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≡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 α-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 α-70 Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC≡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 α-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≡C-CH 2 -CH 3 ). This protein shows no detectable reduction of the internal alkyne 2-butyne (H 3 C-C≡C-CH 3 ). In contrast, substitution of the nearby α-191 Gln residue by alanine, in combination with the α-70 Ala substitution, does result in significant reduction 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves sideon 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.

Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

“…This result is consistent with expectations for the substitution of residues predicted to be proximal to the FeMo-co active site. Recent mutagenic, spectroscopic, and density functional theoretic studies have led to a prevailing view that the binding sites for N 2 , azide, acetylene, and hydrazine (N 2 H 4 ) are located on the Fe2-Fe3-Fe6-Fe7 face of FeMo-co (7,8,12,24,29,46). We observe that most modifications involving R284 and F388 (on the Fe2-Fe4-Fe5-Fe6 face) or Q193 and H197 (near Fe6 and Fe2, respectively) lead to substantial losses in N 2 reduction activities.…”

Insights into substrate binding at FeMo-cofactor in nitrogenase from the structure of an α-70Ile MoFe protein variant

“…This model predicted that substitution of a-191 Gln by a-191 Ala might allow 2-butyne to fit into a position to bind side-on to Fe6. The a-191 Ala substituted MoFe protein was found to reduce 2-butyne at considerable rates, also locating the substrate binding site to Fe6 [18]. Finally, a density functional theory study favors binding of alkynes such as propargyl alcohol to Fe6 [19].…”

“…The findings with the a-70 Ala , a-70 Gly , and a-70 Ile substituted MoFe proteins were interpreted as evidence that the residue at position a-70 controls access to FeMo-cofactor for binding of both alkyne and nitrogenous substrates, suggesting that substrate binding occurs at a specific FeS cluster face of FeMo-cofactor (Fe atoms 2, 3, 6, 7) [18]. Subsequent studies on a state of the a-70 Ala MoFe protein with either the substrate propargyl alcohol and propargyl amine trapped suggested that a specific Fe atom (number 6) might be the site of propargyl alcohol binding [19].…”

“…For e.g., the a-70 Ala MoFe protein is found to reduce larger alkynes such as propyne (HC"C-CH 3 ) and propargyl alcohol (HC"C-CH 2 -OH), whereas these compounds are only poor substrates in the wild-type MoFe protein [17]. Likewise, the nitrogenous substrate hydrazine (H 2 N-NH 2 ) can be reduced at much higher rates by the a-70 Ala MoFe protein when compared to the wild-type MoFe protein [18]. In the a-70 Gly MoFe protein, 1-butyne (HC"C-CH 2 -CH 3 ) is found to be a substrate, whereas this compound is only reduced at very low rates by the wild-type MoFe protein.…”

“…To explain these results, both 1-butyne and 2-butyne were modeled into the wild-type structure bound sideon to Fe6 of FeMo-cofactor [18], which indicated 2-butyne cannot bind because of steric overlap with the side chain of a-191 Gln . This model predicted that substitution of a-191 Gln by a-191 Ala might allow 2-butyne to fit into a position to bind side-on to Fe6.…”