TY - JOUR
T1 - Alkyne substrate interaction within the nitrogenase MoFe protein
AU - Dos Santos, Patricia C.
AU - Mayer, Suzanne M.
AU - Barney, Brett M.
AU - Seefeldt, Lance C.
AU - Dean, Dennis R.
N1 - Funding Information:
This work was supported by NIH Grant R01-GM59087 to L.C.S. and D.R.D. P.C.D. acknowledges the Advance-VT for a postdoctoral fellowship.
PY - 2007/11
Y1 - 2007/11
N2 - Nitrogenase catalyzes the biological reduction of N2 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-70Val 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-70Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC≡C-CH3), 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-70Val residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC≡C-CH2-CH3). This protein shows no detectable reduction of the internal alkyne 2-butyne (H3C-C≡C-CH3). In contrast, substitution of the nearby α-191Gln residue by alanine, in combination with the α-70Ala 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.
AB - Nitrogenase catalyzes the biological reduction of N2 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-70Val 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-70Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC≡C-CH3), 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-70Val residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC≡C-CH2-CH3). This protein shows no detectable reduction of the internal alkyne 2-butyne (H3C-C≡C-CH3). In contrast, substitution of the nearby α-191Gln residue by alanine, in combination with the α-70Ala 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.
KW - Alkyne reduction
KW - Mechanism
KW - Metalloenzyme
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U2 - 10.1016/j.jinorgbio.2007.05.007
DO - 10.1016/j.jinorgbio.2007.05.007
M3 - Article
C2 - 17610955
AN - SCOPUS:35348871332
SN - 0162-0134
VL - 101
SP - 1642
EP - 1648
JO - Journal of Inorganic Biochemistry
JF - Journal of Inorganic Biochemistry
IS - 11-12
ER -