title: 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 DeltanifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [Fe(4)S(4)]-like cluster pairs in DeltanifH 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 DeltanifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the alpha(2)beta(2)-tetrameric MoFe protein, i.e., one P-cluster is formed in one alphabeta dimer before the other in the second alphabeta dimer.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.
authors.forename: Chi Chung
authors.initials: CC
authors.lastname: Lee
authors.forename: Michael A
authors.initials: MA
authors.lastname: Blank
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Fay
authors.forename: Janice M
authors.initials: JM
authors.lastname: Yoshizawa
authors.forename: Yilin
authors.initials: Y
authors.lastname: Hu
authors.forename: Keith O
authors.initials: KO
authors.lastname: Hodgson
authors.forename: Britt
authors.initials: B
authors.lastname: Hedman
authors.forename: Markus W
authors.initials: MW
authors.lastname: Ribbe
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology*; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D013057:Spectrum Analysis; D013997:Time Factors; D014965:X-Rays
chemical_list: D008670:Metals; D008983:Molybdoferredoxin