Localization of a catalytic intermediate bound to the FeMo-cofactor of nitrogenase.
Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation) as well as the reduction of a number of alternative substrates, including acetylene (HC identical with CH) to ethylene (H2C=CH2). It is known that the metallocluster FeMo-cofactor located within the nitrogenase MoFe protein component provides the site of substrate reduction, but the exact site where substrates bind and are reduced on the FeMo-cofactor remains unknown. We have recently shown that the alpha-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site; alpha-70 approaches one face of the FeMo-cofactor, and when valine is substituted by alanine at this position, the substituted nitrogenase is able to accommodate a reduction of the larger alkyne propargyl alcohol (HC identical with CCH(2)OH, propargyl-OH). During this reduction, a substrate-derived intermediate can be trapped on the FeMo-cofactor resulting in an S = 1/2 spin system with a novel electron paramagnetic resonance spectrum. In the present work, trapping of the propargyl-OH-derived or propargyl amine (HC identical with CCH(2)NH(2), propargyl-NH(2))-derived intermediates is shown to be dependent on pH and the presence of histidine at position alpha-195. It is concluded that these catalytic intermediates are stabilized and thereby trapped by H-bonding interactions between either the-OH group or the-NH(3)(+)group and the imidazole epsilon-NH of alpha-195(His). Thus, for the first time it is possible to establish the location of a bound substrate-derived intermediate on the FeMo-cofactor. Refinement of the binding mode and site was accomplished by the use of density functional and force field calculations pointing to an eta(2) coordination at Fe-6 of the FeMo-cofactor.
Igarashi
Robert Y
RY
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USA.
Dos Santos
Patricia C
PC
Niehaus
Walter G
WG
Dance
Ian G
IG
Dean
Dennis R
DR
Seefeldt
Lance C
LC
D000114:Acetylene; D000409:Alanine / Q000737:chemistry; D001395:Azotobacter; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007501:Iron; D007700:Kinetics; D008280:Magnetics; D008956:Models, Chemical; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry*; D009591:Nitrogenase / Q000737:chemistry*; D011485:Protein Binding; D014633:Valine / Q000737:chemistry
D007093:Imidazoles; D008983:Molybdoferredoxin; D006639:Histidine; C029899:imidazole; D007501:Iron; D009591:Nitrogenase; D014633:Valine; D000114:Acetylene; D000409:Alanine