XAS spectroscopy reveals X-ray-induced photoreduction of free and protein-bound B12 cofactors
XAS spectroscopy reveals X-ray-induced photoreduction of free and protein-bound B12 cofactors
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DOI：
10.1107/s0909049500006336
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发表时间：
2000-07-01
影响因子：
2.5
通讯作者：
Kratky, C
中科院分区：
文献类型：
--
作者：
Champloy, F;Gruber, K;Kratky, C
具有 B-12 辅因子的几种蛋白质的晶体结构显示，辅因子的 Co 原子与其“下层”配体（通常是来自组氨酸残基的蛋白质衍生咪唑）之间存在异常长的轴向键。使用以下辅因子衍生物进行 X 射线吸收光谱 (XAS) 实验，以检查键伸长是否可能是由于 X 射线诱导的辅因子钴中心还原所致：水钴胺、氰钴胺、甲钴胺、5'-脱氧腺苷钴胺和钴胺素。在 100 K 的水/甘油或水/海藻糖玻璃中对每种辅因子进行了研究，它们既作为未结合的游离物质，又与谷氨酸变位酶的蛋白质成分结合。在用能量 7.76 keV 的 X 射线彻底（10 分钟）照射之前和之后，收集每个样品在钴吸收边缘周围的 XAS 数据。虽然钴胺素 (II) 胺、甲基钴胺素和 5'-脱氧腺苷钴胺素的 XAS 光谱在照射前后相同（在实验误差内），但无论是游离状态还是蛋白质结合状态，含有水钴胺素和氰钴胺素的样品的光谱在照射后发生了显着变化。辐照样品的光谱彼此相似，并且与还原的钴(II)胺的光谱相似但不相同。讨论了这些观察结果对于解释 B-12 蛋白晶体学中观察到的“长”轴向 Co-N 键的影响。
Crystal structures of several proteins with a B-12 cofactor show abnormally long axial bonds between the cofactor's Co atom and its 'lower' ligand, which is typically a protein-derived imidazole from a histidine residue. X-ray absorption spectroscopy (XAS) experiments were carried out with the following cofactor derivatives to examine the question of whether the bond elongation might be due to an X-ray-induced reduction of the cofactor's cobalt centre: aquocobalamin, cyanocobalamin, methylcobalamin, 5'-desoxyadenosylcobalamin and cob(II)alamin. Each cofactor was investigated at 100 K in a water/glycerol or water/trehalose glass, both as unbound free species and bound to the protein components of the enzyme glutamate mutase. XAS data were collected for each sample around the cobalt absorption edge before and after exhaustive (10 min) irradiation with X-rays of energy 7.76 keV. While XAS spectra for cob(II)alamin, methylcobalamin and 5'-desoxyadenosylcobalamin were the same (within experimental error) before and after irradiation, both in the free and protein-bound state, the spectra of samples with aquocobalamin and cyanocobalamin changed substantially upon irradiation. The spectra of the irradiated samples resembled each other and were similar - but not identical - to the spectrum of the reduced cob(II)alamin. The implications of these observations for the interpretation of the 'long' axial Co-N bonds observed crystallographically in B-12 proteins are discussed.