Localization of a Catalytic Intermediate Bound to the FeMo-cofactor of Nitrogenase

Abstract Nitrogenase catalyzes the biological reduction of N2 to ammonia (nitrogen fixation) as well as the reduction of a number of alternative substrates, including acetylene (HC≡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 α-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site; α-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≡CCH2OH, 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≡CCH2NH2, propargyl-NH2)-derived intermediates is shown to be dependent on pH and the presence of histidine at position α-195. It is concluded that these catalytic intermediates are stabilized and thereby trapped by H-bonding interactions between either the–OH group or the–group and the imidazole ϵ-NH of α-195His. 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 η2 coordination at Fe-6 of the FeMo-cofactor.