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Nonenzymatic synthesis of the P-cluster in the nitrogenase MoFe protein: evidence of the involvement of all-ferrous [Fe4S4](0) intermediates.

  • Author(s): Rupnik, Kresimir
  • Lee, Chi Chung
  • Wiig, Jared A
  • Hu, Yilin
  • Ribbe, Markus W
  • Hales, Brian J
  • et al.

Published Web Location Commons 'BY' version 4.0 license

The P-cluster in the nitrogenase MoFe protein is a [Fe8S7] 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 [Fe4S4]-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 [Fe4S4]-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 [Fe4S4](+) state of the clusters. MCD spectroscopy reveals this reduction results in all four 4Fe clusters being converted into the unusual, all-ferrous [Fe4S4](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 [Fe4S4] 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.

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