Spectroelectrochemical Investigation of Intramolecular and Interfacial Electron-Transfer Rates Reveals Differences Between Nitrite Reductase at Rest and During Turnover

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• 作者：艁ukasz Krzemi艅ski ; Lionel Ndamba ; Gerard W. Canters ; Thijs J. Aartsma ; Stephen D. Evans ; Lars J. C. Jeuken
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：September 28, 2011
• 年：2011
• 卷：133
• 期：38
• 页码：15085-15093
• 全文大小：983K
• 年卷期：v.133,no.38(September 28, 2011)
• ISSN：1520-5126

文摘

A combined fluorescence and electrochemical method is described that is used to simultaneously monitor the type-1 copper oxidation state and the nitrite turnover rate of a nitrite reductase (NiR) from Alcaligenes faecalis S-6. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labeled enzyme molecules immobilized on modified gold electrodes, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by F枚rster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. The homotrimeric structure of the enzyme is reflected in heterogeneous interfacial electron-transfer kinetics with two monomers having a 25-fold slower kinetics than the third monomer. The intramolecular electron-transfer rate between the type-1 and type-2 copper site changes at high nitrite concentration (鈮?20 渭M), resulting in an inhibition effect at low pH and a catalytic gain in enzyme activity at high pH. We propose that the intramolecular rate is significantly reduced in turnover conditions compared to the enzyme at rest, with an exception at low pH/nitrite conditions. This effect is attributed to slower reduction rate of type-2 copper center due to a rate-limiting protonation step of residues in the enzyme鈥檚 active site, gating the intramolecular electron transfer.