 - 8642]。在这项工作中，通过进行保守的血红素口袋突变Gln64Asn，Ht cyt c(552)的（1）H核磁共振超精细位移模式发生了显著改变。突变体（Ht Q64N）显示出的血红素超精细位移模式与结构同源的铜绿假单胞菌细胞色素c(551)非常相似，后者在第64位是天冬酰胺且具有单一的血红素轴向甲硫氨酸构象。核磁共振分析表明，Ht Q64N中的天冬酰胺64被定位为与轴向甲硫氨酸61相互作用，而野生型Ht cyt c(552)中的谷氨酰胺64则不然。还发现Ht Q64N中的血红素轴向甲硫氨酸不具有流动性，并且其取向与铜绿假单胞菌细胞色素c(551)中的相似。这些结果表明，与轴向甲硫氨酸的外周相互作用在决定细胞色素c中轴向甲硫氨酸的取向和血红素电子结构方面起着重要作用。
