Stepwise formation of P-cluster in nitrogenase MoFe protein

Abstract

The P-cluster of nitrogenase is one of the most complex biological metallocenters known to date. Despite the recent advances in the chemical synthesis of P-cluster topologs, the biosynthetic mechanism of P-cluster has not been well defined. Here, we present a combined biochemical, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine-structure investigation of the maturation process of P-clusters in Δ nifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [Fe 4 S 4 ]-like cluster pairs in Δ nifH MoFe protein are indeed the precursors to P-clusters, which can be reductively coupled into the mature [Fe 8 S 7 ] structures in the presence of Fe protein, MgATP, and dithionite. Moreover, our observation of a biphasic maturation pattern of P-clusters in Δ nifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the α 2 β 2 -tetrameric MoFe protein, i.e., one P-cluster is formed in one αβ dimer before the other in the second αβ dimer.