Requirement of homocitrate for the transfer of a 49V-labeled precursor of the iron-vanadium cofactor from VnfX to nif-apodinitrogenase

• Published in: The Journal of biological chemistry Vol. 276; no. 6; pp. 4522 - 4526
• Main Authors: Ruttimann-Johnson, C, Rangaraj, P, Shah, V K, Ludden, P W
• Format: Journal Article
• Language: English
• Published: United States 09.02.2001
• Subjects: Azotobacter vinelandii - enzymology; Azotobacter vinelandii - metabolism; Bacterial Proteins - metabolism; Metalloproteins - metabolism; Nitrogenase - metabolism; Tricarboxylic Acids - metabolism
• ISSN: 0021-9258; 1083-351X

A vanadium- and iron-containing cluster has been shown previously to accumulate on VnfX in the Azotobacter vinelandii mutant strain CA11.1 (DeltanifHDKvnfDGK::spc). In the present study, we show the homocitrate-dependent transfer of (49)V label from VnfX to nif-apodinitrogenase in vitro. This transfer of radiolabel correlates with acquisition of acetylene reduction activity. Acetylene is reduced both to ethylene and ethane by the hybrid holodinitrogenase so formed, a feature characteristic of alternative nitrogenases. Structural analogues of homocitrate prevent the acetylene reduction ability of the resulting dinitrogenase. Addition of NifB cofactor (-co) or a source of vanadium (Na(3)VO(4) or VCl(3)) does not increase nitrogenase activity. Our results suggest that there is in vitro incorporation of homocitrate into the V-Fe-S cluster associated with VnfX and that the completed cluster can be inserted into nif-apodinitrogenase. The homocitrate incorporation reaction and the insertion of the cluster into nif-apodinitrogenase (alpha(2)beta(2)gamma(2)) do not require MgATP. Attempts to achieve FeV-co synthesis using extracts of other FeV-co-negative mutants were unsuccessful, showing that earlier steps in FeV-co synthesis, such as the steps requiring VnfNE or VnfH, do not occur in vitro.