Biomimetic Environment to Study E. coli Complex I through Surface-Enhanced IR Absorption Spectroscopy

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• 作者：S茅bastien Kriegel ; Taro Uchida ; Masatoshi Osawa ; Thorsten Friedrich ; Petra Hellwig
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：October 14, 2014
• 年：2014
• 卷：53
• 期：40
• 页码：6340-6347
• 全文大小：396K
• ISSN：1520-4995

文摘

In this study complex I was immobilized in a biomimetic environment on a gold layer deposited on an ATR-crystal in order to functionally probe the enzyme against substrates and inhibitors via surface-enhanced IR absorption spectroscopy (SEIRAS) and cyclic voltammetry (CV). To achieve this immobilization, two methods based on the generation of a high affinity self-assembled monolayer (SAM) were probed. The first made use of the affinity of Ni-NTA toward a hexahistidine tag that was genetically engineered onto complex I and the second exploited the affinity of the enzyme toward its natural substrate NADH. Experiments were also performed with complex I reconstituted in lipids. Both approaches have been found to be successful, and electrochemically induced IR difference spectra of complex I were obtained.