CIRCULAR-DICHROISM AND X-RAY SPECTROSCOPIES OF AZOTOBACTER-VINELANDIINITROGENASE IRON PROTEIN - MGATP AND MGADP INDUCED PROTEIN CONFORMATIONAL-CHANGES AFFECTING THE [4FE-4S] CLUSTER AND CHARACTERIZATION OF A [2FE-2S] FORM

Nucleotide interactions with nitrogenase are a central part of the mec hanism of nitrogen reduction, Previous studies have suggested that MgA TP or MgADP binding to the nitrogenase iron protein (Fe protein) induc e protein conformational changes that control component protein dockin g, interprotein electron transfer, and substrate reduction, In the pre sent study, we have investigated the effects of MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the propertie s of the [4Fe-4S] cluster using circular dichroism (CD) and x-ray abso rption spectroscopies, Previous CD and magnetic CD studies on nitrogen ase Fe protein suggested that binding of either MgATP or MgADP to the Fe protein resulted in identical changes in the CD spectrum arising fr om transitions of the [4Fe-4S](2+) cluster, We present evidence that M gADP or MgATP binding to the oxidized nitrogenase Fe protein results i n distinctly different CD spectra, suggesting distinct changes in the environment of the [4Fe-4S] cluster, The present results are consisten t with previous studies such as chelation assays, electron paramagneti c resonance, and NMR, which suggested that MgADP or MgATP binding to t he nitrogenase Fe protein induced different conformational changes, Th e 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 resu lt of a partial conversion from a [4Fe-4S] cluster to a [2Fe-2S] clust er, 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+) clu ster in Fe protein was apparent from absorption, CD, and electron para magnetic resonance data, Fe K-edge x-ray absorption spectra of the oxi dized Fe protein revealed no changes in the structure of the [4Fe-4S] cluster upon MgATP binding to the Fe protein, The present results reve al that MgATP or MgADP binding to the oxidized state of the Fe protein result in different con formational changes in the environment around the [4Fe-4S] cluster.