The Substrate-Bound Type 2 Copper Site of Nitrite Reductase: The Nitrogen Hyperfine Coupling of Nitrite Revealed by Pulsed EPR

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• 作者：Maria Fittipaldi ; Hein J. Wijma ; Martin P. Verbeet ; Gerard W. Canters ; Edgar J. J. Groenen ; and Martina Huber
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：November 22, 2005
• 年：2005
• 卷：44
• 期：46
• 页码：15193 - 15202
• 全文大小：375K
• 年卷期：v.44,no.46(November 22, 2005)
• ISSN：1520-4995

文摘

A pulsed electron paramagnetic resonance study has been performed on the type 2 copper siteof nitrite reductase (NiR) from Alcaligenes faecalis. The H145A mutant, in which histidine 145 is replacedby alanine, was studied by ESEEM and HYSCORE experiments at 9 GHz on frozen solutions. Thismutant contains a reduced type 1 copper site which allowed a selective investigation of the type 2 site ofH145A and of its nitrite-bound form H145A (NO₂^-). The experiments yielded hyperfine and quadrupoleparameters of the remote nitrogens of two of the histidines in the type 2 copper site of the protein andrevealed the changes of these values induced by substrate binding (¹⁴NO₂^- and ¹⁵NO₂^-). The HYSCOREexperiments displayed a signal of ¹⁵NO₂^- bound to H145A, from which hyperfine parameters of thenitrite nitrogen were estimated. The small isotropic hyperfine coupling, 0.36 MHz, of the nitrite nitrogen(¹⁴N) suggests that the substrate binds in an axial position to the copper in the type 2 site and that themolecular orbital containing the unpaired electron extends onto the substrate. This and other changes inthe EPR parameters occurring after nitrite binding suggest a change in electronic structure of the site,which most likely prepares the site for the catalytic reaction. We propose that this change is essential forthe reaction to occur.