Stepwise Formation of P-Cluster in Nitrogenase MoFe Protein

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-11, Vol.106 (44), p.18474-18478
• Main Authors: Lee, Chi Chung, Blank, Michael A., Fay, Aaron W., Yoshizawa, Janice M., Hu, Yilin, Hodgson, Keith O., Hedman, Britt, Ribbe, Markus W.
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatase; Atoms; Azotobacter vinelandii; Azotobacter vinelandii - enzymology; Biochemistry; Biological Sciences; Biosynthesis; Dimers; Electrons; Enzymes; Fourier transformations; Light; Metals - chemistry; Models, Molecular; Molybdoferredoxin - metabolism; Mutation - genetics; Protein precursors; Proteins; Spectrum Analysis; Synchrotron radiation; Time Factors; X-Rays
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0909149106

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 ΔnifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [Fe₄S₄]-like cluster pairs in ΔnifH MoFe protein are indeed the precursors to P-clusters, which can be reductively coupled into the mature [Fe₈S₇] structures in the presence of Fe protein, MgATP, and dithionite. Moreover, our observation of a biphasic maturation pattern of P-clusters in ΔnifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the α₂β₂-tetrameric MoFe protein, i.e., one P-cluster is formed in one αβ dimer before the other in the second αβ dimer.