Structural Biology of multi-heme protein from ammonia oxidizing bacterium

(1)细胞色素c-554和细胞色素c-552的晶体学研究。研究了细胞色素c-554和c-552的结晶条件。虽然细胞色素c-552制备得到了无定形沉淀，但细胞色素c-554制备不出晶体。(2)多血红素蛋白内部和内部电子转移的机制。一般认为，“NOH”是羟胺氧化还原酶将羟胺氧化为亚硝酸盐的中间产物。这有力地表明分子中两个电子转移的两个步骤是精确地发生的。然而，羟胺氧化还原酶分子中只有血红素c作为假基，这是一个电子传递组分。1998年，我们试图在晶体结构的基础上阐明羟胺氧化还原酶中电子转移的分子机制。我们发现酶是一个三聚体，每个单体在分子中有8个血红素c，表明酶在分子中总共有24个血红素c。血红素在分子中排列成环状，形成血红素簇。一种是三血红素簇，由结合底物的血红素c P460和两个血红素c组成。另一种是双血红素簇。该单体分子中有一个三血红素簇、两个双血红素簇和一个血红素c，表明电子传递是三血红素簇*双血红素簇*双血红素簇。本研究已阐明了其特有的血红素排列。

(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.