Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes
Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes
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DOI：
10.1074/jbc.m110.132340
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发表时间：
2010-07-30
影响因子：
4.8
通讯作者：
Gardner, Paul R.
中科院分区：
文献类型：
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作者：
Gardner, Anne M.;Cook, Matthew R.;Gardner, Paul R.
在体外和肝细胞中研究了细胞红蛋白（Cygb）作为一氧化氮双加氧酶（NOD）的功能。抗坏血酸和细胞色素b(5)支持高NOD活性。Cygb-NOD活性显示抗坏血酸、细胞色素b(5)、NO和O-2的k - M值分别为0.25 mM、0.3 μ M、40 nM和20 μ M， k（cat）为0.5 s（-1）。抗坏血酸和细胞色素b(5)还原氧化的Cygb-NOD中间体，其表观二级速率常数分别为1000 M-1 s（-1）和3 × 10(6) M-1 s（-1）。在表达人Cygb的大鼠肝细胞中，Cygb-NOD活性显示出相似的kcat值为1.2 s(-1), k -m（NO）为40 nM， k(cat)/ k -m(NO) （k'(NOD)）值为3 × 10(7) M-1 s(-1)，表明了催化的效率。NO以[NO]/[O-2] 1:500的比例抑制活性，并限制催化周转。该活性被CO竞争性抑制，被氰化物缓慢灭活，与微粒体NOD活性不同。Cygb-NOD为no敏感的乌头酶提供保护。结果确定了Cygb的NOD功能，并证明了抗坏血酸和细胞色素b(5)作为还原剂的作用。
Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b(5) were found to support a high NOD activity. Cygb-NOD activity shows respective K-m values for ascorbate, cytochrome b(5), NO, and O-2 of 0.25 mM, 0.3 mu M, 40 nM, and similar to 20 mu M and achieves a k(cat) of 0.5 s(-1). Ascorbate and cytochrome b(5) reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 M-1 s(-1) and 3 x 10(6) M-1 s(-1), respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar kcat of 1.2 s(-1), a K-m(NO) of 40 nM, and a k(cat)/K-m(NO) (k'(NOD)) value of 3 x 10(7) M-1 s(-1), demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O-2] ratios > 1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b(5) as reductants.