Stepwise formation of P-cluster in nitrogenase MoFe protein.
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.
Lee
Chi Chung
CC
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.
Blank
Michael A
MA
Fay
Aaron W
AW
Yoshizawa
Janice M
JM
Hu
Yilin
Y
Hodgson
Keith O
KO
Hedman
Britt
B
Ribbe
Markus W
MW
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
D008670:Metals; D008983:Molybdoferredoxin