Ryle M J, Lanzilotta W N, Seefeldt L C, Scarrow R C, Jensen G M
Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
J Biol Chem. 1996 Jan 19;271(3):1551-7. doi: 10.1074/jbc.271.3.1551.
Nucleotide interactions with nitrogenase are a central part of the mechanism of nitrogen reduction. Previous studies have suggested that MgATP or MgADP binding to the nitrogenase iron protein (Fe protein) induce protein conformational changes that control component protein docking, interprotein electron transfer, and substrate reduction. In the present study, we have investigated the effects of MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the properties of the [4Fe-4S] cluster using circular dichroism (CD) and x-ray absorption spectroscopies. Previous CD and magnetic CD studies on nitrogenase Fe protein suggested that binding of either MgATP or MgADP to the Fe protein resulted in identical changes in the CD spectrum arising from transitions of the [4Fe-4S]2+ cluster. We present evidence that MgADP or MgATP binding to the oxidized nitrogenase Fe protein results in distinctly different CD spectra, suggesting distinct changes in the environment of the [4Fc-4S] cluster. The present results are consistent with previous studies such as chelation assays, electron paramagnetic resonance, and NMR, which suggested that MgADP or MgATP binding to the nitrogenase Fe protein induced different conformational changes. The CD spectrum of a [2Fe-2S]2+ form of the nitrogenase Fe protein was also investigated to address the possibility that the MgATP- or MgADP-induced changes in the CD spectrum of the native enzyme were the result of a partial conversion from a [4Fe-4S] cluster to a [2Fe-2S] cluster. No evidence was found for a contribution of a [2Fe-2S]2+ cluster to the CD spectrum of oxidized Fe protein in the absence or presence of nucleotides. A novel two-electron reduction of the [2Fe-2S]2+ cluster in Fe protein was apparent from absorption, CD, and electron paramagnetic resonance data. Fe K-edge x-ray absorption spectra of the oxidized Fe protein revealed no changes in the structure of the [4Fe-4S] cluster upon MgATP binding to the Fe protein. The present results reveal that MgATP or MgADP binding to the oxidized state of the Fe protein result in different conformational changes in the environment around the [4Fe-4S] cluster.
核苷酸与固氮酶的相互作用是氮还原机制的核心部分。先前的研究表明，MgATP或MgADP与固氮酶铁蛋白（Fe蛋白）结合会诱导蛋白质构象变化，从而控制组分蛋白对接、蛋白间电子转移和底物还原。在本研究中，我们使用圆二色光谱（CD）和X射线吸收光谱研究了MgATP或MgADP与棕色固氮菌固氮酶Fe蛋白结合对[4Fe-4S]簇性质的影响。先前对固氮酶Fe蛋白的CD和磁CD研究表明，MgATP或MgADP与Fe蛋白结合会导致[4Fe-4S]2+簇跃迁引起的CD光谱发生相同变化。我们提供的证据表明，MgADP或MgATP与氧化态的固氮酶Fe蛋白结合会导致明显不同的CD光谱，这表明[4Fc-4S]簇的环境发生了明显变化。目前的结果与先前的研究如螯合测定、电子顺磁共振和核磁共振一致，这些研究表明MgADP或MgATP与固氮酶Fe蛋白结合会诱导不同的构象变化。还研究了固氮酶Fe蛋白的[2Fe-2S]2+形式的CD光谱，以探讨MgATP或MgADP诱导的天然酶CD光谱变化是否是[4Fe-4S]簇部分转化为[2Fe-2S]簇的结果。在不存在或存在核苷酸的情况下，未发现[2Fe-2S]2+簇对氧化态Fe蛋白的CD光谱有贡献。从吸收、CD和电子顺磁共振数据可以明显看出Fe蛋白中[2Fe-2S]2+簇的新型双电子还原。氧化态Fe蛋白的Fe K边X射线吸收光谱显示，MgATP与Fe蛋白结合后[4Fe-4S]簇的结构没有变化。目前的结果表明，MgATP或MgADP与Fe蛋白的氧化态结合会导致[4Fe-4S]簇周围环境发生不同的构象变化。
