Johnson J L, Rajagopalan K V
J Biol Chem. 1977 Mar 25;252(6):2017-25.
Treatment of rat liver sulfite oxidase with trypsin leads to loss of ability to oxidize sulfite in the presence of cytochrome c as electron acceptor. Ability to oxidize sulfite with ferricyanide as acceptor is undiminished, while sulfite leads to O2 activity is partially retained. Gel filtration of the proteolytic products has led to the isolation of two major fragments of dissimilar size derived from sulfite oxidase. The smaller fragment has a molecular weight of 9500 and appears to be monomeric when detached from sulfite oxidase. It contains the heme in its cytochrome b5 structure, has no sulfite oxidase activity, and is reducible with dithionite but not with sulfite. The heme fragment can mediate electron transfer between pig liver microsomal NADH cytochrome b5 reductase and cytochrome c. The larger fragment has a molecular weight of 47,400 under denaturing conditions but elutes from Sephadex G-200 as a dimer. It contains no heme but retains all of the molybdenum and the modified sulfite-oxidizing capacity present in the proteolytic mixture. All of the EPR properties of the molybdenum center of native sulfite oxidase are retained in the molybdenum fragment. The molybdenum center is a weak chromophore with an absorption sectrum suggestive of coordination with sulfur ligands. Reduction by sulfite generates a spectrum attributable to molybdenum (V). Spectra of oxidized and sulfite-reduced preparations are sensitive to anions and pH. NH2-terminal analysis of native sulfite oxidase and the two tryptic fragments has permitted the conclusion that the sequence represented by the heme fragment is the NH2 terminus of native enzyme. These studies have demonstrated that the two cofactor moieties of sulfite oxidase are contained in distinct domains which are covalently held in contiguity by means of an exposed hinge region. Isolation of functional heme and molybdenum domains of sulfite oxidase after tryptic cleavage has demonstrated conclusively that the cytochrome b5 region of the molecule is required for electron transfer to the physiological acceptor, cytochrome c.
用胰蛋白酶处理大鼠肝脏亚硫酸盐氧化酶，会导致其在以细胞色素c作为电子受体时氧化亚硫酸盐的能力丧失。以铁氰化物作为受体氧化亚硫酸盐的能力未受影响，而亚硫酸盐导致的O2活性部分得以保留。对蛋白水解产物进行凝胶过滤，已分离出两个大小不同的主要片段，它们源自亚硫酸盐氧化酶。较小的片段分子量为9500，从亚硫酸盐氧化酶上分离后似乎呈单体形式。它在其细胞色素b5结构中含有血红素，没有亚硫酸盐氧化酶活性，能用连二亚硫酸盐还原但不能用亚硫酸盐还原。血红素片段可介导猪肝微粒体NADH细胞色素b5还原酶与细胞色素c之间的电子转移。较大的片段在变性条件下分子量为47400，但从Sephadex G - 200上洗脱时为二聚体。它不含血红素，但保留了蛋白水解混合物中所有的钼以及修饰后的亚硫酸盐氧化能力。天然亚硫酸盐氧化酶钼中心的所有电子顺磁共振特性都保留在钼片段中。钼中心是一种弱发色团，其吸收光谱表明与硫配体配位。亚硫酸盐还原会产生归因于钼（V）的光谱。氧化和亚硫酸盐还原制剂的光谱对阴离子和pH敏感。对天然亚硫酸盐氧化酶和两个胰蛋白酶片段进行氨基末端分析，得出结论：血红素片段所代表的序列是天然酶的氨基末端。这些研究表明，亚硫酸盐氧化酶的两个辅因子部分包含在不同的结构域中，这些结构域通过一个暴露的铰链区共价相连。胰蛋白酶切割后分离出亚硫酸盐氧化酶的功能性血红素和钼结构域，最终证明分子的细胞色素b5区域对于向生理受体细胞色素c的电子转移是必需的。
