Fujii Sotaro, Oki Hiroya, Kawahara Kazuki, Yamane Daisuke, Yamanaka Masaru, Maruno Takahiro, Kobayashi Yuji, Masanari Misa, Wakai Satoshi, Nishihara Hirofumi, Ohkubo Tadayasu, Sambongi Yoshihiro
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Osaka, Japan.
Protein Sci. 2017 Apr;26(4):737-748. doi: 10.1002/pro.3120. Epub 2017 Mar 6.
Thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c' (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit-subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit-subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit-subunit interactions with conservation of the ligand-binding ability.
We report the X-ray crystal structure of cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme-related and subunit-subunit interactions, which were confirmed by a mutagenesis study. The ligand-binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand-binding ability. This study furthers the understanding of the stability and function of cytochromes c.
嗜热嗜热栖热菌细胞色素c'（PHCP）比嗜温的嗜硫红假单胞菌细胞色素c'（AVCP）表现出更高的热稳定性，AVCP具有同型二聚体结构和配体结合能力。为了了解热稳定的PHCP蛋白的热稳定性机制和配体结合能力，首先确定了PHCP的晶体结构。它形成了同型二聚体结构，PHCP和AVCP之间的主链均方根偏差（rmsd）值为0.65 Å。在PHCP结构中，六个特定残基似乎加强了与血红素相关的和亚基-亚基相互作用，这些在AVCP结构中并不保守。亚基-亚基相互作用改变的PHCP变体比血红素相关相互作用改变的变体更严重地去稳定化。PHCP结构进一步揭示了一个配体结合通道和一个五配位血红素，这与在AVCP蛋白中观察到的情况一样。光谱研究清楚地表明一些配体与PHCP蛋白结合。得出的结论是，嗜热菌的二聚体PHCP通过与血红素相关的和亚基-亚基相互作用有效地稳定下来，同时保留了配体结合能力。
我们报道了嗜热嗜热栖热菌细胞色素c'（PHCP）的X射线晶体结构。PHCP的高热稳定性归因于与血红素相关的和亚基-亚基相互作用，这通过诱变研究得到证实。通过分光光度法检测了PHCP的配体结合能力。PHCP在保留配体结合能力时获得了热稳定性。这项研究进一步加深了对细胞色素c的稳定性和功能的理解。
