Open AccessNifEN-B complex of Azotobacter vinelandii is fully functional in nitrogenase FeMo cofactor assembly
Author(s) -
Jared A. Wiig,
Yilin Hu,
Markus W. Ribbe
Publication year - 2011
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.1102773108
Subject(s) - azotobacter vinelandii , nitrogenase , cofactor , electron paramagnetic resonance , chemistry , biosynthesis , stereochemistry , bioinorganic chemistry , metalloprotein , biochemistry , site directed spin labeling , crystallography , nitrogen fixation , enzyme , physics , organic chemistry , nuclear magnetic resonance , membrane , nitrogen
Assembly of nitrogenase FeMoco is one of the key processes in bioinorganic chemistry. NifB and NifEN are two essential elements immediately adjacent to each other along the biosynthetic pathway of FeMoco. Previously, an 8Fe-precursor of FeMoco was identified on NifEN; however, the identity of the biosynthetic intermediate on NifB has remained elusive to date. Here, we present a combined biochemical and spectroscopic investigation of a His-tagged NifEN-B fusion protein ofAzotobacter vinelandii . Our data from the EPR and activity analyses confirm the presence of the 8Fe-precursor in the NifEN entity of NifEN-B; whereas those from the metal, EPR, and UV/Vis experiments reveal the presence of additional [Fe4 S4 ]-type cluster species in the NifB entity of NifEN-B. EPR-, UV/Vis- and metal-based quantitative analyses suggest that the newly identified cluster species in NifEN-B consist of both SAM-motif (CXXXCXXC)- and non-SAM-motif-bound [Fe4 S4 ]-type clusters. Moreover, EPR and activity experiments indicate that the non-SAM-motif [Fe4 S4 ] cluster is a NifB-bound intermediate of FeMoco assembly, which could be converted to the 8Fe-precursor in a SAM-dependent mechanism. Combined outcome of this work provides the initial insights into the biosynthetic events of FeMoco on NifB. More importantly, the full capacity of NifEN-B in FeMoco biosynthesis demonstrates the potential of this fusion protein as an excellent platform for further investigations of the role of NifB and its interaction with NifEN during the process of FeMoco assembly.