Structural Biology of multi-heme protein from ammonia oxidizing bacterium

(1)细胞色素c-554和细胞色素c-552的结晶研究。虽然从细胞色素c-552制备得到了无定形沉淀物，但从细胞色素c-554中没有得到晶体。(2)多血红素蛋白中电子内和电子间转移的机制。一般认为，在羟胺氧化还原酶将羟胺氧化成亚硝酸根的过程中，会产生“NOH”。这有力地表明，分子中两个电子转移的两个步骤是准确发生的。然而，羟胺氧化还原酶在分子中只有氯化血红素c作为辅基，这是一个电子转移成分。1998年，我们试图从晶体结构的角度来阐明羟胺氧化还原酶中电子转移的分子机制。我们发现该酶是一个三聚体，每个单体分子中含有8个血红素c，表明该酶分子中共有24个血红素c。在分子中，亚铁血红素以亚铁血红素簇的形式排列成环。一种是三个亚铁血红素簇，由一个底物结合的亚铁血红素c、p460和两个亚铁血红素c组成；另一个是双亚铁血红素簇。单体分子中含有一个三个亚铁血红素簇、两个双亚铁血红素簇和一个亚铁血红素C簇，表明分子中的电子转移是三亚铁血红素簇*双亚铁血红素簇*双亚铁血红素簇。本研究阐明了血红素特有的排列方式。

(1)Crystalographic studies on cytochrome c-554 and cytochrome c-552.We investigated the conditions for cyrstallization of cytochrome c-554 and c-552. Although the amorphous precipitates were obtained from cytochrome c-552 preparation, no crystals could not be prepared from cytochrome c-554.(2)Mechanism of intra- and inter-electron transfers in the multi-heme protein.It is generally accepted that the "NOH" is produced as an intermediate in the oxidation of hydroxylamine to nitrite by the hydroxylamine oxidoreductase. This strongly suggest that the two steps of two-electron transfer in the molecule is precisely occurred. However, the hydroxylamine oxidoreductase has only hemes c as prosthetic groups in the molecule which is one electron transfer component. In 1998, we tried to elucidate the molecular mechanism of electron transfer in the hydroxylamine oxidoreductase on the basis of the crystal structure. We found that the enzyme is a trimer and each monomer has 8 hemes c in the molecule, indicating that the enzyme has totally 24 hemes c in the molecule. The hemes are aligned to form a ring as heme-cluster in the molecule. One is triple heme-cluster which is composed to be a substrate-binding heme c, P460, and two hemes c. Another is double heme-cluster. The monomer has one triple-heme cluster, two double heme-clusters and one heme c in the molecule, suggesting that the electron transfer is triple-heme cluster * double heme-cluster * double heme-cluster. The characteristic heme arrangement has been elucidated in the present study.