Evidence for a dynamic role for homocitrate during nitrogen fixation: the effect of substitution at the {alpha}-Lys 426} position in MoFe-protein of Azotobacter vinelandii
Résumé
Although it is generally accepted that the active site of nitrogenase is located on the iron-molybdenum cofactor, the exact site(s) of N 2} binding and reduction remain the subject of continuing debate, with both Mo and Fe atoms being suggested as key players. The current consensus favours binding of acetylene and some other non-biologically relevant substrates to the central Fe atoms of the FeMo cofactor (Dos Santos, P.C., et al. (2005) Acc. Chem. Res, 38, 208 - 214). The reduction of N 2} is however a more demanding process than reduction of these alternative substrates because it has a much higher activation energy and does not bind until three electrons have been accumulated on the enzyme. The possible conversion of bidentate to monodentate homocitrate on this three electron-reduced species has been proposed to free up a binding site for N 2} on the Mo atom. One of the features of this hypothesis is that {alpha}-lysine426 facilitates chelate ring opening and subsequent orientation of the monodentate homocitrate by forming a specific H-bond to the homocitrate -CH 2}CH 2}CO 2} -} carboxylate group. In support of this concept, we show that substitution of {alpha}-lysine426 can selectively perturb N 2} reduction without affecting acetylene reduction. We interpret our experimental observations in the light of a detailed molecular mechanics modelling study of the wild type and altered nitrogenase MoFe-proteins.
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