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J. Biol. Chem., Vol. 276, Issue 6, 4522-4526, February 9, 2001

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Requirement of Homocitrate for the Transfer of a ⁴⁹V-Labeled Precursor of the Iron-Vanadium Cofactor from VnfX to nif-apodinitrogenase^*

Carmen Rüttimann-Johnson, Priya Rangaraj, Vinod K. Shah, and Paul W. Ludden

From the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

A vanadium- and iron-containing cluster has been shown previously to accumulate on VnfX in the Azotobacter vinelandii mutant strain CA11.1 (nifHDKvnfDGK::spc). In the present study, we show the homocitrate-dependent transfer of ⁴⁹V 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 (Na₃VO₄ or VCl₃) 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 (₂₂₂) 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.

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