Mayr Stefan, Latkoczy Christopher, Krüger Martin, Günther Detlef, Shima Seigo, Thauer Rudolf K, Widdel Friedrich, Jaun Bernhard
Laboratory of Organic Chemistry, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
J Am Chem Soc. 2008 Aug 13;130(32):10758-67. doi: 10.1021/ja802929z. Epub 2008 Jul 22.
Microbial mats collected at cold methane seeps in the Black Sea carry out anaerobic oxidation of methane (AOM) to carbon dioxide using sulfate as the electron acceptor. These mats, which predominantly consist of sulfate-reducing bacteria and archaea of the ANME-1 and ANME-2 type, contain large amounts of proteins very similar to methyl-coenzyme M reductase from methanogenic archaea. Mass spectrometry of mat samples revealed the presence of two nickel-containing cofactors in comparable amounts, one with the same mass as coenzyme F430 from methanogens (m/z = 905) and one with a mass that is 46 Da higher (m/z = 951). The two cofactors were isolated and purified, and their constitution and absolute configuration were determined. The cofactor with m/z = 905 was proven to be identical to coenzyme F430 from methanogens. For the m/z = 951 species, high resolution ICP-MS pointed to F430 + CH2S as the molecular formula, and LA-ICP-SF MS finally confirmed the presence of one sulfur atom per nickel. Esterification gave two stereoisomeric pentamethyl esters with m/z = 1021, which could be purified by reverse phase HPLC and were subjected to comprehensive NMR analysis, allowing determination of their constitution and configuration as (17(2)S)-17(2)-methylthio-F430 pentamethyl ester and (17(2)R)-17(2)-methylthio-F430 pentamethyl ester. The corresponding diastereoisomeric pentaacids could also be separated by HPLC and were correlated to the esters via mild hydrolysis of the latter. Equilibration of the pentaacids under acid catalysis showed that the (17(2)S) isomer is the naturally occurring albeit thermodynamically less stable one. The more stable (17(2)R) isomer (80% at equilibrium) is an isolation artifact generated under the acidic conditions necessary for the isolation of the cofactors from the calcium carbonate-encrusted mats.
在黑海冷甲烷渗漏处采集的微生物垫利用硫酸根作为电子受体，将甲烷进行厌氧氧化（AOM）生成二氧化碳。这些微生物垫主要由硫酸盐还原细菌以及ANME - 1和ANME - 2型古菌组成，含有大量与产甲烷古菌的甲基辅酶M还原酶非常相似的蛋白质。微生物垫样品的质谱分析显示存在两种含量相当的含镍辅因子，一种与产甲烷菌的辅酶F430质量相同（m/z = 905），另一种质量高46 Da（m/z = 951）。分离并纯化了这两种辅因子，确定了它们的组成和绝对构型。已证明m/z = 905的辅因子与产甲烷菌的辅酶F430相同。对于m/z = 951的物种，高分辨率电感耦合等离子体质谱（ICP - MS）表明分子式为F430 + CH₂S，激光烧蚀电感耦合等离子体扇形场质谱（LA - ICP - SF MS）最终证实每个镍存在一个硫原子。酯化反应生成了两种m/z = 1021的立体异构五甲酯，可通过反相高效液相色谱（HPLC）纯化，并进行全面的核磁共振（NMR）分析，从而确定它们的组成和构型分别为(17(2)S)-17(2)-甲硫基 - F430五甲酯和(17(2)R)-17(2)-甲硫基 - F430五甲酯。相应的非对映异构五酸也可通过HPLC分离，并通过后者的温和水解与酯相关联。五酸在酸催化下的平衡表明，(17(2)S)异构体是天然存在的，尽管在热力学上不太稳定。更稳定的(17(2)R)异构体（平衡时为80%）是在从碳酸钙包裹的微生物垫中分离辅因子所需的酸性条件下产生的分离假象。
