Structure of the native cysteine-sulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8 A resolution.
Structure of the native cysteine-sulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8 A resolution.
复制标题
以 2.8 A 分辨率精制肠球菌 NADH 过氧化物酶的天然半胱氨酸-磺酸氧化还原中心的结构。
DOI：
10.1021/bi961037s
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Hol,WG
中科院分区：
文献类型：
--
作者：
Yeh,JI;Claiborne,A;Hol,WG
为了获得具有天然Cys 42-次磺酸氧化还原中心的黄素蛋白NADH过氧化物酶的晶体结构，采用了一种将晶体减少暴露于环境氧气和在−160 °C下收集数据相结合的策略。天然酶的结构，以2.8 μ m分辨率进行了描述，这些结果最终建立的Cys 42-次磺酸作为过氧化物酶的功能性非黄素氧化还原中心的存在，并提供了第一个结构的任何天然存在的蛋白质次磺酸。Cys 42-次磺酸原子Cα−Cβ−Sγ−O大致定义了一个平面排列，它平行于FAD异咯嗪的表面堆叠，并且位于距离FAD-C4 A仅3.3 nm的位置。His 10-Nε2与次磺酸氧形成氢键，距离为3.2 π。尽管在早期野生型过氧化物酶结构中鉴定的非天然Cys 42-磺酸衍生物的一个氧原子（OX 1）被认为代表天然Cys 42-次磺酸氧[Stehle，T.，艾哈迈德，S。一、Claiborne，A.，& Schulz，G. E.（1991）J.Mol. Biol.221，1325 - 1344]，该结构表明次磺酸氧不占据该位置，也不像OX 1那样与Cys 42-N氢键结合。天然Cys 42-次磺酸结构与两电子还原型谷胱甘肽还原酶的比较提供了一个洞察观察到的次磺酸FAD电荷转移相互作用与野生型和His 10突变过氧化物酶。最近在酶的停流分析中观察到的E·NADH中间体的模型[起重机，E. J.，III，Parsonage，D.，普尔湖B.，& Claiborne，A.（1995）Biochemistry 34，14114 - 14124]也被用来帮助分析次磺酸还原的化学机理。
In order to obtain the crystal structure of the flavoprotein NADH peroxidase with its native Cys42-sulfenic acid redox center, a strategy combining reduced exposure of crystals to ambient oxygen and data collection at −160 °C was applied. The structure of the native enzyme to 2.8 Å resolution is described; these results conclusively establish the existence of the Cys42-sulfenic acid as the functional non-flavin redox center of the peroxidase and provide the first structure for any naturally occurring protein-sulfenic acid. The Cys42-sulfenic acid atoms Cα−Cβ−Sγ−O roughly define a planar arrangement which is stacked parallel to thesiface of the FAD isoalloxazine and positions the sulfenyl oxygen atom only 3.3 Å from FAD-C4A. His10-Nε2 contributes a hydrogen bond to the sulfenic acid oxygen, at a distance of 3.2 Å. Although one oxygen atom (OX1) of the non-native Cys42-sulfonic acid derivative identified in the earlier wild-type peroxidase structure was taken to represent the native Cys42-sulfenic acid oxygen [Stehle, T., Ahmed, S. A., Claiborne, A., & Schulz, G. E. (1991)J.Mol. Biol.221, 1325−1344], this structure shows that the sulfenic acid oxygen does not occupy this position, nor is it hydrogen-bonded to Cys42-N as was OX1. Comparison of the native Cys42-sulfenic acid structure with that of two-electron reduced glutathione reductase provides an insight into the sulfenic acid FAD charge-transfer interaction observed with both wild-type and His10 mutant peroxidases. A model of the E·NADH intermediate recently observed in stopped-flow analyses of the enzyme [Crane, E. J., III, Parsonage, D., Poole, L. B., & Claiborne, A. (1995)Biochemistry34, 14114−14124] has also been generated to assist in analyzing the chemical mechanism of sulfenic acid reduction.