Alkyne substrate interaction within the nitrogenase MoFe protein

Abstract

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC triple bond CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein alpha-70(Val) residue, whose side chain approaches one Fe-S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of alpha-70(Val) by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC triple bond C-CH(3)), whereas, substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the alpha-70(Val) residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC triple bond C-CH(2)-CH(3)). This protein shows no detectable reduction of the internal alkyne 2-butyne (H(3)C-C triple bond C-CH(3)). In contrast, substitution of the nearby alpha-191(Gln) residue by alanine, in combination with the alpha-70(Ala) substitution, does result in significant reduction of 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves side-on binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein.

Figure 1. FeMo-cofactor and its environment

(panel A) A stereoview of FeMo-cofactor and the side chains of α-70^Val and α-191^Gln is shown. Fe atoms 2, 3, 6, and 7 are labeled. R-homocitrate is shown in stick format at the back of the view bound to Mo. (panel B) Same view as panel A with 2-butyne (cyan) placed into position for interaction with Fe atom 6. Van der Waals surfaces are shown around the 2-butyne and the side chain of α-191^Gln. The steric overlap between the 2-butyne and the side chains of α-70^Val and α-191^Gln is apparent. Coordinates are from the protein database file 1M1N.pdb with carbon shown in gray, oxygen in red, iron in rust, sulfur in yellow, molybdenum in magenta, and nitrogen in dark blue. The atom of unknown identity (X) at the center of FeMo-cofactor is shown in green.

Figure 2

Structures of substrates used in this study.

Figure 3. Reduction of short chain alkynes by α–70 MoFe protein variants

The specific activity (nmol product/min/mg MoFe protein) is plotted against the partial pressure of the indicated substrate. Argon makes up the remaining gas to 1 atm total pressure. Substrates: (panel A) acetylene; (panel B) propyne; and (panel C) 1-butyne. The MoFe protein variants are: α-70^Val wild-type (-■-), α-70^Ala (-▲-) and α-70^Gly (-●-). Fits of the data to the Michaelis-Menten equation are shown (lines).

Figure 4. The effect of 2-butyne-1-ol and 2-butyne-1,4-diol on diazotrophic growth of A. vinelandii expressing MoFe protein variants

Cells expressing the MoFe protein variants include: α-191^Ala, α-70^Ala, and α-191^Ala/α-70^Ala. The indicated strain was cultured on agar plates made with Burk’s media lacking a fixed nitrogen source and including the following additions: no addition; + 2-butyne-1-ol, 10 mM 2-butyne-1-ol; and + 2-butyne-1,4 diol, 10 mM 2-butyne-1,4 diol.

Figure 5. Reduction of 2-butyne by α

-70^Ala/α-191^Ala MoFe protein. The specific activity for 2-butyne reduction (nmol of cis-2-butene produced/min/mg MoFe protein) is plotted against the partial pressure of the substrate, 2-butyne. The remaining gas is argon with a final total pressure of 1 atm. The data are fit to a straight line.