Nonenzymatic Synthesis of the P-Cluster in the Nitrogenase MoFe Protein: Evidence of the Involvement of All-Ferrous [Fe(4)S(4)](0) Intermediates

[Image: see text] The P-cluster in the nitrogenase MoFe protein is a [Fe(8)S(7)] cluster and represents the most complex FeS cluster found in Nature. To date, the exact mechanism of the in vivo synthesis of the P-cluster remains unclear. What is known is that the precursor to the P-cluster is a pair of neighboring [Fe(4)S(4)]-like clusters found on the ΔnifH MoFe protein, a protein expressed in the absence of the nitrogenase Fe protein (NifH). Moreover, incubation of the ΔnifH MoFe protein with NifH and MgATP results in the synthesis of the MoFe protein P-clusters. To improve our understanding of the mechanism of this reaction, we conducted a magnetic circular dichroism (MCD) spectroscopic study of the [Fe(4)S(4)]-like clusters on the ΔnifH MoFe protein. Reducing the ΔnifH MoFe protein with Ti(III) citrate results in the quenching of the S = (1)/(2) electron paramagnetic resonance signal associated with the [Fe(4)S(4)](+) state of the clusters. MCD spectroscopy reveals this reduction results in all four 4Fe clusters being converted into the unusual, all-ferrous [Fe(4)S(4)](0) state. Subsequent increases of the redox potential generate new clusters. Most significantly, one of these newly formed clusters is the P-cluster, which represents approximately 20–25% of the converted Fe concentration. The other two clusters are an X cluster, of unknown structure, and a classic [Fe(4)S(4)] cluster, which represents approximately 30–35% of the Fe concentration. Diamagnetic FeS clusters may also have been generated but, because of their low spectral intensity, would not have been identified. These results demonstrate that the nitrogenase P-cluster can be generated in the absence of NifH and MgATP.