Molecular Recognition in Nitrogenase Catalysis and Two Proton-Relay Pathways from P-Cluster to M-Center

Abstract

Based upon the known substrate and inhibitor specificities of nitrogenases containing FeMo-cofactors with various homocitrate and malate analogs (Ludden et al., 1993), the following inferences can be made: (1) the M-center must be a cage with molecular-sieve-shape selectivity for binding substrates and inhibitors inside or/and outside the cage (Tsai, Wan, 1995). (2) With homocitrate-nitrogenase, there are two H⁺-transport pathways through the [Mo]-bound m-(R)-homocitrate and mediated by either αH442 onto the [Mo]-site or by αH195 onto the [Fe2]-site. The highly conserved αH442 may also serve as electron entry. Proton-transport through αH442 leads to high H₂-evolution activity, insensitive to CO inhibition, whereas H⁺-transport through αH195 leads to fairly high H₂-evolution activity, sensitive to CO inhibition. With improperly oriented γ- and β-carboxylates, as in FeMoco with (S)-citramalate, H₂-evolution activity is lost. (3) N₂ may coordinate double-end-on heptanuclearly to the [Mo-3Fe, 3Fe]-site in monocapped octahedral coordination, and thus binds more strongly than C₂H₂ coordinating double-end-on-plus-double-side-on to the [4Fe,2Fe]-site inside the cage. Reductive deuteration of μ6-C₂H₂ in D₂O inside the cage will give only cis-C₂H₂D₂, whereas reductive deuteration of μ-C₂H₂ at the [2Fe]-site outside the cage will give much lower cis selectivity. So, with the strongly competing N₂, reductive deuteration inside the cage will be strongly inhibited, while μ-C₂H₂ reduction at the [2Fe]-site will not, thus decreasing the cis-trans ratio. (4) With citrate-nitrogenase, the N₂-reduction activity drops to only <10% and H₂ evolution is sensitive to CO, indicating that only the second proton-transport pathway is operating.

This work is supported by a National Key Research Project on Symbiotic Nitrogen Fixation and by the National Natural Science Foundation of China.