UC Irvine

The P-cluster of the nitrogenase MoFe protein is a [ Fe₈ S₇] cluster that mediates efficient transfer of electrons to the active site for substrate reduction. Arguably the most complex homometallic FeS cluster found in nature, the biosynthetic mechanism of the P-cluster is of considerable theoretical and synthetic interest to chemists and biochemists alike. Previous studies have revealed a biphasic assembly mechanism of the two P-clusters in the MoFe protein upon incubation with Fe protein and ATP, in which the first P-cluster is formed through fast fusion of a pair of [ Fe₄ S₄]⁺ clusters within 5 min and the second P-cluster is formed through slow fusion of the second pair of [ Fe₄ S₄]⁺ clusters in a period of 2 h. Here we report a VTVH MCD and EPR spectroscopic study of the biosynthesis of the slow-forming, second P-cluster within the MoFe protein. Our results show that the first major step in the formation of the second P-cluster is the conversion of one of the precursor [ Fe₄ S₄]⁺ clusters into the integer spin cluster [ Fe₄ S_3-4]^α, a process aided by the assembly protein NifZ, whereas the second major biosynthetic step appears to be the formation of a diamagnetic cluster with a possible structure of [ Fe₈ S_7-8]^β, which is eventually converted into the P-cluster.