Abstract
Nitrogenase catalyzes biological dinitrogen fixation, the reduction of N(2) to 2NH(3). Recently, the binding site for a non-physiological alkyne substrate (propargyl alcohol, HC triple bond C-CH(2)OH) was localized to a specific Fe-S face of the FeMo-cofactor approached by the MoFe protein amino acid alpha-70(Val). Here we provide evidence to indicate that the smaller alkyne substrate acetylene (HC triple bond CH), the physiological substrate dinitrogen, and its semi-reduced form hydrazine (H(2)N-NH(2)) interact with the same Fe-S face of the FeMo-cofactor. Hydrazine is a relatively poor substrate for the wild-type (alpha-70(Val)) MoFe protein. Substitution of the alpha-70(Val) residue by an amino acid having a smaller side chain (alanine) dramatically enhanced hydrazine reduction activity. Conversely, substitution of alpha-70(Val) by an amino acid having a larger side chain (isoleucine) significantly lowered the capacity of the MoFe protein to reduce dinitrogen, hydrazine, or acetylene.
Highlights
Biological reduction of dinitrogen (N2) is catalyzed by nitrogenase, a two-component metalloenzyme
Dinitrogen and Alkyne Reduction Site—Previous studies indicated that propargyl alcohol can access the nitrogenase active site and be trapped there when the MoFe protein ␣-70Val residue is substituted by alanine
Mechanistic Implications and Conclusions—In this work we demonstrate that the size of the amino acid side chain at position ␣-70 in the MoFe protein, which is located directly over a specific Fe-S face of the FeMo-cofactor, controls the ability of nitrogenase to reduce N2, acetylene, and hydrazine
Summary
Materials and Protein Purification—All reagents, unless specified otherwise, were obtained from Sigma-Aldrich and were used as received. Azotobacter vinelandii strains DJ995 (wild-type ␣-70Val), DJ1373 (␣-70Ile), and DJ1310 (␣-70Ala) were grown and nitrogenase proteins were expressed as described previously (13, 14, 20 –22). The ␣-70Val (wild-type), ␣-70Ala, and ␣-70Ile MoFe proteins, each containing a seven-His addition on the ␣-subunit, were purified by a zinc affinity purification protocol as described earlier [20]. Dinitrogen, Acetylene, and Proton Reduction Assays—Activity assays for dinitrogen reduction were performed in 1-ml liquid volumes in vials with 9 ml of total volume using established protocols [13, 23] for 10 min at 30 °C in assay buffer with a MgATP regenerating system (5 mM ATP, 30 mM phosphocreatine, 100 mM MOPS, pH 7.0, 1.2 mg/ml bovine serum albumin, 0.2 mg/ml creatine phosphokinase, 6 mM MgCl2, and 9 mM dithionite).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
