Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase.

The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as αß(γ) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases , previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N to NH, with no significant difference in selectivity over obligatory/parasitic H formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H and NH formation (under Ar or N), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein-protein communication in biological N fixation in .